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Purification and Characterization of Beta-Protein Variants of 20S Proteasomes of the Haloarchaeon Haloferax volcanii

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Purification and Characterization of Beta-Protein Variants of 20S Proteasomes of the Haloarchaeon Haloferax volcanii
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WRIGHT, AMY JOY
Copyright Date:
2008

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Active sites ( jstor )
Amino acids ( jstor )
Archaea ( jstor )
Dehydrogenases ( jstor )
Gels ( jstor )
Genomics ( jstor )
Plasmids ( jstor )
Proteins ( jstor )
Purification ( jstor )
Yeasts ( jstor )
City of Jacksonville ( local )

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University of Florida
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University of Florida
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Copyright Amy Joy Wright. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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5/31/2008
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496806887 ( OCLC )

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PURIFICATION AND CHARAC TERIZATION OF BETA-PROTEIN VARIANTS OF 20S PROTEASOMES OF THE HALOARCHAEON Haloferax volcanii By AMY JOY WRIGHT A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Amy Joy Wright

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This work is dedicated to my family, Steve, Sandi, and Barry Wright. Their love, laughter, support, and sacrifice have been consta nt. Because of this, in all aspects of my life, I am the luckiest person in the world. I am truly grateful.

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ACKNOWLEDGMENTS I thank Dr. Julie A. Maupin-Furlow for allowing me to work in her lab and for her discussions and patience. I also thank my committee members, Dr. Madeline Rasche and Dr. Richard Lamont, for their insights and their time. I thank my lab mates for their suggestions and for making the lab an enjoyable place to work. I also thank Moshe Mevarech for kindly providing the plasmid encoding malate dehydrogenase used in the proteinase activity studies. iv

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES..........................................................................................................vii LIST OF ABBREVIATIONS..........................................................................................viii ABSTRACT.......................................................................................................................ix CHAPTER 1 INTRODUCTION........................................................................................................1 Overview.......................................................................................................................1 Haloferax volcanii Background....................................................................................2 Proteasomal Roles in the Cell.......................................................................................4 Proteasome Structure....................................................................................................6 Proteasomal Activity..................................................................................................13 20S Proteasomal Processing.......................................................................................14 Proteasomal Assembly................................................................................................23 2 PURIFICATION AND CHARACTERIZATION OF BETA-PROTEIN VARIANTS OF 20S PROTEASOMES OF THE HALOARCHAEON Haloferax volcanii.......................................................................................................................31 Introduction.................................................................................................................31 Methods and Materials...............................................................................................32 Materials..............................................................................................................32 Strains, Media, and Plasmids...............................................................................33 Cloning and Site-Directed Mutagenesis..............................................................33 DNA Purification and Transformation................................................................34 Synthesis and Purification of -protein Mutant 20S Proteasomes......................35 Synthesis and Purification of Malate Dehydrogenase from Haloarcula marismortui......................................................................................................36 Immunoanalysis...................................................................................................37 Protein and Peptide Hydrolysis Assays...............................................................37 Results.........................................................................................................................39 Processing Mechanisms of Variant -proteins (-His, -Thr1Ala-His, and -Thr1Ser-His) in H. volcanii.............................................................................39 v

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Assembly of 20S Proteasome with -protein Variants........................................42 Peptide Hydrolyzing Activity of -protein Variants...........................................44 Protein Hydrolyzing Activity of -protein Variants............................................45 Kinetics of -protein Variants.............................................................................48 Discussion...................................................................................................................49 3 CONCLUSIONS........................................................................................................70 LIST OF REFERENCES...................................................................................................72 BIOGRAPHICAL SKETCH.............................................................................................82 vi

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LIST OF FIGURES Figure page 1-1 General structure of 20S proteasomes......................................................................29 1-2 Model of location of -subunits in yeast 20S proteasomes......................................30 2-1 Partial purification of the variant -proteins............................................................58 2-2 Immunoanalysis of Ni 2+ -Sepharose purified protein fractions of H. volcanii expressing -His, -Thr49Ser-His, and -Thr49Ala-His proteins...........................59 2-3 Levels of -subunit propeptide processing in 20S proteasomes containing the -protein variants.........................................................................................................60 2-4 Processing of the -Thr49Ala-His variant...............................................................61 2-5 Assembly and suggested processing levels of the -His variants............................62 2-6 Assembly of the variant -proteins..........................................................................63 2-7 Stability of the -protein variants.............................................................................64 2-8 Purity of -His and -Thr49Ala-His variants..........................................................65 2-9 Peptidase activity of the -protein variants..............................................................66 2-10 Proteinase activity of the -protein variants.............................................................67 2-11 Kinetics of the -protein variants -His and -Thr49Ala-His.................................68 2-12 Hanes-Wolff plot for 20S proteasomes containing the -Thr49Ala-His variant.....69 vii

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LIST OF ABBREVIATIONS AAA... A TPases a ssociated with various cellular a ctivities Amc.-amino-4-methylcoumarin CPcore particle DEAE.diethylaminoethyl DMSOdimethylsulfoxide DTTdithiothreitol -IFN..gamma interferon HRPhorseradish peroxidase MES(2-N-morpholino) ethanesulfonic acid MHC Imajor histocompatibility complex class I Ntn.N-terminal nucleophile PANproteasome-activating nucleotidase PGPH.peptidylglutamyl-peptide hydrolyzing PRPP..5-phosphoribosyl-1-pyrophosphate PVDF.polyvinylidene difluoride RP..regulatory particle SDS-PAGEsodium dodecyl sulphate-polyacrylamide gel electrophoresis TCA.trichloroacetic acid U.units viii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science PURIFICATION AND CHARACTERIZATION OF BETA-PROTEIN VARIANTS OF 20S PROTEASOMES OF THE HALOARCHAEON Haloferax volcanii By Amy Joy Wright May 2006 Chair: Julie A. Maupin-Furlow Major Department: Microbiology and Cell Science During the final stages of 20S proteasome maturation, the NH2-terminal propeptides of -type subunits are autocatalytically cleaved to expose an active site threonine residue. Currently it is unclear whether the -subunits of archaeal proteasomes are processed via an interor intramolecular mechanism. The haloarchaeon Haloferax volcanii is an ideal model to readily examine assembly of 20S proteasomes in vivo since a genetic exchange system is available. This organism is simple compared to its eukaryal counterpart and is comparable in a number of cellular mechanisms. In this study, the active site threonine residue at amino acid position 49 of the H. volcanii -protein was modified to alanine, as well as serine. The modified -proteins (-His, -Thr49Ala-His, -Thr49Ser-His) were separately expressed with C-terminal polyhistidine tags in H. volcanii and purified by Ni2+-Sepharose chromatography. Protein fractions were analyzed by Western blot using polyclonal antibodies raised against 1, 2, and . The ix

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results revealed that all three -His tagged proteins assembled with 1, 2, and expressed from the genome. The -subunit expressed from the genome was processed in all three samples; however, differences were observed in the extent of the processing of the -His variants. The -His protein appeared to be completely processed. In contrast, only a fraction of the -Thr49Ser-His proteins were cleaved, while the -Thr49Ala-His proteins did not appear to be processed. Assembly of full 20S proteasomal complexes containing the -His variants occurs as demonstrated by gel filtration, although these variants are shown to be unstable and dissociate under certain conditions. The peptidase and proteinase activities of the -His and -Thr49Ala-His variants were examined in more detail. Specific peptide hydrolyzing activity was detected for these purified -variants using N-Succ-LLVY-7-amino-4-methylcoumarin (Amc) as a fluorogenic substrate. The -His variant proteasome showed cooperative kinetics with the peptide substrate as evidenced by sigmoidal kinetics, while the -Thr49Ala-His modified 20S proteasomes displayed Michaelis-Menten kinetics. Protein hydrolyzing activity, using bovine insulin B chain as a substrate, was also detected for 20S proteasomes with the -His and -Thr49Ala-His modified proteins. These findings are important for elucidating proteasomal assembly, processing, and catalytic activity in archaea. Results from this study provide evidence that -protein processing might occur by an intramolecular mechanism, as full assembly of 20S proteasomes occurs with the -His protein variants, yet genomic encoded -subunits do not appear to process the -Thr49Ala-His variants. This study also demonstrates by kinetic analyses the cooperativity of 20S proteasomes in H. volcanii, as well as the loss of cooperativity when the active site residues are modified. x

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CHAPTER 1 INTRODUCTION Overview Proteasomes are molecular machines designed for controlled proteolysis. These energy-dependent proteases are found in all domains of life (Dahlmann et al., 1989). This fact reflects their importance in protein turnover. The diversity of proteasomal substrates reveals the central role proteasomes have in many cellular responses, including stress response, cell cycle control, and metabolic adaptation (Bochtler et al., 1999). Proteasomes are also important in mammalian specific immune response, where they act to provide peptides to the major histocompatibility complex class I (MHC I) for display by hydrolyzing antigens (Bochtler et al., 1999). Although there is variety in the number of subunit isoforms forming 20S proteasomes among organisms, the general structure of the 20S proteasome (the catalytic core) remains similar from archaea to humans (Lwe et al., 1995; Groll et al., 1997; Unno et al., 2002; Groll et al., 2003). Because of this conserved architecture, the simplicity of archaeal proteasomal systems relative to eukarya make archaea, such as H. volcanii, attractive models for eukaryotic systems. H. volcanii is also an ideal system because of useful genetic tools available for the organism, such as a transformation system (Cline et al., 1989), shuttle vectors with antibiotic resistance (Lam and Doolittle 1989; Holmes and Dyall-Smith 1990; Holmes et al., 1994), and a gene knockout system (Bitan-Banin et al., 2003). In general, despite being prokaryotes, archaea are ideal models for eukaryotic systems due to similarities in in their genetic mechanics (DNA 1

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2 replication, transcription, and translation) and protein quality control systems (proteasomes and chaperones) compared to their eubacterial counterparts. In fact, fundamental understanding of basic energy-dependent proteolysis has been advanced due to archaeal homologs of eukaryotic 26S proteasomes (Baumeister et al., 1998; Maupin-Furlow et al., 2001). In eukaryotes and archaea, assembly of 20S proteasomes appears to occur in two steps: the formation of preproteasome complexes, where the -rings and unprocessed -rings combine, and finally the dimerization of two half-proteasomes at the -ring interface to form 20S proteasome complexes. Cleavage of the -propeptides yields mature 20S proteasomes (Kisselev et al., 2000; Kwon et al., 2004). In archaea, the question still remains whether -protein processing occurs via an intraor intermolecular mechanism. This project will examine proteasomal processing and assembly relative to eukaryal proteasomal systems by using an archaeal proteasome model and in vivo assays. Also, this project will investigate the effects of site-directed mutagenesis at the active site threonine residue on peptidase and proteinase activity. This is the first report of such a construct made in H. volcanii. Knowledge from this study will help clarify proteasomal structure and function within the cell. Haloferax volcanii Background The archaeon, Haloferax volcanii, belongs to the family Halobacteriaceae and was isolated from the Dead Sea in the early 1970s (Mullakhanbhai and Larsen 1975). Originally described as Halobacterium volcanii, this pleomorphic microorganism is a moderate halophile and requires salt concentrations of 2-3 M NaCl , as well as 0.1-0.2 M magnesium for viability, yet it can also survive under high magnesium concentrations of

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3 1.4-1.5 M (Mullakhanbhai and Larsen 1975; Oren 2002). There are three groups of halophiles that have been described: aerobic archaea, anaerobic methanogenic archaea, and halophilic bacteria (Kamekura 1998). The majority of the extreme halophilic enzymes that have been characterized so far are from the family Halobacteriaceae (Madern et al., 2000), and more recently from the halobacterium Salinibacter ruber, which belongs to the family Crenotrichaceae (Oren et al., 2002). It is interesting to note that the activity of some enzymes purified from archaeal halophiles decreases with increasing salt concentrations (Madern et al., 2000). For example, the activity of dihydrolipoamide dehydrogenase from H. volcanii has been shown to decline with excessive salt concentrations (Mevarech et al., 1977; Jolley et al., 1996). In contrast, however, the activities of most haloarchaeal proteins are optimal at high salt. For example, the activity of -galactosidase from Haloferax alicantei is optimal at 4 M NaCl (Holmes et al., 1997). In addition, the tetramer, malate dehydrogenase, from Haloarcula marismortui has been shown to dissociate into inactive monomers if incubated at salt conditions below 2 M, with subsequent reassociation into active tetramers when quickly incubated in high salt conditions (Mevarech et al., 2000). Continual incubation of the malate dehydrogenase monomers in low salt solution decreases the amount of tetramers that can reassociate in high salt solutions (Mevarech et al., 2000). It has also been shown that 20S proteasomes from H. volcanii that dissociate in low salt can rapidly reassociate into full active proteasomes upon incubation in a high salt buffer (2 M NaCl) (Wilson et al., 1999). The type of salt can be significant. For example, the activity of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase from H. volcanii increases in high KCl

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4 concentrations, but decreases in excessive NaCl concentrations (Bischoff and Rodwell 1996). The ability of H. volcanii and other haloarchaea to survive such harsh conditions relies on the delicate balance between a high salt environment and high intracellular concentrations of KCl salt that nears saturation (Lanyi 1974; Oren 1986, 2002; Madern et al., 2000; Oren et al., 2002). One manner in which these halophiles tolerate the hypersaline cytosol is the excess of acidic amino acids and low content of basic amino acid residues, as well as a low proportion of hydrophobic amino acids found in the halophilic proteins (Lanyi 1974; Oren 1986; Madern et al., 2000; Mevarech et al., 2000). This anomaly is a signature found among halophiles which is offset by a higher than normal content of ‘borderline’ hydrophobic amino acids serine and threonine (Lanyi 1974). It is argued that the high negative surface charge of the prevalent acidic amino acid residues in a halophilic protein renders the protein more soluble in high salt conditions, where the charge is neutralized by tightly bound water dipoles (Madern et al., 2000). Apparently, the requirement for high salt conditions is due to a low affinity of salt binding to different sites on the surface of the folded protein, thus stabilizing its active conformation (Madern et al., 2000). Proteasomal Roles in the Cell 20S proteasomes are compartmentalized proteases that regulate protein turnover. The containment of active sites within the barrel-like proteasome prevents destruction of proteins not intended for hydrolysis, as entry into 20S proteasomes is tightly regulated (Wenzel and Baumeister 1995). The proteasome plays a role in cell stress response, as it is involved in degrading misfolded and denatured proteins (Finley et al., 1987; Kostova and Wolf 2003). In eukaryotes, proteins that are destined to be secreted are translocated

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5 in an unfolded state to the endoplasmic reticulum, where in this organelle, the proteins are modified and folded into their active conformations (Haigh and Johnson 2002; Kostova and Wolf 2003). In the endoplasmic reticulum, discrimination between folded and misfolded proteins occurs, with the misfolded proteins being retranslocated back into the cytoplasm and tagged with ubiquitin with subsequent degradation by the 26S proteasome (Ellgaard et al., 1999; Kostova and Wolf 2003). In addition to “housekeeping” functions, the proteasome is also involved in regulating cell cycle control (King et al., 1996; Pagano 1997). For example, as yeast cells enter the S phase of cell division, there is a requirement for the ubiquitin-proteasome system to degrade cyclin-dependent kinases and cyclin-dependent kinase inhibitors, which encourage the cell to proliferate and which inhibit the cell from dividing in response to some type of cell stress, respectively (Pagano 1997). This certain phase of the cell cycle is just one example. Proteasomes also act to provide antigenic peptides for display by MHC I by substituting constitutive proteasomal subunits for immunoproteasome subunits and degrading antigens within the cell (Goldberg and Rock 1992; Michalek et al., 1993; Rock et al., 1994). Also, 20S proteasomes have been found to degrade short-lived proteins, such as transcription factor inhibitors (Hershko and Ciechanover 1998). For example, nuclear factor B is an inducible transcription factor involved in the central immune, stress, and developmental process (Hershko and Ciechanover 1998). With the cell under certain antigenic stress, the inhibitor of nuclear factor B is degraded via the ubiquitin pathway, which includes 20S proteasomes (Hershko and Ciechanover 1998). In general, 20S proteasomes hydrolyze substrate proteins processively into peptides of varying length (4 to 25 amino acids long) that are ultimately hydrolyzed to free amino acids by downstream

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6 proteases and peptidases and subsequently used for de novo protein synthesis (Akopian et al., 1997; Kisselev et al., 1998, 1999b). Proteasome Structure The 26S proteasome is an ATP-dependent eukaryotic protease that consists of a 20S core particle (CP) or 20S proteasome and one or two 19S regulatory particles (RP) that recognize ubiquinated substrates (Coux et al., 1996). These RP or proteasomeassociated ATPases belong to the AAA family ( A TPases a ssociated with various cellular a ctivities) (Neuwald et al., 1999; Ogura and Wilkinson 2001). The RP of yeast have been shown to be composed of two multisubunit substructures consisting of a lid and a base. There are several roles that belong to these RP, some of which include substrate recognition, unfolding, and translocation into the 20S proteolytic chamber (Maupin-Furlow et al., 2003). AAA proteins are also known to act as chaperones that “proofread” after initial binding of a substrate and determine which proteins are to be refolded, disaggregated, or degraded (Wickner et al., 1999). Archaeal homologs of these ATPases are known as proteasome-activating nucleotidases (PANs), where in H. volcanii, there are two known to date (PanA and PanB) (Reuter et al., 2004). Much detail about the structure of the catalytic 20S proteasome is known due to X-ray diffraction studies, and the overall architecture of the 20S CP is conserved among the three domains of life (Lwe et al., 1995; Groll et al., 1997; Unno et al., 2002; Groll et al., 2003). Briefly, there are fourteen -type subunits and fourteen -type subunits that oligomerize to form four heptameric rings, which in turn stack to form a cylindrical barrel-shaped protease (Groll et al., 2003). The proteolytically inactive -type rings form on the top and bottom of the barrel, while the two -type rings join to form the catalytic

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7 core (Figure 1-1) (Bochtler et al., 1999). Both the -type and -type subunits have the same fold (Lwe et al., 1995; Groll et al., 1997), where they are both composed of a four-layer + structure with antiparallel five-stranded -sheets and three -helices. This structure is characteristic of the family N-terminal nucleophile (Ntn) hydrolases in which the 20S proteasomes belong (Smith et al., 1994; Brannigan et al., 1995; Duggleby et al., 1995; Lwe et al., 1995; Oinonen et al., 1995). A feature that does seem to distinguish eukaryal proteasomes from prokaryotic proteasomes, however, has been shown with these crystal structure data. 20S proteasomes from Thermoplasma show an opening of about 1.3-nm wide in the -rings (Lwe et al., 1995). Also, electron microscopy data of 20S proteasomes that were incubated with gold-labeled substrates show that this opening is in fact an entry site (Wenzel and Baumeister 1995). In yeast, however, there is no obvious substrate entry site (Groll et al., 1997), but it has been shown that the yeast proteasome was crystallized in the closed form, where the N-terminal tails of the -subunits seemed to form a plug, where these tails in the archaeal proteasome structure are disordered giving the appearance of an open gate (Zwickl 2002). To clarify the differences between the openings, nine N-terminal amino acid residues were deleted from the 3-subunit of the yeast 20S proteasome (Groll et al., 2000). This mutation created a disorder among other -subunits and thus formed an opening in yeast 20S proteasomes similar to the Thermoplasma 20S proteasome. In general, however, only unfolded proteins can pass through this narrow passageway (Wenzel and Baumeister 1993). While found in Eukarya, Archaea, and Gram-Positive Actinomycetales (Dahlmann et al., 1989; Tamura et al., 1995), the number of different 20S proteasomes produced in a

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8 cell varies among these organisms. For example, yeast cells and nematodes (i.e., Caenorhabditis elegans) produce only one type of 20S proteasome (Maupin-Furlow et al., 2003), while higher eukarotes synthesize constitutive and ancillary 20S proteasomes. In addition to the “housekeeping” proteasome, for example, the vertebrate immunoproteasome is expressed when induced by the cytokine -interferon (-INF) (Tanaka and Kasahara 1998; Van den Eynde and Morel 2001). Also, within a rat skeletal muscle cell, 20S proteasomes can be divided into six subtypes (Dahlmann et al., 2001). Two of the three major subtypes are constitutive 20S proteasomes, while two of the three minor subtypes that are expressed are immunoproteasomes (Dahlmann et al., 2001). This is in contrast to 20S proteasomes found in spleen cells, where the majority are immunoproteasomes (Eleuteri et al., 1997; Cardozo and Kohanski 1998; Dahlmann et al., 2000). A small amount of constitutive 20S proteasomes are found in spleen cells (Dahlmann et al., 2000). This is not surprising in that the spleen is an important organ in the immune response of eukaryotes. It is, however, feasible that many subtypes can form, as a partial exchange of constitutive subunits with immuno-subunits does occur (Dahlmann et al., 2001). It is possible that plants also produce multiple 20S proteasomes, as up to 23 different and -type genes have been identified (Fu et al., 1998). Two different 20S proteasomes are synthesized in the haloarchaeon Haloferax volcanii, with differing -subunit composition (i.e., 1---1 and 1---2), that is to say either only 1-subunits are present in the -rings or that 1 and 2-subunits are present perhaps each in their own ring. (Kaczowka and Maupin-Furlow 2003). Different subunits of 20S proteasomes are found among all domains of life. The crystal structure of yeast 20S proteasome reveals that it contains seven different, yet

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9 related -type and -type subunits (i.e., two copies each of 1 to 7 and 1 to 7) while still maintaining the conserved barrel-like architecture (Groll et al., 1997). The distribution of these and -type subunits is not random, but is ordered with each subunit having a distinct location defined by their neighboring subunits (Kopp et al., 1993, 1995). In higher eukaryotes, an auxiliary 20S proteasome (e.g., immunoproteasome) is formed when additional subunits (1i, 2i, 5i) are expressed (Groettrup et al., 2001). These subunits replace the closely related -subunits of the constitutively expressed proteasomal -subunits. Bacterial 20S proteasomes are much simpler in that they are formed with a single -type and single -type subunit (De Mot et al., 1999). The bacterium, Rhodococcus erythropolis, is an exception, however, because two -type and two -type subunits present in the cell are synthesized to form a single 20S proteasome (Zhl et al., 1997b). To detemine this, Zhl et al. (1997b) used an E. coli BL21 (DE3) expression system that allowed the coexpression of the genes encoding the and -subunits in all possible combinations (Zhl et al., 1997b). These genes were found to be arranged in two separate operons with the 1and 2-subunits being 81.6 percent identical and the 1and 2-subunits being 86.5 percent identical (Tamura et al., 1995; Zhl et al., 1997b). Also in this study, 20S proteasomes were purified from R. erythropolis for comparison. With the availability of four proteasomal subunits, there are many models of assembly that can be made; however, several models were ruled out based on pore gradient electrophoresis. This techinique yields sufficient resolution to discriminate among four different constructs that differ in theoretical masses by 6 kDa (Zhl et al., 1997b). When the products of two constructs (1-2 and 2-1) were mixed together, two distinct bands

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10 were found; however, the proteasome purified from R. erythropolis showed a single sharp band between the two bands on the non-denaturing gradient gel. It was therefore concluded that a single population of proteasomes exists within R. erythropolis that is built from all four subunits; however, whether the populations consist of homo-oligomeric rings formed by 1, 2, 1, and 2 or random placement of subunits still remains to be determined (Zhl et al., 1997b). The only archaeon to date shown to synthesize three different 20S proteasomal proteins (1, 2, and ) is H. volcanii (Wilson et al., 1999). This study used a purified 1/ proteasome to construct DNA-hybridization probes to isolate the corresponding genes, psmA and psmB, respectively. It was found that a second DNA fragment hybridized to the -subunit DNA probe on the H. volcanii genome. This gene was cloned and designated psmC. Alignment of psmA and psmC deduced protein sequences with other known sequences revealed a high identity to the -type proteins that included a highly conserved N-terminal extension that is characteristic of -type subunits. To identify if the 2-subunit was expressed, the purification methods were revised to increase the yield, including harvesting H. volcanii cells at mid-log to stationary phase and concentrating purified fractions with chymotrypsin-like activity by dialysis, as opposed to precipitating 20S proteasomes from a dilute protein sample. The proteasomes purified from this revised protocol catalyzed peptide hydrolysis with N-Succ-LLVY-Amc as a substrate similar to the 1/ proteasome, although SDS-PAGE analysis of the newly purified proteasome revealed an additional band migrating at 34.5 kDa. Internal protein sequences of this extra band were identical to the deduced protein sequence of the PsmC (or 2) protein.

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11 The role of the 2-subunit was shown to be a 20S proteasomal subunit of H. volcanii (Kaczowka and Maupin-Furlow 2003). This study included separately expressing the 1, 2, or -proteins with histidine tags in H. volcanii. The histidine tagged proteins of recombinant H. volcanii strains were separately purified by Ni 2+ -affinity and gel filtration chromatography. Purification of the 2-His proteins by affinity chromatography and gel filtration revealed that the 2-protein copurified with high molecular mass complexes of 600 kDa. The 600 kDa complexes that copurified with the 2-His subunit also harbored chymotrypsin-like activity comparable to the same activity of 1/ proteasomes purified from wild type H. volcanii. In addition, these recombinant complexes contained 1, 2 (with and without a histidine tag), and -subunits, as determined by Western blot analysis with polyclonal antibodies against 1, 2, and , and were similar in structure to wild type 1/ proteasomes, as determined by transmission electron microscopy. For comparison, the recombinant H. volcanii strains expressing 1-His were also used (Kaczowka and Maupin-Furlow 2003). The expressed recombinant protein was purified in a similar manner to the 2-His protein. Similar to 2-His proteins, the 1-His proteins were purified in high molecular mass complexes (600 kDa). These complexes also contained 1, 2, and -subunits, as determined by Western blot analysis, with chymotrypsin-like peptidase activity and architecture similar to wild type H. volcanii 20S proteasomes. Likewise, when unprocessed -His proteins were expressed from recombinant H. volcanii, high molecular mass complexes formed with 1, 2, and -specific proteins with full chymotrypsin-like activity and expected 20S proteasomal

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12 structure. These results suggest that 2-proteins associate with 1 and -proteins to form full active 20S proteasomal complexes in H. volcanii. The interactions between the 1and 2-proteins were analyzed by purifying 1-220S proteasomes from stationary phase wild type H. volcanii, along with purified 20S proteasomes that contained the 1and -proteins from a mutant H. volcanii strain that does not encode the psmC gene (i.e., the 2-subunit), which served as a control (Kaczowka and Maupin-Furlow 2003). It was discovered during this study that low levels of CaCl 2 stablized 20S proteasomes from H. volcanii in the absence of salt without any effect to peptidase activity. This finding allowed for the two previously described complexes to be separated by native gels when pre-incubated in 10 mM CaCl 2 . These two 20S proteasomes migrated as two distinct bands on a native gel suggeting that they were two different complexes. Both of these complexes hydrolyzed N-Succ-LLVY-Amc at comparable rates. It was suggested that 1-220S proteasomes are the proteasomes found in H. volcanii grown to stationary phase. The subunit topology of the two -subunits (1 and 2) was also examined as independent rings and in 20S proteasomal complexes (Kaczowka and Maupin-Furlow 2003). Wild type purified 20S proteasomes (1-2-), 20S proteasomes expressed from an H. volcanii strain with the psmC gene (2-protein) deleted, and recombinant proteasomes with -His proteins were separately cross-linked with glutaraldehyde under conditions that encouraged dimer formation and analyzed by Western blot. The latter 20S proteasomal complex was used since these were likely to contain a mixture of 20S proteasomes with all possible -subunit combinations. The 1 and 2-proteins were also separately purified from recombinant E. coli and used as controls. Using an anti-2

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13 antibody, a distinct band appeared for 1-2proteasomes, as well as proteasomes that contained -His, that was not detected in proteasomes containing 1 and -subunit ring formations. The signal for this detected band also migrated similarly to the 2-protein expressed from recombinant E. coli. A separate band specific for 1-proteins was also detected for all three 20S proteasomes examined and was found to migrate analogously to the 1-dimer expressed from E. coli. Also, 1/-dimers were found for all three 20S proteasomes examined and 2/-dimers were detected for all 20S proteasomes except those that expressed only 1 and -subunits. Therefore, it was concluded that an 1-220S proteasome in H. volcanii is composed primarily of 1-1 contacts and 2-2 contacts in which one -ring is composed of 1-subunits and the other -ring is comprised of 2-subunits. It is likely that in H. volcanii 1-subunits and 2-subunits form separate homo-heptameric rings. Proteasomal Activity It has been shown in eukaryotic proteasomes that three different types of peptidase activities exist: peptidylglutamyl-peptide hydrolyzing, trypsin-like, and chymotrypsin-like, where substrate cleavage occurs on the carboxyl side of acidic, basic, and hydrophobic amino acid residues, respectively (Orlowski 1990; Chen and Hochstrasser 1996; Arendt and Hochstrasser 1997; Heinemeyer et al., 1997; Dick et al., 1998). In some archaea, it appears that only chymotrypsin-like peptidase activity is present as shown in T.acidophilum (Wenzel and Baumeister 1993, 1995) and H. volcanii (Wilson et al., 1999). It has also been shown that the M. thermophila and Methanococcus jannaschii 20S proteasomes have three cleavage specificities like eukaryal 20S proteasomes, although M. jannaschii appears to have limited trypsin-like activity (Maupin-Furlow et

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14 al., 1998; Wilson et al., 2000). The bacterium, Rhodococcus, appears to have only chymotryptic-like activity, although a small amount of trypsin-like activity was detected (Zhl et al., 1997b). In eukaryotes, only three of the seven -type subunits are active (1, 2, 5) (Groll et al., 1997) with activities described as peptidylglutamyl-peptide hydrolyzing (PGPH), trypsin-like, and chymotrypsin-like, respectively (Orlowski 1990; Heinemeyer et al., 1997). Interestingly, the other inactive -subunits still maintain a high conservation of backbone geometry (Groll et al., 1999). It is still unknown, however, why only three of the -type subunits are active whereas the others are not; however, it is speculated that inactivity of these subunits is due to the lack of processing to expose an N-terminal threonine residue; that is to say, processing does occur for two of the four inactive -subunits, however, at a different amino acid residue that does not form an available N-terminal threonine active site (Groll et al., 1997, 1999). For example, from crystal structure data of yeast 20S proteasomes, it was determined that 3and 4-subunits are unprocessed and 6and 7-subunits are intermediately processed, that is to say not processed at the threonine active site residue, but rather at a glutamine and a threonine ten and eight amino acids upstream of the active site threonine residue, respectively (Schmidtke et al., 1996; Groll et al., 1997). Essentially, in eukaryotes, there are six total active -subunits in the 20S complex, whereas prokaryotic 20S proteasomes, which have been examined, harbor fourteen total active -subunits. 20S Proteasomal Processing 20S proteasomes belong to a family of N-terminal nucleophile (Ntn) hydrolases (Brannigan et al., 1995; Seemller et al., 1995). These hydrolases contain an N-terminal

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15 amino acid residue (e.g. cysteine, serine, or threonine) that acts as a nucleophile during catalysis (Souciet et al., 1988; Choi et al., 1992; Seemller et al., 1995; Heinemeyer et al., 1997). In 20S proteasomes, this active residue is formed by the N-terminal threonine residue of the mature -type subunits (Seemller et al., 1995). The -type subunits are synthesized as inactive precursors with an N-terminal propeptide that is autocatalytically cleaved during 20S proteasomal assembly (Kisselev et al., 2000). This cleavage generates the active site N-terminal threonine residue (Zhl et al., 1997b). The other three currently known Ntn hydrolases include glutamine 5-phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase (Souciet et al., 1988; Smith et al., 1994), penicillin acylase (Choi et al., 1992; Duggleby et al., 1995), and aspartylglucosaminidase (Fisher et al., 1993; Oinonen et al., 1995), with cysteine, serine, and threonine as N-terminal active site residues, respectively. For all Ntn hydrolases, a processing step is required for the exposure of the N-terminal amino group and the maturation of the enzyme (Bochtler et al., 1999). The fact that threonine acts as the active site residue was from studies that involved crystal structure analysis of the Thermoplasma 20S proteasome, where it was complexed with the inhibitor acetyl-Leu-Leu-norleucinal (LLnL) calpain inhibitor I, a potent inhibitor of chymotrypsin-like activity of 20S proteasomes (Lwe et al., 1995; Seemller et al., 1995). In addition, mutational studies have been done to show the importance of the active site threonine residue. Replacing threonine with serine does not appear to affect the rates of hydrolysis of small fluorogenic peptide substrates, but the rate of degrading larger peptide and protein substrates is significantly lowered by this replacement or substitution (Seemller et al., 1995; Maupin-Furlow et al., 1998; Kisselev

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16 et al., 2000). Also, with this mutation of the -subunit, autocatalytic propeptide processing is inefficient (Chen and Hochstrasser 1996; Seemller et al., 1996; Heinemeyer et al., 1997). Just the opposite, however, occurs when changing the threonine residue to cysteine. A 20S proteasome with a cysteine residue at the active site cannot hydrolyze any substrates, but autocatalytic processing does occur (Seemller et al., 1996). With Thermoplasma 20S proteasomes in recombinant E. coli, deletion or mutation of the threonine active site residue to alanine resulted in -subunits that were folded correctly and that fully assembled into a proteasome, but these variant proteasomes were found to be inactive using peptide and protein substrates (Seemller et al., 1995; Kisselev et al., 2000). It has also been shown in yeast that the exchange of threonine for alanine results in inactive proteasomes (Chen and Hochstrasser 1996; Arendt and Hochstrasser 1997; Heinemeyer et al., 1997; Dick et al., 1998). This is also true for 20S proteasomes of the methanoarchaeon Methanosarcina thermophila studied in recombinant E. coli (Maupin-Furlow et al., 1998). Evidence that the amino group of the threonine residue serves as an active site is from a study where the propeptides were deleted from the 1-subunits in a eukaryotic proteasome (Arendt and Hochstrasser 1999). These mutant proteasomes are defective for PGPH peptidase activity. N-terminal sequencing of the 1-subunit was unsuccessful on several attempts, most likely due to an N-terminal blockage. In eukaryotes, the most common N-amino modification is acetylation (Bradshaw et al., 1998). Construction of double mutants with a deletion of the 1-subunit propeptide and deletion of the N-acetyltransferase I gene yielded proteins that were compatible with N-terminal sequencing, which revealed the predicted N-terminal sequence (Arendt and Hochstrasser

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17 1999). This confirms that the N-terminus was acetylated and that deletion of the N-acetyltransferase I gene completely restores PGPH activity within the 1-subunit. -type subunits destined to be the active subunits involved in proteolysis are often synthesized in an inactive precursor form with N-terminal extensions of variable lengths (propeptides) that are autocatalytically removed, which in turn allows for the formation of active -type subunits. This processing event is linked to assembly of a full 20S proteasome. Currently, in archaea, it is unclear as to the mechanism of processing of the -type prosequences; that is whether or not the mechanism proceeds by intraor intermolecular mechanisms, or whether both apply. Processing of the -type prosequences has been shown to occur via an intramolecular manner in mammalian and yeast 20S proteasomes (Brannigan et al., 1995; Schmidtke et al., 1996; Groll et al., 1999). For example, in the study of mammalian 20S proteasomes (Schmidtke et al., 1996), LMP2, a 1i-subunit, that upon induction with -IFN, replaces the constitutively expressed 1-subunit forming an immunoproteasome, was mutated at the threonine active site to alanine. Separately, another alanine mutant of this subunit was made at the conserved glycine that precedes the threonine residue. With these mutations, the subunits were not correctly processed, that is to say processed at sites within the prosequence other than the processing consensus motif, although they were incorporated into a full 20S proteasome with active, processed -subunit neighbors. These results suggest that processing of -subunit propeptides occurs via an intramolecular mechanism, as incorporation of the mutated 1i-subunit into full 20S proteasomes with processed -subunits was not hindered, yet processing of this subunit was absent. The 1-subunit harbors the PGPH activity of the 20S proteasome in

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18 eukaryotes (Heinemeyer et al., 1997). Exchange of this subunit for the corresponding immunoproteasome subunit LMP2 (1i) reduces the PGPH activity of 20S proteasomes (Kuckelkorn et al., 1995). Proteasomes containing the mutated LMP2 subunit that were not completely processed also did lower the PGPH activity (Schmidtke et al., 1996). In one study, it has been shown in yeast that the active proteasomal subunits (1, 2, and 5) are processed independently of each other and that mutating the Lys33 residue in the 5-subunit to alanine or arginine does not affect the processing of 1or 2-subunits, although this subunit itself is not processed (Groll et al., 1999). The lysine 33 residue of the 5-subunit is mutated instead of the threonine active site residue because cell viability is diminished with a threonine to alanine mutation in this subunit (Groll et al., 1999). Lys33 is an important conserved residue among proteasomes and is involved in catalysis (Lwe et al., 1995; Seemller et al., 1995). This residue participates either indirectly by stablizing and providing an environment for the correct orientation of active site residues or acts directly as a proton acceptor for the hydroxyl group of the active site threonine residue by its -amino group (Lwe et al., 1995; Seemller et al., 1995). Both Lys33 and the active site threonine residue are necessary residues for peptide hydrolysis (Seemller et al., 1995) and also for autocatalytic processing of the propeptide of the -subunit (Seemller et al., 1996). Also, a mutation in 1or 2-subunits (exchanging the active site threonine for alanine) separately or as double mutants does not affect the processing of the other active subunits, although they themselves are not processed (Groll et al., 1999). Interestingly, it would appear implausible that processing events occur intermolecularly in eukaryotic proteasomes, as the active sites are nearly 30 apart from each other, so it would not be feasible for a

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19 neighboring subunit to process another propeptide other than its own (Groll et al., 1999). The inactive -subunits (6 and 7), however, do appear to be processed by the active -subunits, as these inactive subunits do not contain the threonine active site residue at which processing occurs autocatalytically (Heinemeyer et al., 1997; Groll et al., 1999). A mutation in 1 (active site threonine residue to alanine), a mutation in 5 (lysine 33 to alanine), and a mutation in both 1 and 5 (active site threonine residue to alanine and lysine 33 to alanine, respectively) do not affect the processing of 6-subunit (Groll et al., 1999); however, a significantly longer propeptide remains attached to the 6-subunit with a mutation in the 2-subunit (active site threonine residue to alanine). This is evidence that the 2-subunit is responsible for processing the inactive 6-subunit and this was also found true for the 7-subunit. Whether this intermolecular cleavage of inactive subunits, however, serves to remove bulky protrusions rather than being integrally involved in intramolecular processing remains to be determined (Zwickl 2002). In addition, Heinemeyer et al. (1997) showed in yeast that inactive -subunits are processed by neighboring active -subunits by mutational analysis and comparison to a model of -subunit arrangement in human 20S proteasomes (Kopp et al., 1997). As shown in Figure 1-2, the inactive 7-subunit is located next to two active subunits, 1 and 2. This proximity lends to availability of active -subunits to process the inactive 7-subunit. For example, it was shown by creating an active site mutant (threonine to alanine) of the 1-subunit that this subunit is not involved in prosequence cleavage of 7. The 7-subunit is still completely processed to its ‘wild-type’ form when the 1-subunit is inactive. Whenever 2-subunit is inactivated by site-directed mutatagenesis at the

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20 active site (threonine to alanine), the 7-subunit is cut somewhat upstream of where it would normally be cut (i.e., immunoanalysis in this study showed additional amino acids present). This processing must have occurred by the next available active -subunit, 1, where its location lends to a different level of processing relative to the 2-subunit. Inactivation of 1 alone does not affect 7 maturation, and a double mutation inactivating 1 and 2 still does not affect 7 processing, as the 5 subunit acts to cleave the propeptide a few more amino acids upstream of where 2 would cleave due to its location further from the 7-subunit. Therefore these results suggest that the trans-processing events of the inactive -subunits occurs between the two -rings and that the length of the resulting cleavage is determined by the position of the closest active site (Heinemeyer et al., 1997). Another study suggests that in recombinant E. coli with Thermoplasma acidophilum genes that encode proteasomal proteins, -protein processing occurs by intermolecular means via in vitro studies which relied upon acid denaturation of the 20S proteasomal proteins prior to assembly (Seemller et al., 1996). This study utilized a mutation at lysine 33 of the -subunit, as well as a mutation at the threonine active site. In this study, inactive, unprocessed proteasomes with a mutation at lysine 33 of the -subunit were mixed with fully active and processed proteasomes with a Thr1Ser mutation and disassembled at a low pH. The mixture was then dialyzed at neutral pH to encourage refolding and proteasome assembly. About 5-10 percent of processed inactive subunits were detected by N-terminal sequencing (Seemller et al., 1996). Autocatalytic processing of Thr1Ser in itself is partially deficient and therefore does not process other subunits with much efficiency because while this mutant is just as proteolytically active

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21 as the wild type, it still is not as adequate as threonine in acting as a nucleophile (Seemller et al., 1995; Kisselev et al., 2000). Also in this study, in vivo coexpression of inactive Lys33Ala mutants with wild type and -subunits from recombinant E. coli resulted in about 80-90 percent processing of inactive subunits (Seemller et al., 1996). In Rhodococcus, the prosequence of -subunits appears to act in trans (Zhl et al., 1997a). In this study, a 72 residue 1-propeptide was expressed in E. coli and purified under denaturing conditions (6 M urea) and refolded by stepwise dilution of urea by dialysis. When this 1-propeptide was added exogenously to an assembly mixture of -subunits and -subunits with the prosequence deleted, a rapid increase in proteolytic activity occurred within at short period of time when using N-Succ-LLVY-Amc as a peptide substrate; whereas, the same assembly mixture (-subunits and -subunits with the propeptide deleted from the protein) without the exogenously added 72 residue 1-propeptide did not show any significant activity. This study also showed that the exogenous propeptide is most efficient at a molar ratio (1:1), as adding a molar excess of propeptide does not increase the reaction rate. In addition, holoproteasomes were formed with the exogenous propeptide present with the mutant -subunits, where the presence of full proteasomal complexes was reduced without the propeptide present. Also, when the amount of exogenous propeptide was decreased, the amount of half proteasomes present increased. These results suggest that the propeptide in bacteria acts in an intermolecular manner. The prosequence of -proteins differ among all three domains of life in length and sequence (Seemller et al., 1996). Common among organisms in the active -subunits, however, is a glycine residue that precedes the conserved active site threonine residue

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22 (Seemller et al., 1995, 1996; Schmidtke et al., 1996). Speculations have been made as to the role of the propeptide of the -subunit. It is commonly assumed that the propeptide is involved in withholding catalytic activity before assembly of 20S proteasomes has occurred. The propeptide may also be involved in protection against coor post-translational modifications. In yeast, it has been shown that the 1-subunit propeptide is involved in prevention of coor post-translational acetylation of the threonine active residue and thereby prevents inactivation of the subunit (Groll et al., 1999; Jger et al., 1999). For example, Groll et al. (1999) replaced the propeptide of the -subunit with ubiquitin and showed the loss of PGPH activity of these mutants. The fused ubiquitin does not hinder the threonine active site because ubiquitin can be cleaved by ubiquitin C-terminal hydrolases to expose an N-terminal threonine residue (Wilkinson 1997). N-terminal sequencing could not be done due to a blockage that upon mass spectrocopy analysis was determined to be an acetyl group (Groll et al., 1999). In addition, the propeptide appears to be required for assembly of 20S proteasomes in some organisms. For example, it has been shown in yeast that the propeptide of the 5-subunit is required for the incorporation of the 5-subunit into 20S proteasomes (Chen and Hochstrasser 1996). In this study, deletion of the 5-subunit prosequence leads to a failure of the 5-subunit to assemble into a full 20S proteasome complex (Chen and Hochstrasser 1996). In addition, deletion of the 5-subunit in yeast 20S proteasomes leads to cell death; therefore the 5 propeptide and subunit appear to be required for the initial folding and maturation of full 20S proteasomes (Chen and Hochstrasser 1996).

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23 Also, in Rhodococcus it appears that the propeptide of the -subunit is required for 20S proteasomal assembly (Zhl et al., 1997a; Kwon et al., 2004). Kwon et al. (2004) have shown by crystallography studies that the -subunit propeptides appear to provide more contact surface area, which facilitates rapid assembly of Rhodococcus 20S proteasomes; whereas, without the propeptides, the assembly process is extremely slow. In archaea, however, it appears that -subunit propeptides are dispensible in regard to 20S proteasomal assembly, at least as shown by in vitro studies. Re-emphasizing that propeptides are not essential for archaeal proteasomal assembly, Grziwa et al. (1994) showed in vitro that assembled Thermoplasma proteasomes can be completely dissociated and reassociated without the need for a propeptide. Also, Zwickl et al. (1994) have shown in E. coli that co-expression of and mutant -subunits lacking a propeptide from Thermoplasma still do assemble and form full active 20S proteasomes. With 20S proteasomes from H. volcanii expressed in E. coli, it was shown that the removal of the high salt buffer (2 M NaCl) dissociates the fully active assembled 20S proteasomes into monomers and that rapid reassociation occurs upon incubation in a high salt buffer (Wilson et al., 1999). This is more evidence that the propeptide in archaea is not essential for assembly, as processed monomer -subunits are able to assemble into full active 20S proteasomes. Proteasomal Assembly The events of 20S proteasomal assembly also differ among the domains of life. In eukaryotes and archaea, assembly appears to occur in two steps: 1) a scaffold of -rings forms to which 2) -subunits anchor themselves. In eukaryotes, a very ordered system must be orchestrated in order to assemble the fourteen different subunits in their specific

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24 positions (Kopp et al., 1997). A series of events ensue where initially a platform of -rings is formed, which provides the correct docking sites for -subunits (Zwickl et al., 1994; Gerards et al., 1997). In eukaryotes, the docking of the -subunits occurs in two steps (Nandi et al., 1997). The first three -subunits that are incorporated into the complex are 2, 3, and 4. The other four -subunits are integrated at a later stage. After all -subunits have been anchored to the -rings, the dimerization of the two immature half proteasomes occurs with subsequent propeptide processing to form a mature 20S proteasome (Schmidtke et al., 1996; Nandi et al., 1997). If formation of the immunoproteasome is underway (upon cytokine induction), the exchange of the induced subunits against the constitutive subunits occurs at the precursor level (Nandi et al., 1997). In archaea, it appears that the -rings act as chaperones in proteasomal assembly (Seemller et al., 1996). Mature -rings are not found in the absence of -rings as determined by separately producing and purifying -ring structures and -subunits of M. thermophila from recombinant E. coli (Maupin-Furlow et al., 1998). In addition, crystallographic data of recombinant Archaeoglobus fulgidus proteasomal proteins shows that the -ring alone has the same conformation as the -ring assembled in a full 20S proteasome, except that the N-terminal region is disordered in the full proteasome structure, whereas the N-terminal region is structured and ordered within the -rings alone (Groll et al., 2003). This suggests a requirement of -rings forming a stable framework for the -subunits to form upon. Additional factors that act as chaperones appear to be required for eukaryotic proteasomal assembly and are only temporarily associated with the newly formed 20S

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25 complex (Schmidtke et al., 1997; Ramos et al., 1998). For example, Schmidtke et al. (1997) have shown that in mammalian proteasomes, chaperone hsc73 is present with precursor complexes, while it is not found co-purified with mature 20S proteasomes. In yeast, Ump1 is an important chaperone involved in eukaryotic proteasomal maturation. Ramos et al. (1998) have shown that in Saccharomyces cerevisiae Ump1 is not only associated with maturing proteasomes, but is required. Ump1 is thought to assist in coordinating -protein processing and is found only in 20S proteasome precursor complexes. Deletion of the gene encoding Ump1 results in unprocessed -subunits and impaired assembly. Cells that lack Ump1 also are hypersensitive to various stresses and do not sporulate. Ump1, upon completion of its critical role, is internalized as a substrate and degraded. A mammalian homolog of the yeast chaperone Ump1 factor has also been found described as hUmp1 or POMP (proteasome maturation protein) (Burri et al., 2000; Witt et al., 2000). Similar to yeast cells, this mammalian proteasomal chaperone is not found in the mature form of assembled 20S proteasomes, although it is associated with the precursor forms (Burri et al., 2000). In addtion, this protein may function in the formation of an immunoproteasome, as it was shown by Northern blot analysis that the expression of the POMP-specific mRNA occurred after -IFN cytokine induction (Witt et al., 2000). Other mammalian chaperone factors have been recently found that appear to facilitate the formation of the -rings by acting as a scaffold (Hirano et al., 2005). These heterodimers are designated PAC1 and PAC2 for proteasome assembling chaperone and homologs are ubiquitously present in mammals (Hirano et al., 2005). Once no longer

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26 needed, they are degraded by 20S proteasomes similar to Ump1 in yeast cells (Hirano et al., 2005). Archaeal 20S proteasomes have also been shown to assemble similar to eukaryal 20S proteasomes. In Thermoplasma, -subunits expressed in E. coli alone can form seven-membered rings, whereas, mature or unprocessed -subunits do not appear to form any ordered structure (Zwickl et al., 1994). Fully assembled and active 20S proteasomes form if the and -genes are coexpressed in E. coli, with or without the propeptide present on -subunits (Zwickl et al., 1992). Also, fully formed 20S proteasomes can assemble in vitro as shown by Maupin-Furlow et al. (1998), when purified -rings and -subunits with the propeptide from M. thermophila were produced separately in E. coli. Mature -subunits were not found unless there were -rings present, and evidence for -ring structures was not found among the independently produced -subunits (Maupin-Furlow et al., 1998). Also, as shown in H. volcanii, 1and 2-subunits that were produced separately in E. coli would assemble into ring formations upon dialysis into a high salt buffer that mimicked the high ionic strength in the cytosol of H. volcanii (Kaczowka and Maupin-Furlow 2003). Therefore, it appears the -subunits form a ring structure platform for the -subunits to build upon. The assembly pathway of the 20S proteasome of the bacterium R. erythropolis has been characterized. It has been shown that R. erythropolis proteasomes are composed of two and two -subunits and was also shown that these subunits assemble into a single 20S complex (Zhl et al., 1997a). Unlike eukaryotic or archaeal assembly, bacterial -proteins alone do not form -rings, but rather remain monomeric (Zhl et al., 1997b). The model proposed for assembly includes expression of monomeric and -proteins,

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27 where the -proteins possess prosequences, and the formation of two / heterodimers to subsequently form the characteristic proteasomal rings. Although not completely proven, the existence of / heterodimers is very likely since neither nor -subunits alone can form any assembled product (Zhl et al., 1997a). Upon formation of the two immature half proteasome complexes, there is an association between the two precursor complexes to form a pre-holoproteasome complex that is processing-competent. Subsequent processing of the -subunits yields a full mature 20S proteasome (Zhl et al., 1997b). The assembly and processing of 20S proteasomes have been studied extensively across all domains of life. It has been found among the three domains that some manners in which 20S proteasomes are formed are similar; however, there are also some differences. For example, the assembly of 20S proteasomes is similar between eukaryotic and archaeal cells; however, differences in the processing of -subunits remains to be determined. Currently, it is unclear as to the mechanism of processing of archaeal 20S proteasomal -subunits. Many studies have been done to elucidate this action; however, most research has revolved around in vitro studies with recombinant E. coli. The focus of this study was to examine the effects of an active site mutation on assembly and activity of 20S proteasomes, as well as determine the mechanism of processing in the haloarchaeon H. volcanii. This organism is an ideal model to study since a genetic system is in place and it is comparable to its eukaryal counterpart in a few cellular mechanisms. Also, its 20S proteasome is less complex compared to eukaryotic 20S proteasomes, which makes the analysis simpler. The specific objectives of this study included modifying the threonine active site residue of -subunits in 20S proteasomes of

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28 H. volcanii along with the addition of a C-terminal histidine tag to aid in purification. These -His subunit variants were expressed in H. volcanii and purified for analysis of processing mechanisms of the -subunits in vivo. This is the first report of a 20S proteasomal complex expressed in vivo in archaea that contains -subunits with a modified active site along with genomic encoded -subunits.

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29 Figure 1-1. General structure of 20S proteasomes. A) Vertical GRASP representation of Thermoplasma acidophilum 20S proteasome. B) Horizontal sphere model of T. acidophilum 20S proteasome. From Bochtler, M., Ditzel, L., Groll, M., Hartmann, C., and Huber, R. (1999). The proteasome. Annu. Rev. Biophys. Biomol. Struct. 28, 295-317, Figure 2. Reprinted, with permission, from the Annual Review of Biophysics and Biomolecular Structure, Volume 28 1999 by Annual Reviews www.annualreviews.org .

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30 Figure 1-2. Model of location of -subunits in yeast 20S proteasomes. In the eukaryotic 20S proteasome, the inactive -subunit, 7, is located next to two active -subunits, 1 and 2 (PGPH and trypsin-like activities, respectively). Activity of 5-subunits is chymotrypsin-like. The arrow represents the -ring interface. Active -subunits are shaded. Adapted from Heinemeyer, W., Fischer, M., Krimmer, T., Stachon, U., and Wolf, D. H. (1997). The active sites of the eukaryotic 20S proteasome and their involvement in subunit precursor processing. J. Biol. Chem. 272, 25200-25209.

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CHAPTER 2 PURIFICATION AND CHARACTERIZATION OF BETA-PROTEIN VARIANTS OF 20S PROTEASOMES OF THE HALOARCHAEON Haloferax volcanii Introduction Proteasomes are energy-dependent proteases found in all domains of life and are important for general protein turnover within the cell (Dahlmann et al., 1989). The architecture of 20S proteasomes is conserved among organisms (Lwe et al., 1995; Groll et al., 1997; Unno et al., 2002; Groll et al., 2003). Although different species, even within the same domain of life, produce different types of and -subunits, structurally these subunits are related and form a barrel-like structure (Lwe et al., 1995; Groll et al., 1997). Fourteen -subunits and fourteen -subunits join together to form four heptameric rings (Groll et al., 2003). The -rings are found on the ends of 20S proteasomes, while two -rings are sandwiched in the central catalytic chamber of the proteasome (Bochtler et al., 1999). The proteasomes are placed in the Ntn hydrolase family, as they only use one amino acid residue as a nucleophile in a proteolytic attack against substrates (Brannigan et al., 1995; Seemller et al., 1995). In the case of 20S proteasomes, the active site residue is a threonine; although, cysteine and serine residues have been described in other enzymes of this family (Seemller et al., 1995). Like all enzymes in the family of Ntn hydrolases, there is a processing step that exposes the active site residue (Bochtler et al., 1999). There is still a question as to how the propeptides of the -subunits of 20S proteasomes are processed; that is to say, whether or not the processing follows an intra31

PAGE 42

32 or intermolecular mechanism. It has been shown in different domains of life to occur by either mechanism. Haloarchaea, such as H. volcanii, serve as simple models to study the processing of -subunits of 20S proteasomes of archaea by in vivo methods because useful genetic systems are available. In addition, many fundamental processes within an archaeal cell (e.g., DNA replication, transcription, and translation, as well as proteasomal and chaperone systems) are comparable to the more complicated eukaryal cell, which makes the analysis of 20S proteasomes easier. For example, eukaryotic cells produce fourteen different subunits that form a twenty-eight subunit 20S proteasome; whereas, in H. volcanii, only three different subunits are produced (Groll et al., 1997; Wilson et al., 1999). The aim of this study is to determine how the propeptide of the -subunit is removed in H. volcanii by in vivo studies. A plasmid encoding the -protein with a C-terminal linker sequence and a histidine tag was shuttled into H. volcanii. Also, site-directed mutants of the -histidine tagged protein were made at the threonine active site (T49A and T49S) and were separately shuttled via plasmids into H. volcanii. The -protein variants were separately expressed and purified to homogeneity and analyzed for processing and assembly. Also, this study evaluated the effects of the -Thr49Ala-His mutation on peptide and protein hydrolyzing activity of 20S proteasomes. Methods and Materials Materials Biochemicals were purchased from Sigma-Aldrich (St. Louis, MO). Other organic and inorganic analytical grade chemicals were from Fisher Scientific (Atlanta, GA). Restriction endonucleases were from New England BioLabs (Beverly, MA).

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33 Diethylaminoethyl (DEAE)-cellulose resin was from Sigma-Aldrich. SnakeSkin dialysis tubing was from Pierce Biotechnology, Inc. (Rockford, IL). Hybond-P membranes and ECL-Plus Detection System used for immunoblot analysis were from Amersham Pharmacia Biotech (Piscataway, NJ). X-ray film was from Research Products International Corporation (Mt. Prospect, IL). Strains, Media, and Plasmids Strains and plasmids used in this study are summarized in Table 2-1. E. coli strains were grown in Luria-Bertani (LB) medium (37C, 200 rpm). H. volcanii DS70 was grown in complex medium (ATCC 974) at 37C. Media was supplemented with 100 mg of ampicillin, 50 mg of kanamycin, or 0.1 mg of novobiocin per liter as needed. Cloning and Site-Directed Mutagenesis Plasmids encoding the -His variant proteins (pJAM202-1.1 and pJAM202-2.1) were constructed as follows: the plasmid encoding the psmB gene (-protein) from H. volcanii with a linker and a C-terminal polyhistidine tag (pJAM621) was cut with BglII and SacII to generate a 464-bp fragment that was Vent polished. The resulting fragment was subcloned into pLITMUS28 at a XhoI site, which was blunt end polished with Klenow polymerase. The orientation of the insert in the resulting plasmid (pJAM697a) was analyzed by a digestion with NdeI and EcoRI. Site-directed mutations at the threonine active site (alanine and serine) were incorporated by PCR amplification with pJAM697a as template DNA and the following primers: 5’-TGGTCGCTCCGGTCTTCGTCTC-3’ (threonine 49 to alanine) and 5’-TGGTGCTTCCGGTCTTCGTCTC-3’ (threonine 49 to serine), along with 5’-CTTACCTCATATGCGTACCCCGACTC-3’, which introduces an NdeI site. The PCR product of 155-bp was cut with NdeI and re-cloned into pJAM697a at the

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34 NdeI/PshAI site to create pJAM698-2.4 and pJAM699-2 (-Thr49Ser-His and -Thr49Ala-His, repectively). Both pJAM698-2.4 and pJAM699-2 were cut with NdeI/AatII to isolate a 240-bp fragment and ligated separately into pJAM621 at NdeI/AatII sites. The resulting plasmids, pJAM553 and pJAM554 (-Thr49Ser-His and -Thr49Ala-His, repectively), were cut with NdeI/BlpI that generated a 825-bp fragment that was cloned into the shuttle plasmid pJAM202 cut with NdeI/BlpI. The resulting plasmids were designated pJAM202-1.1 and pJAM202-2.1 (-Thr49Ser-His and -Thr49Ala-His, repectively). Site-directed mutations were confirmed by DNA sequencing (DNA Synthesis Core Laboratory, University of Florida). Ligation products of site-directed mutants into shuttle plasmid pJAM202 were confirmed by PCR using the following primers: 5’-GCGATATCGATGCCCTTAAGTACAA-3’ and 5’-GCCAATCCGGATATAGTTCCTCC-3’ and were also confirmed by DNA restriction maps and sequencing. DNA Purification and Transformation Plasmid DNA was isolated using Qiagen plasmid miniprep kit (Valencia, CA). DNA fragments were eluted from an 0.8% SeaKem GTG agarose (FMC Bioproducts, Rockland, ME) gel in 1X TAE buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8.5) using Qiaquick gel extraction kit (Qiagen). Shuttle plasmids were transformed into H. volcanii DS70 strain according to the method of Cline et al. (1989). Plasmids transformed into E.coli strains were transformed with 100 L of chemically competent cells and 2 L of plasmid DNA and incubated on ice for 20 min. The DNA/cell mixture was transferred to a 37C water bath for 5 min then re-incubated on ice for 2 min. Then, 1 mL of LB medium was added to the DNA/cell mixture and incubated at 37C for 90 min. The cells

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35 were pelleted and plated on selective plates corresponding to the resistance marker on the plasmid. The plates were incubated overnight at 37C. Synthesis and Purification of -protein Mutant 20S Proteasomes H. volcanii DS70 strains separately transformed (Cline et al., 1989) with the plasmids containing the -protein variants (-His, -Thr49Ala-His, and -Thr49Ser-His) were grown in complex medium (ATCC 974) supplemented with 0.1 g/mL novobiocin at 37C. Cells were harvested at stationary phase (A 600 ~ 2 to 3 U) by centrifugation at 7,800 g for 30 min at 4C. Cell pellets were frozen at C. Once thawed on ice, cell pellets were resuspended in 20 mM Tris-HCl, pH 7.2 with 2 M NaCl (Buffer A) and lysed by passage through a French pressure cell at 20,000 psi. Cell free extract was clarified by centrifugation 17,000 g for 20 min at 4C twice and filtered with 0.8, 0.45, and 0.22 m-pore size filters, respectively, (Nalge Nunc International, Rochester, NY) prior to application to a Ni 2+ -Sepharose 6 Fast Flow (His Trap FF) (Amersham Pharmacia Biotech) column equilibrated with Buffer A including 5 mM imidizole (equilibration buffer). The column was washed with two times the column volume with equilibration buffer followed by two times the column volume washes with Buffer A containing 60 mM imidizole. His-tagged proteins were eluted with Buffer A containing 500 mM imidizole and collected in 0.5 mL fractions. Protein concentration of fractions was determined by Bradford protein assay using bovine serum albumin as a standard (Bradford 1976). The fractions that contained significant amounts of protein were pooled and dialyzed overnight at 4C into 80% AS Buffer A (2.2 M ammonium sulfate, 1.5 M NaCl, 20 mM Tris-HCl, pH 7.2) with 20% AS Buffer B (1.0 M ammonium sulfate, 2.7 M NaCl, 20 mM Tris-HCl, pH 7.2) using SnakeSkin dialysis tubing with a 3,500 molecular

PAGE 46

36 weight cut-off (MWCO). The dialyzed sample was applied to DEAE-cellulose equilibrated in 80% AS buffer A and 20% AS buffer B. Peak fractions were analyzed for peptidase activity using N-Succ-LLVY-Amc (Sigma-Aldrich) as a peptide substrate. Fractions with peptide hydrolyzing activity were pooled and dialyzed overnight at 4C into Buffer A using SnakeSkin dialysis tubing (3,500 MWCO). Purity of purified fractions was determined by separating on a 12% reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel with the following low range molecular mass standards (Bio-Rad, Hercules, CA): phosphorylase (97.4 kDa), serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa) and stained with Coomassie R-250. Samples used in activity assays were stored at 4C. Samples used for SDS-PAGE gels and Western blots were stored at C. Assembly of purified -protein variants (-His, -Thr49Ala-His, and -Thr49Ser-His) was determined by loading Ni 2+ -Sepharose purified samples on a Superose 6 HR 10/30 (Amersham Pharmacia) column equilibrated in Buffer A with 1 mM dithiothreitol (DTT). Molecular mass standards were run on the same column equilibrated in 20 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1 mM DTT. Standards included serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), -amylase (200 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa). Synthesis and Purification of Malate Dehydrogenase from Haloarcula marismortui The plasmid encoding malate dehydrogenase from H. marismortui, p-hMDH2 (Cendrin et al., 1993) (kindly provided by Moshe Mevarech), was transformed into E. coli Rosetta (DE3) (Novagen). Transformants were grown as described (Cendrin et al.,

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37 1993) with 0.1 g per liter ampicillin. The initial purification steps of Cendrin et al. (1993) were followed as described. After the final ammonium sulfate concentration was brought to 2.5 M and centrifuged (12,000 g, 15 min), the sample was dialyzed into 2 M NaCl with 20 mM Tris-HCl, pH 7.2. The purity of purified recombinant malate dehydrogenase was determined by separation on a 12% SDS-PAGE gel and a standard enzymatic assay containing 4 M NaCl, 20 mM Tris-HCl, pH 8, 1mM oxaloacetate, and 0.1 mM NADH, as described by Cendrin et al. (1993). Immunoanalysis Ni 2+ -Sepharose and DEAE-cellulose purified -protein variants (-His, -Thr49Ala-His, and -Thr49Ser-His) were separated on a 12% reducing SDS-PAGE gel and blotted to a Hybond-P polyvinylidene difluoride (PVDF) membrane by using 10 mM (2-N-morpholino) ethanesulfonic acid (MES) at pH 6.0 with 10% (vol/vol) methanol. The 1, 2, and -proteins were detected using anti-proteasomal polyclonal primary antibodies (1 and diluted 1:5000, 2 diluted 1:3500) (Kaczowka and Maupin-Furlow 2003) and horseradish peroxidase-conjugated (HRP) anti-rabbit immunoglobulin (H+L) antibody raised in goat (Southern Biotechnology Associates, Inc., Birmingham, AL) (diluted 1:5000). The histidine-tagged -protein variants were also detected using anti-histidine-tag antibody conjugated to HRP (U.S. Biological, Swampscott, MA) (diluted 1:5000). Chemiluminescent signal was detected by X-ray film using ECL-Plus Detection System following manufacturer’s instructions (Amersham Pharmacia Biotech). Protein and Peptide Hydrolysis Assays Chymotrypsin-like peptide-hydrolyzing activity was assayed by measuring the release of fluorgenic substrate, 7-amino-4-methylcoumarin (Amc), conjugated to N-Succ

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38 LLVY (N-Succ-LLVY-Amc) as previously described (Wilson et al., 1999). Purified 20S proteasome (0.1 g) per 300 l of Buffer A (2 M NaCl-Tris buffer) was incubated with 20 M Succ-LLVY-Amc substrate with 6% (vol/vol) dimethylsulfoxide (DMSO) at 65C at intervals of 0 to 120 min. Specific activities were reported as nanomoles of product released per min per mg of protein. Protein hydrolyzing activity (Wilson et al., 1999) was measured with bovine oxidized insulin B chain (Sigma-Aldrich) as a substrate. Purified proteasome (0.2 g) and 145 M substrate, in 1 mL final volume with 2 M NaCl, 20 mM Tris-HCl, pH 7.2 buffer and 6% (vol/vol) DMSO, were incubated at 60C. Samples of 10 L were removed from the reaction mixture at time intervals of 0 to 75 min and were added to 100 L of cold 0.1 M sodium phosphate buffer (pH 6.8) to stop the hydrolysis reaction, and 50 L of fluorescamine (0.3 mg per mL of acetone) was added according to Akopian et al. (1997). The mixture was thoroughly vortexed for 1 min and the volume was increased to 1 mL by addition of dH 2 O. The amount of free -amino groups released upon peptide bond hydrolysis were measured by fluorescence with an excitation wavelength of 370 nm and an emission wavelength of 480 nm with leucine as the standard (Udenfriend et al., 1972) (Aminco-Bowman series 2 luminescence spectrometer; Spectronic Instruments, Rochester, NY). Specific activities were reported as nanomoles of leucine equivalents per h per mg of protein. Protein hydrolzying activity was also assayed using recombinant malate dehydrogenase from H. marismortui (Cendrin et al., 1993). Assay conditons included a reaction mixture containing 1 g of PanA purified from H. volcanii (Reuter et al., 2004), 0.2 g of purified -His variant, and 0.5 g malate dehydrogenase in a 1 M NaCl, 100

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39 mM Tris-HCl, pH 8 buffer including 1 mM MgCl 2 and 1 mM ATP that was incubated at 40C overnight. Control reaction mixtures were included and are described as follows: 1 g of PanA alone in buffer, 0.5 g of malate dehydrogenase alone in buffer, and 0.2 g of -His variant alone in buffer. To determine the levels of malate dehydrogenase, protein hydrolyzing activity of the -His variant using fluorescamine was analyzed as described previously, as well as separated on a 12% SDS-PAGE gel. Results Processing Mechanisms of Variant -proteins (-His, -Thr1Ala-His, and -Thr1Ser-His) in H. volcanii A plasmid reported in a previous study (pJAM202) was used to synthesize the H. volcanii 20S proteasomal -protein with a C-terminal linker (KLAAALE), which joined a polyhistidine tag (Kaczowka and Maupin-Furlow 2003). In this study, the active site threonine residue of this previously modified -protein (-His), was changed to alanine and serine by site-directed mutagenesis. These modified -proteins (-His, -Thr49Ala-His, -Thr49Ser-His) were separately expressed in H. volcanii via shuttle plasmids and purified by Ni 2+ -Sepharose chromatography. The residue number for threonine (49) is from a DNA-deduced amino acid sequence and N-terminal sequencing of the -protein of H. volcanii (Wilson et al., 1999). Purified samples were separated on a 12% reducing SDS-PAGE gel (Figure 2-1). Extraneous bands at the top and bottom of the gel are non-specific to the -His variants and appear to bind non-specifically to the Ni 2+ -Sepharose matrix, as these protein bands are also found in the control wild type strain, H. volcanii DS70, that did not express the -His variant proteins (data not shown). Approximate sizes of 1, 2, and genomic -protein based on SDS-PAGE are known from a previous study (37.5, 34.5, 30 kDa, respectively) (Wilson et al., 1999) and

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40 it appears that all three variant -proteins (-His, -Thr49Ala-His, and -Thr49Ser-His) assembled with these three proteins. This is based on the detection of protein bands of 38-40 kDa, 35-38 kDa, and 29-31 kDa common to all three -His variant samples, which is consistent with 1, 2, and genomic -subunits (Figure 2-1). Differences between the estimated masses of the subunits in this study might be due to differences in the gel and the gel-running conditions. To confirm these bands, however, a Western blot was performed with polyclonal antibodies against 1, 2, and -proteins (Figures 2-2 and 2-3). Indeed, the bands suspected to be 1, 2, and genomic -proteins are present in the purified samples based on cross-reactivity with the anti-1, 2, or antibodies of bands which migrated at the positions noted above. Additional bands that cross-reacted with the antiantibody were also detected (Figure 2-3). In the -His and -Thr49Ser-His variants (Figure 2-3, Lanes 1 and 2), the extra band appears to migrate just below 31 kDa at what is suspected to be processed -proteins. This band is not present in the -Thr49Ala-His sample (Figure 2-3, Lane 3), although another band that cross-reacted with the antiantibody that migrates just above 31 kDa is present at what is suspected to be unprocessed -subunits. To confirm the identity of these extra bands, Ni 2+ -Sepharose and DEAEcellulose purified -His and -Thr49Ala-His samples were analyzed by Western blot with anti-polyhistidine antibody (Figure 2-4). Results reveal that the “extra” bands seen in Figure 2-1 are histidine tagged -proteins based on cross-reactivity with antiand anti-histidine tag antibodies. Based on the migration patterns, the -His variant appears to be processed, while -Thr49Ala-His does not appear to be processed (Figure 2-4). Estimated molecular masses of the -His and -Thr49Ala-His variants are 24.5 kDa and 26.3 kDa, respectively (Figure 2-4). Analysis of the calculated molecular mass

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41 of the -protein variants from a deduced protein sequence reveals that a processed -His variant with a linker and polyhistidine tag present would have an approximate molecular mass of 22 kDa, while the -Thr49Ala-His variant would have an approximate molecular mass of 27 kDa. The genomic encoded -subunit estimated molecular mass is about 20 kDa (completely processed). It would be expected that a processed -His variant with a seven amino acid linker and a polyhistidine tag would run slightly slower (about 2 kDa larger) than the genomic encoded -subunit, as seen in Figure 2-5. Also, it is expected that an unprocessed -variant protein, such as -Thr49Ala-His, with a linker and a polyhistidine tag would migrate even slower (about 7 kDa larger than the genomic encoded -subunit). This is also shown in Figure 2-5. The acidic nature of these halophilic proteins, however, makes the comparison between the apparent molecular mass and the calculated molecular mass from an SDS-PAGE gel difficult, as these proteins run slightly slower in an SDS-PAGE gel (Izotova et al., 1983). With longer exposure using the antiantibody, there are clearly different levels of processing with the -Thr49Ser-His variant (Figure 2-3, Lane 2), as there are bands seen for what is suggested to be completely unprocessed or completely processed -Thr49Ser-His variant based on migration patterns and cross-reactivity with antiand anti-polyhistidine antibodies. In addition, the bands found in this sample are analogous to both the unprocessed -Thr49Ala-His variant and the processed -His variant (Figure 2-5), suggesting that a percentage of propeptides in the -Thr49Ser-His variant are completely processed with a percentage that are completely unprocessed. This suggests that processing levels of the -subunit are reduced with a substitution of serine for the threonine active site residue.

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42 Assembly of 20S Proteasome with -protein Variants The Ni 2+ -Sepharose purified -protein variants were further purified by Superose 6 gel filtration chromatography to determine if 20S proteasome complexes assembled with the -protein variants (Figure 2-6). The estimated molecular masses of the -variant complexes were as follows: the -His complex was 995 kDa, -Thr49Ser-His was 1,352 kDa, and -Thr49Ala-His was 976 kDa. The ratio of genomic encoded -subunits to -Thr49Ala-His variant subunits appears to be about 3:1 as determined by separating the variant 20S proteasomes on a 12% SDS-PAGE gel stained with Coomassie Blue, visualizing with the VersaDoc 1000 Imaging System (Bio-Rad, Hercules, CA), and analyzing band density with Quantity One 1-D analysis software (Bio-Rad, Hercules, CA). The ratio of genomic encoded -subunits to -His variant subunits appears to be 1:1. The estimated molecular masses of the -His and -Thr49Ala-His variants appear to be comparable, although it is speculated that some propeptides are still present within the -Thr49Ala-His variant. The molecular mass would not be expected to change dramatically because of these propeptides due to the higher amount of processed genomic encoded -subunits present in the -Thr49Ala-His variant, as demonstrated by densitometry analysis. The dramatic difference in the estimated molecular mass of -Thr49Ser-His variants, as compared to the -His and -Thr49Ala-His variants, might be due to the limitation posed by the lack of commercially available protein standards above 669 kDa. Although molecular masses of the variant 20S proteasomes are in the linear range of separation for the Superose 6 column, the estimated molecular masses of the -His variants were determined by extrapolation of the standard curve (Firgure 2-6).

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43 The calculated molecular mass of processed -His variants with the linker and histidine tag in a 1:1 ratio with processed genomic encoded -subunits was 679 kDa. The calculated molecular mass of the -Thr49Ser-His variant with the linker and histidine tag was 698 kDa, assuming half of the -Thr49Ser-His variants were processed and half were unprocessed. The calculated molecular mass of the -Thr49Ala-His variants with the linker and histidine tag was 689 kDa, assuming a 3:1 ratio of processed genomic encoded -subunits to unprocessed -Thr49Ala-His proteins. The significant difference between the calculated and estimated molecular masses might be due to the histidine tags present on the C-terminal side of the variant -subunits. It is possible that these tags might have changed the conformation of the 20S proteasome complex that altered the interaction of the complex with the column matrix that might otherwise not have occurred if the tags were not present. Also, it is possible that the histidine tags themselves are interacting with the matrix and ultimately retarding the variant 20S complexes. In addition, the variant 20S proteasomes from the halophile H. volcanii require high salt conditions for stability; therefore, the column was equilibrated in a high salt buffer (2 M NaCl). It is possible, however, that under these buffer conditions that the variant 20S proteasomes interacted with the column matrix and separation was not based simply on size exclusion. Lastly, the standard curve does not contain proteins with a molecular mass above 669 kDa, as they are not commercially available, so the values for the -variants in this study are extrapolated from this curve. In essence, results suggest that these variant proteasomes do assemble into full 20S complexes. The stability of these variant proteasomes (-Thr49Ser-His and -Thr49Ala-His), however, is lowered considerably, as compared to -His variants. The proteasomes

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44 of the -Thr49Ser-His and -Thr49Ala-His variants dissociate when purified by hydroxylapatite chromatography with a 10 mM to 420 mM sodium phosphate gradient (Figure 2-7) and peptidase activity is decreased by about half after a week of storage at 4C in Buffer A (2 M NaCl, 20 mM Tris-HCl, pH 7.2) compared to the -His variant control (data not shown). Due to difficulty in stability of the -Thr49Ser-His and -Thr49Ala-His variants, further studies of the variant -proteins only included the -Thr49Ala-His variant with the -His variant as a control. Modifications to the protocol were similar to purification methods of Kaczowka and Maupin-Furlow (2003) and included purification of the Ni 2+ -Sepharose purified -His and -Thr49Ala-His variants on a DEAE-cellulose column. These fractions were previously shown to contain purified 20S proteasomes (Kaczowka and Maupin-Furlow 2003). Peptide Hydrolyzing Activity of -protein Variants Peptide hydrolyzing activity of 20S proteasomes in H. volcanii cleaves at the carboxyl end of hydrophobic residues, such as tyrosine, similar to chymotrypsin-like activity. A fluorogenic small-peptide substrate (N-Succ-LLVY-Amc) was used to monitor peptide bond hydrolysis by measuring the amount of fluorescence upon release of Amc. Specific peptidase activity for the -His and -Thr49Ala-His variants in the purified 20S proteasome complexes (Figure 2-8) was 310 and 135 nmoles of product produced per min per mg of protein, respectively, in Buffer A at 65C (Figure 2-9). This corroborates a previous specific activity reported for H. volcanii 20S proteasomes with N-Succ-LLVY-Amc as substrate that was around 350 nmoles of product produced per min per mg of protein (Wilson et al., 1999). This activity was found to be optimal at 60-70C in 2 M NaCl with a broad pH optimum at 7.0 to 9.3 (Wilson et al., 1999); therefore,

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45 under the same reaction conditions, the activities for the wild type 20S proteasome from H. volcanii and the variant proteasome (-His) are comparable. It does not appear that the addition of a histidine tag affects peptide hydrolyzing activity. The activity value for the -Thr49Ala-His variant is most likely from the genomic encoded -subunits, as it has been shown extensively that the substitution of threonine for alanine in other 20S proteasomes does not catalyze they hydrolysis of peptides or proteins (Seemller et al., 1995; Chen and Hochstrasser 1996; Arendt and Hochstrasser 1997; Heinemeyer et al., 1997; Dick et al., 1998; Maupin-Furlow et al., 1998; Kisselev et al., 2000). Also, densitometry measurements in this current study have shown that the ratio of genomic encoded -subunits to -Thr49Ala-His variant subunits is about 3:1, so activity displayed is most likely due to the genomic encoded -subunits, as the activity of -Thr49Ala-His is 44 percent of -His. Protein Hydrolyzing Activity of -protein Variants The specific activity of the purified 20S proteasome complexes containing the -variants, -His and -Thr49Ala-His, was determined using bovine insulin B chain (30 amino acids) as a substrate. The -His variant hydrolyzed insulin B chain with a specific activity of 464 nmoles of leucine equivalents per h per mg of protein (Figure 2-10). The -Thr49Ala-His mutant hydrolyzed insulin B chain with a specific activity of 190 nmoles of leucine equivalents per h per mg of protein (Figure 2-10). Previous activity results show that 1/-proteasomes purified from H. volcanii have a specific activity of 82 nmoles of leucine equivalents per h per mg of protein under the same reaction conditions (Wilson et al., 1999). The six-fold difference of activity between the wild type 1/-proteasome and the -His variant may be due to the methods employed in purification.

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46 The protocol used by Wilson et al., (1999) required more steps to purify the native proteasome from H. volcanii, as well as temperatures that would encourage denaturation. The -His variant required fewer steps in purification. Adding a histidine tag to the -protein allowed for only two steps in the purification process (Ni 2+ -Sepharose and DEAE-cellulose chromatography). Although the peptide hydrolyzing activity between wild type 20S proteasomes of H. volcanii and -His variant 20S proteasomes is comparable, differences in the proteinase activity might also be due to a low level of contaminating proteins still present in the preparation after purification of wild type 20S proteasomes. It is possible that the background levels of contaminating proteins were just high enough that the bovine insulin chain B could not compete with these other proteins in wild type H. volcanii 20S proteasomes. It might also be feasible that the histidine tags used in this study altered the conformation of 20S proteasomes such that access was limited in regard to protein versus peptide substrates. The protein hydrolyzing activity of the variant -Thr49Ala-His protein is 2.4-fold lower than that of -His; however, it is still significant. Since insulin B chain is only 33 amino acids long, it is possible that like N-Succ-LLVY-Amc substrate used for peptide hydrolyzing studies, insulin B chain might be small enough to avoid the potential or predicted steric hinderance of the propeptides in the -Thr49Ala-His sample to access the genomic encoded -subunits. As shown before, H. volcanii 20S proteasomes do not hydrolyze bovine -casein in 2 M NaCl with 20 mM Tris-HCl, pH 7.2 at 60C (Wilson et al., 1999). This is also the case with the -variants of this study (-His and -Thr49Ala-His) (data not shown). This is surprising since other archaea, such as T. acidophilum and M. thermophila can

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47 hydrolyze bovine -casein in less concentrated salt solutions at denaturing temperatures, including 60C (Dahlmann et al., 1992; Maupin-Furlow et al., 1998). It is possible, however, that even at denaturing temperatures, this substrate aggregates in the high salt conditions that are required for the 20S proteasome of H. volcanii to remain intact (2 M NaCl) (Wilson et al., 1999). Malate dehydrogenase of H. marismortui has been extensively studied and from these studies, it was concluded that this halophilic protein tolerates a higher concentration of salt by hydrated salt ions that associate with the abundant acidic residues on the protein surface so that the salt concentration of the solvation layer is higher than that of the bulk solvent (Bonete et al., 1994; Madern et al., 1995; Ebel et al., 1999). The molecular mass of this 303 amino acid protein from H. marismortui is about 33 kDa (Cendrin et al., 1993). Purified 20S proteasomes of H. marismortui are able to use malate dehydrogenase as a substrate in protein hydrolysis (Franzetti et al., 2002). Since no large proteins have been defined as substrates for H. volcanii, malate dehydrogenase from H. marismortui is an ideal substrate to study because even though it is purified from a halophile, it itself is not stable in salt concentrations below 2 M (Franzetti et al., 2002). Below 2 M salt concentrations, malate dehydrogenase begins to denature at 40C and remains unfolded even when returned to a 2 M KCl buffer (Franzetti et al., 2002). Also, it was found that the unfolded protein did not aggregate in low salt conditions (Franzetti et al., 2002). It was concluded that malate dehydrogenase purified from H. marismortui is a better substrate if it undergoes denaturation, as opposed to being presented as a fully denatured polypeptide chain (Franzetti et al., 2002). It therefore seemed fitting to use this protein as a substrate for the H. volcanii 20S proteasomes with the -protein variants

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48 produced in this study. Malate dehydrogenase from H. marismortui was overexpressed in E. coli and purified, as described by Cendrin et al. (1993), with modifications described in the Methods and Materials section. The -His and -Thr49Ala-His containing 20S proteasomes did not hydrolyze recombinant malate dehydrogenase under optimal conditions for H. volcanii activity (2 M NaCl, 60-70C). Even under various conditions that would encourage malate dehydrogenase to unfold and not aggregate, including low salt (1 M NaCl), where H. volcanii 20S proteasomes have been shown to retain 50% optimal activity (Wilson et al., 1999), and low temperatures (40C), the -His variants still did not hydrolyze the substrate (data not shown). Furthermore, malate dehydrogenase was not degraded when PanA purified from H. volcanii (Reuter et al., 2004) was included in a 5:1 ratio with 20S proteasomes and incubated overnight at 40C in 1 M NaCl as determined by SDS-PAGE analysis (data not shown). Also, it has been recently observed that 20S proteasomes of H. volcanii are stabilized in 10 mM CaCl 2 in the absence of 2 M NaCl without minimal effect to peptidase activity using N-Succ-LLVY-Amc as a substrate (Kaczowka and Maupin-Furlow 2003). The -variants in this study, however, did not hydrolyze bovine -casein even in low salt buffers (i.e., supplemented with 10 mM CaCl 2 , 2 M KCl, 1 M KCl, or 0.5 M KCl) at 60C (data not shown). Kinetics of -protein Variants Kinetic parameters of the -His and -Thr49Ala-His 20S proteasomal variants were analyzed using varying concentrations of N-Succ-LLVY-Amc as a substrate under the same conditions used for determining peptide hydrolyzing activity as described in Methods and Materials. This substrate is cleaved by the chymotryptic-like activity,

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49 which is the best-characterized activity of 20S proteasomes and the only significant activity reported for H. volcanii 20S proteasomes (Wilson et al., 1999). In this study, the -His variant displays a sigmoidal curve of activity with increasing substrate concentration suggesting cooperative kinetics, with an apparent Vmax of 160 nmols per min of product produced per mg of protein and an apparent Km of 5 M (Figure 2-11). It is therefore conceivable that there is cooperative binding of substrate to the multiple (fourteen) active sites of the proteasome, as it has been shown before for eukaryotic 20S proteasomes (Orlowski et al., 1991; Djaballah and Rivett 1992; Stein et al., 1996). In contrast, the -Thr49Ala-His variant follows Michaelis-Menten kinetics with an estimated Vmax of 130 nmols per min of product produced per mg of protein, with an estimated Km of 5.5 M and an estimated kcat of 1.35 sec (Figures 2-11 and 2-12). The absence of cooperative kinetics and instead the presence of a hyberbolic curve of activity with increasing peptide substrate concentrations is most likely due to -Thr49Ala-His variant subunits being interspersed among more prevalent genomic encoded -subunits. It is possible that cooperativity is lost because subunits without threonine active sites (i.e., -Thr49Ala-His variant subunits) are adjacent to genomic encoded -subunits, which disrupts the efficient continual rate of reaction of active subunits for substrate, that is to say, it takes slightly longer for substrate to be “placed” and acted upon with an active subunit when the active site subunit continuum is disrupted with inactive subunits. Discussion While the assembly of and -type subunits differs among the domains of life, the processing of the -subunit propeptide appears to occur at the moment full 20S proteasomes are being formed, that is to say, that just before the two half proteasomal

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50 complexes join together at the -ring interface, the prosequences are autocatalytically cleaved to allow the formation of a full mature 20S proteasome (Schmidtke et al., 1996). This implies that the half 20S complexes contain intact propeptides, where they are cleaved in fully assembled 20S proteasomes. In this study, the propeptides of the genomic -subunits appear to be fully processed exposing an active site threonine residue. It does not appear that -Thr49Ala-His is processed at all since the band that represents processed -subunits is not present in this sample in comparison to -His, which also appears to be completely processed. Instead, another band of slightly higher molecular weight does appear in the -Thr49Ala-His samples that, based on its migration pattern and Western analysis results (Figure 2-4), appears to be unprocessed -Thr49Ala-His. The -Thr49Ser-His sample does appear to be processed, as a comparable band to -His is found in this sample; however, processing levels appear to be reduced with this mutation, as demonstrated by another band present that is comparable to the -Thr49Ala-His variant (Figure 2-5). Also, longer exposures with the antiantibody show varying levels of processing for the -Thr49Ser-His sample, where signals are seen for what is expected to be the processed and unprocessed -His proteins (Figure 2-3). This suggests that processing levels of -Thr49Ser-His variant 20S proteasomes are decreased with this mutation present at the active site. Based on this data, it is most likely that processing of the -His variants in H. volcanii might occur via an intramolecular mechanism. If the mechanism were to occur by intermolecular means, then it is speculated that the genomic -subunit that assembles with the expressed -mutant from the plasmid would process the -variant propeptide; however, no processing appeared to occur with the -Thr49Ala-His sample, so genomic

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51 -subunits do not appear to be processing -proteins that cannot process themselves. Therefore, the results of this study suggest that the most likely processing event in H. volcanii occurs by intramolecular means. It is feasible, however, that processing is abolished because the bond between the conserved glycine and threonine residue is altered when exchanging the active site threonine residue for alanine. It is possible from this alteration that an adjacent subunit cannot cleave this modified bond, that upon different site-directed mutations of the lysine 33 residue to an alanine residue with an intact glycine to threonine active site motif might suggest intermolecular processing; however, this remains to be determined. Also, further analysis of the -His variants, by N-terminal sequencing, would also serve to clarify whether or not processing events did in fact occur. The estimated molecular weight of a fully assembled 20S proteasome purified from H. volcanii with 1 and -subunits was 600 kDa (Wilson et al., 1999). Differences in the molecular weights of the assembled -protein variants compared to this published report is most likely due to the addition of a seven amino acid linker with a polyhistidine tag. In a 20S proteasome, there are fourteen -subunits with a calculated molecular mass of 20 kDa, assuming that there is no propeptide present. The calculated molecular mass of a processed -His variant with a linker and polyhistidine tag is 22 kDa, while the molecular mass of the -Thr49Ala-His variant is calculated to be 27 kDa (with the linker also present). The addition of these extra entities on the -protein would slightly increase the molecular mass by about 1.5 kDa. Differences in calculated and estimated molecular masses are likely due to interactions of the 20S proteasomal variants with the matrix.

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52 Whether it is the histidine tags or other interactions due to a conformation change in the 20S complexes remains to be determined. Peptidase and proteinase activity has been shown before for 20S proteasomes of H. volcanii using N-Succ-LLVY-Amc and bovine insulin B chain, respectively, as substrates (Wilson et al., 1999). The significant presence of peptidase and proteinase hydrolyzing activity of the -Thr49Ala-His variant is most likely due to activity of the genomic encoded -subunits. It appears that the -Thr49Ala-His variant is not incorporated into the 20S proteasomal complexes as high a level as the processed genomic encoded -subunits (Figures 2-4 and 2-5) based on the finding that the ratio of genomic encoded -subunits compared to the -Thr49Ala-His variant is about 3:1. In addition, it is only reasonable that the activity of the -Thr49Ala-His variants is actually from the genomic encoded -subunits, as it has been shown by Kisselev et al. (2000) that alanine cannot function as an efficient active site residue for -subunits, although this data was from in vitro studies with 20S proteasomes that only carried a threonine to alanine mutation without any active -subunits. Also, since there is possibly no processing observed with the -Thr49Ala-His mutant, there would be no active site residue available for activity. Since the substrates used are relatively small, it possible that the substrates were not affected by steric hinderance of the propeptides present and “by-passed” these propeptides to access the genomic -subunit active sites. The proteasome has been shown to degrade unfolded substrates (Wenzel and Baumeister 1993). These substrates used in this study are relatively small, so in regard to the proteasome, they are essentially in an “unfolded” state.

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53 Higher proteinase activity values for -His are also shown in this study as compared to previously reported values of wild type 20S proteasomes of H. volcanii (Wilson et al., 1999). The six-fold difference in activity between the two studies could be due to the purification conditions. The purification methods of Wilson et al. required harsher conditions, such as heat, and more manipulations of the sample to purify a native 20S proteasome from H. volcanii. This could have possibly affected the proteinase activity of the proteasome, whereas the -variants in this study were purified in fewer steps via an affinity tag and thus did not necessitate the use of high temperatures for purification purposes. The peptidase activity of the -His variants, however, is comparable to wild type 20S proteasomes; therefore, it is more likely that the purification scheme employed by Wilson et al. retained a low level of contaminating proteins that competed with insulin chain B as a substrate. Another plausible explanation for such high activity of the -His variants is that the histidine tags present may have altered the conformation of 20S proteasomes. Since the histidine tags are on the C-terminal end of the -protein, they may be causing some type of conformation alterations subsequently modifying the opening or other structural features of the variant 20S proteasomes. Eukaryotic 20S proteasomes have been shown to display cooperative kinetics with varying concentrations of peptide substrate (Orlowski et al., 1991; Djaballah and Rivett 1992; Stein et al., 1996), so it is not surprising that the -His variant also displays cooperative kinetics; however, we demonstrate that the -Thr49Ala-His mutant does not exhibit this type of kinetics. If other active sites within the proteasome promote binding of substrate, it would seem reasonable that the active sites in the -Thr49Ala-His variant (most likely the genomic-encoded -subunits) would also display cooperative kinetics,

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54 although at a reduced rate. Kisselev et al. (1999a) have shown that certain sites can allosterically activate other sites with the same type of activity, as well as sites of different types of activity. Also, a later study showed that inhibitors (which mimic substrate) of PGPH activity stimulated trypsin-like activity of yeast 20S proteasomes, but this activity was not stimulated when the inhibitor could not bind to PGPH sites due to mutant -protein with an uncleaved propeptide (Kisselev et al., 2003). It might be likely that the steric hinderence of the propeptides present may alter the kinetics where they might inhibit the cooperativity of other subunits. Also, the amount of -Thr49Ala-His variant subunits is reduced compared to genomic encoded -subunits, as shown by densitometry studies. This might interfere with the cooperativity of -Thr49Ala-His variant 20S proteasomes, as fewer active sites are present within the 20S complex. In addtion, the cooperativity might be interrupted, as -Thr49Ala-His variant proteins are interspersed among genomic encoded -subunits, hindering the increased affinity for peptide substrate. The disruption in cooperativity upon site-directed mutagenesis is not without precedent, as it has been shown by Chen et al. (1994) that replacing the glutamic acid residue that binds AMP with glutamine results in a loss of cooperativity. It is rational that 20S proteasomes of H. volcanii exhibit cooperativity since there are fourteen active sites that act coordinately together to degrade substrate. It is important that there is communication between the sites to achieve efficient hydrolysis. Also, the fact that 20S proteasomes of H. volcanii are cooperative lends to the idea that the proteasome is a flexible protein molding its conformation to allow for the best conditions in ridding the cell of unwanted proteins.

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55 Table 2-1. Strains and plasmids used in this study. Strain or plasmid (protein) Phenotype or genotype (oligonucleotides used for PCR amplification) Source or reference Strains E. coli DH5 F recA1 endA1 hsdR17(r k m k + ) supE44 thi-1 gyrA relA1 Life Technologies E. coli BL21 (DE3) F ompT [lon]hsdS B (r B m B + ) (an E. coli B strain) with DE3, a prophage carrying the T7 RNA polymerase gene Novagen E. coli GM2163 F ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm-6 hisG4 rfbD1 rpsL 136 dam13::Tn9 xylA5 mtl-1 thi-1 mcrB1 hsdR2 New England Biolabs E. coli Rosetta (DE3) F ompThsdS B (r B m B ) gal dcm (DE3) pRARE 2 (Cam R ) Novagen H. volcanii DS70 Cured of plasmid pHV2 Wendoloski et al., 2001 Plasmids pJAM621 (-His) Km r ; 738-bp fragment generated by PCR amplification from the H. volcanii genome ligated into pET24b using NdeI and HindIII; carries psmB-H6; -His expressed in E. coli (5’-CTTACCTCATATGCGTACCCCGACTC-3’ and 5’-TTTGAAGCTTTTCGAGGCCTTCGAAG-3’) (NdeI and HindIII sites in bold) Kaczowka and Maupin-Furlow 2003 pLITMUS28 Ap r ; 2,823-bp pUC19-derived vector with M13 origin of replication New England Biolabs pJAM697a Ap r ; 464-bp BglII-to-SacII fragment of pJAM621 Vent polished subcloned into blunt end Klenow polished XhoI site of pLITMUS28 This study

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56 Table 2-1. Continued. Strain or plasmid (protein) Phenotype or genotype (oligonucleotides used for PCR amplification) Source or reference Plasmids pJAM698-2.4 Ap r ; 155-bp fragment generated by PCR amplification from pJAM697a cut with NdeI and ligated into NdeI/PshAI site of pJAM697a (5’TGGTGCTTCCGGTCTTCGTCTC-3’) (5’-CTTACCTCATATGCGTACCCCGACTC-3’) (Thr to Ser and NdeI sites are in bold, respectively) This study pJAM699-2 Ap r ; 155-bp fragment generated by PCR amplification from pJAM697a cut with NdeI and ligated into NdeI/PshAI site of pJAM697a (5’TGGTCGCTCCGGTCTTCGTCTC-3’) (5’-CTTACCTCATATGCGTACCCCGACTC-3’) (Thr to Ala and NdeI sites are in bold, respectively) This study pJAM553 Km r ; 240-bp NdeI-to-AatII fragment of pJAM698-2.4 ligated into NdeI/AatII site of pJAM621 This study pJAM554 Km r ; 240-bp NdeI-to-AatII fragment of pJAM699-2 ligated into NdeI/AatII site of pJAM621 This study pJAM202 (-His) Ap r Nv r ; 1,152-bp XbaI-to-DraIII fragment of pJAM621 blunt end ligated with a 9.9-kb BamHI-to-KpnI fragment of pBAP5010; psmB-H6 oriented with rRNA P2; -His expressed in H. volcanii Kaczowka and Maupin-Furlow 2003 pJAM202-1.1 (Thr49Ser-His) Ap r Nv r ; 825-bp NdeI-to-BlpI fragment of pJAM553 cloned into pJAM202; Thr49Ser mutation in psmB; -Thr49Ser-His expressed in H. volcanii This study

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57 Table 2-1. Continued. Strain or plasmid (protein) Phenotype or genotype (oligonucleotides used for PCR amplification) Source or reference Plasmids pJAM202-2.1 (Thr49Ala-His) Ap r Nv r ; 825-bp NdeI-to-BlpI fragment of pJAM554 cloned into pJAM202; Thr49Ala mutation in psmB; -Thr49Ala-His expressed in H. volcanii This study p-hMDH2 (MDH) Ap r ; malate dehydrogenase from H. marismortui cloned into pET11a Cendrin et al., 1993

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58 Figure 2-1. Partial purification of the variant -proteins. SDS-PAGE gel stained with Coomassie Blue of Ni 2+ -Sepharose purified protein fractions of H. volcanii expressing -His (lane 1), -Thr49Ser-His, (lane 2), -Thr49Ala-His (lane 3) proteins.

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59 Figure 2-2. Immunoanalysis of Ni 2+ -Sepharose purified protein fractions of H. volcanii expressing -His, -Thr49Ser-His, and -Thr49Ala-His proteins. Samples were transferred to a PVDF membrane and probed with polyclonal antibodies raised against 1 and 2-subunits. Lanes 1 and 4 are the -His variant, lanes 2 and 5 are the -Thr49Ser-His variant, and lanes 3 and 6 are the -Thr49Ala-His variant.

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60 Figure 2-3. Levels of -subunit propeptide processing in 20S proteasomes containing the -protein variants. Immunoanalysis of Ni 2+ -Sepharose purified protein fractions of H. volcanii expressing -His, -Thr49Ser-His, and -Thr49Ala-His proteins. Samples were transferred to a PVDF membrane and probed with a polyclonal antibody raised against -subunit. Different exposures with the antiantibody show varying levels of processing among -His (lane 1), -Thr49Ser-His (lane2) and -Thr49Ala-His (lane 3) proteins expressed from the plasmid. () processed -subunits expressed from the genome, (*) processed -His subunits expressed from the plasmid, and (<) unprocessed -His subunits expressed from the plasmid.

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61 Figure 2-4. Processing of the -Thr49Ala-His variant. -Thr49Ala-His proteasomal variant propeptide does not appear to be processed. Immunoanalysis of -His (lanes 1 and 3) and -Thr49Ala-His (lane 2) variants (1 g) using anti-His antibody conjugated to HRP. Calculated molecular mass for -His (blot A, lane 1) is 24.5 kDa and for -Thr49Ala-His (blot A, lane 2) is 26.3 kDa.

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62 Figure 2-5. Assembly and suggested processing levels of the -His variants. All three modified -His tagged proteins assemble with 1, 2, and -subunits expressed from the genome. SDS-PAGE gel stained with Coomassie Blue of Ni 2+ -Sepharose purified protein fractions of H. volcanii expressing -His (lane 1), -Thr49Ser-His (lane 2), and -Thr49Ala-His (lane 3) proteins. () denotes possible processed -subunits and () denotes possible unprocessed -His subunits in -Thr49Ala-His and -Thr49Ser-His variants.

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63 Figure 2-6. Assembly of the variant -proteins. Assembly of full 20S proteasomes containing the -protein variants appears to occur. Superose 6 gel filtration chromatographs of Ni 2+ -Sepharose purified fractions of H. volcanii strains expressing -His (a), -Thr49Ser-His (b), and -Thr49Ala-His (c) proteins. Estimated molecular mass of (a) 995 kDa, (b) 1, 352 kDa, and (c) 976 kDa. The standard curve shown includes the -variant proteins (a) -Thr49Ala-His, (b) -His, and (c) -Thr49Ser-His.

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64 Figure 2-7. Stability of the -protein variants. -protein variants (-Thr49Ser-His, and -Thr49Ala-His) are not stable under certain conditions compared to the -His variant proteins. Chromatographs of -protein variants (a) -His, (b) -Thr49Ser-His, and (c) -Thr49Ala-His purified by hydroxylapatite chromatography. Extra peaks indicate that 20S proteasomes are disassembling in a 10 mM to 420 mM sodium phophate gradient. Arrows indicate fractions in that peak that have peptide hydrolyzing activity as described in Methods and Materials. Numbers indicate subunits 1 (1), 2 (2), and (3).

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65 Figure 2-8. Purity of -His and -Thr49Ala-His variants. TCA precipitated -His and -Thr49Ala-His variants (1g) purified by Ni 2+ -Sepharose and DEAE-cellulose chromatography were used to determine specific peptide and protein hydrolyzing activities. SDS-PAGE gel stained with Coomassie Blue of -His (lane 2) and -Thr49Ala-His (lane 3). Lane 1 is the marker.

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66 Figure 2-9. Peptidase activity of the -protein variants. The 20S proteasome -protein variants -His and -Thr49Ala-His exhibit significant peptidase activity. Peptide hydrolysis of Ni 2+ -Sepharose and DEAE-cellulose purified -His and -Thr49Ala-His samples (0.1g) was assayed as described in Materials and Methods using 20 M N-Succ-LLVY-Amc as substrate.

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67 Figure 2-10. Proteinase activity of the -protein variants. The 20S proteasome -protein variants -His and -Thr49Ala-His exhibit significant proteinase activity. Protein hydrolysis of Ni 2+ -Sepharose and DEAE-cellulose purified -His and -Thr49Ala-His samples (0.2g) was assayed as described in Materials and Methods using 145 M bovine insulin chain B as substrate.

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68 Figure 2-11. Kinetics of the -protein variants -His and -Thr49Ala-His. -protein variant -His (open circles) shows sigmoidal kinetics, while the -Thr49Ala-His (closed triangles) variant shows Michaelis-Menten kinetics. Ni 2+ -Sepharose and DEAE-cellulose purified 20S proteasomes containing the -His variant (0.1g) were assayed with varying levels of N-Succ-LLVY-Amc substrate concentrations as described in Materials and Methods.

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69 Figure 2-12. Hanes-Wolff plot for 20S proteasomes containing the -Thr49Ala-His variant.

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CHAPTER 3 CONCLUSIONS There is much debate as to the mechanism of processing (intraor intermolecular) of -subunits of 20S proteasomes among the domains of life. This study suggests, as shown by in vivo experimentation in the haloarchaeon H. volcanii, that the prosequences of the -subunits are most likely processed intramolecularly. It is possible that one method is preferred over the other in different species or that both methods occur within one organism. This remains to be determined. Also, it was shown that the peptidase and proteinase activities of 20S proteasomes with a -His variant protein are comparable to previous studies, if not more active. The increase in proteinase activity could be due to less stringent purification conditions that salvaged the full active form of the variant 20S proteasome. It is also possible that low levels of contaminating proteins present in the wild type preparation compete as 20S proteasomal substrates with bovine insulin chain B. In addition, histidine tags used in this study may have altered the conformation of the variant 20S proteasomes to where access as a protein substrate is limited. Significant but reduced activity is reported in this study with the -Thr49Ala-His variant, although it is most likely due to the genomic encoded -subunits that assembled with the variant protein, since alanine has been previously shown to impede activity. Assembly of full 20S proteasomes still does occur with the -protein variants. The estimated molecular weights of these variant proteasomes differ slightly from wild type 70

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71 20S proteasomes purified from H. volcanii. These increases in mass are most likely due to the addition of a C-terminal histidine tag with a seven amino acid linker and the unprocessed propeptides still inside the -Thr49Ala-His variant proteasome. Although there is full assembly with the variant -subunits, the -Thr49Ala-His sample, however, is shown to be unstable under certain conditions and shows a reduction in activity quite quickly. This study is a contribution to the increasing knowledge of 20S proteasomes. Archaea, in regard to eukarya, are simpler models to study. They also are comparable models to eukarya in that their genetic mechanisms and protein turnover schemes are similar. Elucidating and understanding basic proteasomal structure and functional could lead to important medical advances, as the 20S proteasome is the major degradative pathway in most organisms and dysfunction in the ubiquitin-proteasomal degradative pathway has been shown to be implicated in neurodegenerative diseases of humans, including Parkinson’s and Alzheimer’s disease. Future studies include defining protein substrates and determining the degradation rates with these substrates of the -His and -Thr49Ala-His protein variants. It has been shown by Hortin and Murthy (2002) that synthetic substrates of variable lengths linked to a constant substrate group (Ala-Ala-Phe) and a linear polymer (methoxypolyethylene glycol) are degraded by 20S proteasomes; therefore, it is also desirable to define the size limit of protein substrates of the -His and -Thr49Ala-His protein variants in H. volcanii. In addition, N-terminal sequencing of the -His variants used in this study would aid in defining the levels and site of processing of the -protein.

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BIOGRAPHICAL SKETCH Amy Joy Wright was born in Jacksonville, FL, on October 10, 1980, to Stephen and Saundra Wright. She has one older brother, Barak. She began her schooling at a private British school, Butler’s Court, in Beaconsfield, England. Her family moved back to Jacksonville and she continued her schooling there and graduated high school from Trinity Baptist Academy with high honors. Knowing she wanted to go to college, but unsure of her major, she attended Florida Community College at Jacksonville, where she met her most influential teacher, Dr. Fred Reynolds. Dr. Reynolds was very supportive of her career goals, was encouraging as a professor boosting her esteem as a student, and was a source of academic advice and guidance. After graduating with high honors with an Associate of Arts degree, she attended the University of Florida (UF), where she majored in microbiology and cell science and minored in chemistry. She graduated in May of 2002 and began to work for Dr. Paul Gulig at UF. Her project included expressing and purifying a recombinant surface protein of vaccina virus to ultimately make monoclonal antibodies for use on a fiber optic probe to detect suspected smallpox contamination in the field. After being exposed to a laboratory setting, she decided to begin graduate school to pursue a master’s degree under the direction of Dr. Julie A. Maupin-Furlow. Her project included purifying and characterizing the processing, assembly, and activity of variants of 20S proteasomes of the haloarchaeon Haloferax volcanii. Amy is graduating with a Master of Science degree in microbiology and cell science in May of 2006. 82