Citation
Effect of Charge and Polarity Changes on the Propensity of Inclusion Formation by Mutant SOD1

Material Information

Title:
Effect of Charge and Polarity Changes on the Propensity of Inclusion Formation by Mutant SOD1
Creator:
Roberts, Brittany-Lee
Pate, Kinaree
Brown, Hilda
Workman, Aron
Borchelt, David R.
Publication Date:
Language:
English

Subjects

Subjects / Keywords:
Aggregation ( jstor )
Amyotrophic lateral sclerosis ( jstor )
Cell aggregates ( jstor )
Charge separation ( jstor )
Genetic mutation ( jstor )
Inclusion bodies ( jstor )
Motor neurons ( jstor )
Plasmids ( jstor )
Point mutation ( jstor )
Superoxides ( jstor )
Amino acids
Mutation (Biology)
Polarity (Biology)
Superoxide dismutase
Genre:
Undergraduate Honors Thesis

Notes

Abstract:
The present study investigated the relationship between changes in charge and polarity on the propensity of aggregate formation by mutant SOD1. Two different mutation sites were examined in the present study, E133 and E40. For both sites, point mutations were made to the glutamate residue in order to assess the effect of changes in charge and polarity at these sites on the propensity of inclusion formation. Experimental constructs at the E133 site that showed aggregate formation included the E133V and E133 deletion (both known to cause fALS). The E133M, L, and G did not form inclusions and resembled the negative WT control. Experimental constructs at the E40 site that demonstrated aggregate formation included the E40G and E40V. The E40Q did not form inclusions. These findings demonstrate that specific changes in amino acid charge or polarity are required to induce aggregation of SOD1. These findings also indicate a simple loss of a negative charge does not induce aggregation. ( en )
General Note:
Brittany Roberts awarded Bachelor of Science; Graduated May 7, 2013 summa cum laude. Major: Interdisciplinary Studies, Emphasis/Concentration: Neurobiological Sciences
General Note:
College/School: College of Liberal Arts and Sciences
General Note:
Legacy honors title: Only abstract available from former Honors Program sponsored database.
General Note:
Advisor: Dr. David Borchelt

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Brittany-Lee Roberts, Kinaree Patel, Hilda Brown, Aron Workman and Dr. David Borchelt. 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.

Full Text

PAGE 1

Effect of Charge and Polarity Changes on the Propensity of Inclusion Formation by Mutant SOD1 Brittany Roberts, Kinaree Patel, Hilda Brown, Aron Workman, David R. Borchelt Department of Neuroscience, Center for Translational Research in Neurodegenerative Disease, of Florida, Gainesville, FL 32610

PAGE 2

Abstract The present study investigated the relationship between changes in charge and polarity on the propensity of aggregate formation by mutant SOD1. Two different mutation sites were examined in the present study, E133 and E40. For both sites, point mutations w ere made to the glutamate residue in order to assess the effect of changes in charge and polarity at these sites on the propensity of inclusion formation. Experimental constructs at the E133 site that showed aggregate formation included the E133V and E133 deletion (both known to cause fALS). The E133M, L, and G did not form inclusions and resembled the negative WT control. Experimental constructs at the E40 site that demonstrated aggregate formation included the E40G and E40V. The E40Q did not form inclusio ns. These findings demonstrate that specific changes in amino acid charge or polarity are required to induce aggregation of SOD1 These findings also indicate a simple loss of a negative charge does not induce aggregation.

PAGE 3

Introduction Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease primarily characterized by loss of upper and lower motor neurons. Although most forms of ALS result from an unknown etiology (sporadic ALS or sALS), a small subset of cases demonstrat e dominant patterns of inheritance in specific proteins (familial ALS or fALS). Of these inherited genetic mutations approximately 20% result in Cu Zn superoxide dismutase (SOD1) [ 1 ] the protein responsible for degrading toxic oxygen radicals in the cell cytoplasm. As of now, over 150 genetic mutations in SOD1 have been linked to f ALS { http://alsod.iop.kcl.ac.uk/default.aspx } While the majority of these inherited mutations result from point mutations few fALS mutations are product of early termination resulting in C terminal ly truncated proteins. Principally, SOD1 is located in the cell cytosol; however, trace amounts have been found in the nuclei, peroxisomes, and mitochondria. SOD1 is a homodimer composed of two 153 strands, an active site that binds copper, a binding site for zinc, an electrostatic loop that directs the substr ate into the active site, and an intramolecular disulfide bond between cysteine 57 and cysteine 146 [2], [3 ] The molecular characteristics of mutant SOD1 responsible for disease progression have yet to be identified. Through cell culture and in vitro models, studies have demonstrated that the effect of fALS mutations on the normal enzyme activity of SOD1 varies greatly [4] [8 ] While some mutants are highly unstable [9], [10 ] or inactive [6], [11 ] other mutants retain high levels of activity [3], [8] [12] [14 ] Because some mutants maintain high levels of activity and SOD1 knockout in mice does not induce ALS like symptoms [15 ] it is indicated that fALS mutations in SOD1 cause disease as a result of an acquired toxic property and not loss of functio n.

PAGE 4

Various studies have demonstrated that as a result of fALS mutations in SOD1, proteins have a higher susceptibility to aggregate formation [16] [20 ] T his is common to all identif ied fALS associated mutations [20 ] Although the role of mutant protein aggregates in acquired neurotoxicity has not been defined s tudies have identified a positive correlation between the rate of aggregate formation and the level of human disease progression [10 ], [20 ]. As identified when comparin g the A4V mutation (rapid disease progression and high propensity of aggregate formation) to the H46R mutation (slow disease progression and low propen sity of aggregate formation) [20 ]. To date, the mechanisms involved in aggregate formation in SOD1 are n ot fully understood. Previously, our lab has studied the folding and aggregation of mutant SOD1 by fusing the coding sequence of SOD1 to yellow fluorescent protein (YFP) and then expressing these fusion proteins in cultured cell lines [21] [23 ] Fusion pro teins of WT SOD1 and YFP demonstrate a diffuse distribution in the cell cytoplasm as expected [21], [23 ] On the other hand, when mutations associated with ALS are made in SOD :YFP fused proteins, the protein forms inclusions i n the cell cytoplasm [21]. [23 ] The current study aims to provide a visual analysis of what type of mutatio n causes the mis folding of SOD1. As a result of the repulsive property of negatively charged species, it was hypothesized that loss of negative residue in the SOD1 would induce protein mis folding and thus aggregate formation [ 24], [25] Our findings demonstrate that simple loss of a negative charge does not induce aggregate formation. Methods Generation of mutant SOD plasmids HSOD1 cDNA's with mutations E133G E133L E133M E133V E133Deletion E40G E40Q, and E40V were subcloned into modified pEF BOS vector containing YFP cDN A [20 ] using the

PAGE 5

In Fusion Cloning kit (Clontech, Mountain View, CA; Cat. No. 63961 7). Each mutant was amplified through standard PCR strategies. Once t he PCR product was obtained, it was analyzed on a 1% agarose gel in order to verify a single PCR product. If there were more than one product present on the gel, the desired PCR product was extracted using a gel extraction kit ( ). Upon acquiring the desir ed PCR product an infusion cloning procedure was used to insert the DNA into pEF BOS vector that already contained the YFP cDNA. These plasmids were transformed into NEB p reparations of plasmid DNA for transfection were prepared by CsCl gradient purification. All plasmids were verified by DNA sequence analysis. Transient Transfections Plasmid DNA expressing constructed mutants were transiently transfected into Chinese hamster ovary (CHO) cells. The day before the transfection, the CHO cells were split into 60 mm poly D lysine coated dishes (1 plate for each DNA construct). Upon reaching 95% confluency, cells were transfected with Li in a CO 2 incubator for 24 hours. Representative pictures were then taken using an AMG EVOS fl digital inverted microscope for fluorescence and transmitted light applications. Pictures of both typical cells and cells with aggregates were taken at both 20 and 40 magnification at both 24 and 48 hours. The transient transfections were repeated two more times for each construct with representative pictures taken of random fields. The images f rom multiple transfections were analyzed with cells showing YFP fluorescence, fluorescent inclusions, and fluorescent cells showing condensed morphology counted in a blinded fashion.

PAGE 6

Results Two different mutation sites were examined in the present stu dy, E133 and E 40. For both sites, various point mutations were made to the glutamate residue in order to evaluate the effect of changes in charge and polarity at these sites on the propensity of inclusion formation. Results of experimental mutations were assessed through comparison to a set of YFP tagged controls (figure 1) including WT:YFP (negative control, no inclusion formation ) and A4V:YFP (positive control, high propensity of inclusion formation). As described in methods, t hree consecutive transient transfections were done for each construct and representative pictures were taken at 24 and 48 hours for a quantitative analysis of aggregate formation (Table 1) As noted in our previous work, cells of an unknown condensed morphology were present in this study as well. Cells demonstrating this condensed morphology were quantified into two subsets: those with inclusion formation and those without inclusion formation (Table 1) Figure 1. Mutant SOD1:YFP Controls. CHO cells were transfected with plasmids for each control and representative pictures were taken at 24 and 48 hours. A4V provides a positive control for inclusion formation and WT provides a negative control for inclusion formati on.

PAGE 7

Table 1 Quantitative analysis of aggregation of SOD1 mutants in transfected cells at 24 and 48 hours. At the 133 site, five different mutated plasmids were generated and transiently transfected into the cell line. The SOD1:YFP mutations consisted of a glutamate residue point mutation to: glycine, valine, leucine, methionine, and a deletion. Of the construc ts, the deletion and valine are known fALS associated mutations. On the other hand, glycine, leucine, and methionine were experimental constructs generated in order to assess the relevance of the negative charge on the glutamate residue. As expected, in ce lls expressing the fALS associated mutation s E133deletion SOD1:YFP (Fig. 2I and J) (Table1) and E133V SOD1:YFP (Fig. 2G and H) (Table 1) we observed inclusion formation by 24 hours and an increased amount by 48 hours Conversely cells expressing the exp erimental constructs, E133G SOD1:YFP (Fig. 2A and B) (Table1) E133L SOD1:YFP (Fig. 2C and D) (Table1) and E133M SOD1:YFP (Fig. 2E and F) (Table1) did not demonstrate any obvious inclusion formation by 24 or 48 hours.

PAGE 8

Figure 2 Mutation of glutamate 133 to glycine, leucine, methionine, valine, and a deletion. fALS associated mutations are the only constructs to induce aggregate formation. In order to further assess the loss of charge, various mutations were constructed at the glutamate 40 site (E4 0). Currently there are no known fALS associated mutations related to the

PAGE 9

E40 site and it provided an additional glutamate residue to evaluate experimental constructs. Mutant SOD1:YFP constructs included point mutations from the glutamate residue to: glycine, glutamine, and valine. The E40G SOD1:YFP showed a high propensity of inclusion formation at 24 hours (Fig. 3A) and over 75% of the fluorescent cells contained inclu sions by 48 hours (Table 1) The E40V SOD1:YFP also produced aggregates by 24 hours (Fig. C) ; however, inclusion formation was far less expressed in comparison to the E40G:YFP (Table 1) and A4V:YFP (positive control, Table 1) The E40Q SOD1:YFP construct d id not form any obvious inclusions at 24 or 48 hours (Fig. 3E and F) Figure 3. Mutated SOD1 constructs were transfected into CHO cells and images were taken at 24 and 48 hours. Mutation of glutamate 40 to glycine, glutamine, and valine is depicted.

PAGE 10

Discussion Through visual and quantitative analysis, our study examined relationship between charge and polarity on the propensity of aggregate formation by the fusion of mutant superoxide dismutase 1 (SOD1) to YFP. Our findings indicate specific changes in amino acid charge or polarity are required in order to induce inclusion formation of SOD1 ; however, these specifications are still unknown Our results also demonstrate that a simple loss of a negative charge is not enough to form aggregates in mutant SOD1 There were two negatively charged positions of SOD1 examined in the present study, 40 and 133, b oth of which contain a glutamate (E) residue. For many proteins, aggregation is due to a type of protein: protein interaction in which specific segments of protein interact in a manner similar to stacking. In this arrangement, a single segment of a protein aligns with the same segment of the protein o ver and over again to form a tightly interacting stack. It was original ly believed that the loss of charge may contribute to protein aggregation by reducing the repulsion that could occur between such segments when charged residues try to align and stack. This premise is supported by the fact that all known fALS associated mutations form inclusions [20] and multiple fALS associ ated mutations are found at the E133 position { http://alsod.iop.kcl. ac.uk/default.aspx } One aspect of our study supports this idea by show ing that the complete deletion of glutamate and point mutation to valine (lacking charge) at the 133 site both demonstrated inclusion formation by 24 hours and persistent formation by 48 hours While this seemed promising w e also examined the effect of the E133 residue mutated to alternative amino acids including: methionine, valine, and glycine; all of which did not form any obvious inclusions by 24 or 48 hours and portrayed the sa me characteristics as the WT (negative control). Because SOD1 mutations to leucine and methionine have similar side chains to valine

PAGE 11

(hydrophobic and no charge) and did not form inclusions, it is indicated that the loss of a negat ive charge is not the only characteristic necessary for aggregate formation. It should be d u ly noted that the magnitude of aggregate formation in the E133V and E133deletion were far less than the A4V at 24 and 48 hours. Based on previous studies correlati ng propensity of aggregate formation to rate of disease progression [10], [20], this possibly signifies that these fALS associated mutations should progress at a slower rate in comparison to the rapidly developing A4V. The 40 residue in SOD1 provided an a lternative site to assess the necessity of the glutamate residue. To date, there are no known fALS associated mutations located at the E40 site and it allowed a novel experimental site for our study. Three mutant SOD1 constructs were developed at the E40 s ite including: glycine, glutamine, and valine. Out of the three point mutations made, the E40G and E40V demonstrated inclusion formation. Conversely the E40Q did not form any obvious inclusions by 24 or 48 hours and had characteristics similar to the WT. Normally, the glutamate 40 forms a hydrogen bond with lysine 91. As a result, any change in charge at the 40 site s hould disrupt this bond and destabilize the protein thus resulting in aggregate formation. This is the case in regards to the glycine and va line SOD1 mutations ; however, the mutation from glutamate to glutamine did not express aggregate formation and resembled the WT Because of the lack of inclusion formation by the glutamine mutation at this site and the similarity in structure between glutamate and glutamine we conclude d elements other than charge were necessary in mediating aggregation. In terms of charge, all three constructed mutations should have resulted in inclusion development. Previousl y, several studies have demonstrated the expression of fusion proteins of mutant SOD1 and fluorescent reporter proteins are susceptible to aggregate formation [26 ] [34 ]. Our

PAGE 12

study provides additional corroborative data entailing the behavior of fluorescent proteins tagged to mutant SOD1. Although the toxic properties of mutant SOD1 are still unknown, o ur present study provides insight into the molecular characteristics involved in the formation of inclusions. Acknowledgements Parts of this paper were refe renced from our previous publication [34 ]. We are also thankful to Dr. P. John Hart for his thoughtful contribution.

PAGE 13

References 1. Rosen D.R., Siddique T., Patterson D., Figlewicz D.A., Sapp P., Hentati A., Donaldson D., Goto J., O'Regan J.P., Deng H. X., et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 1993;362:59 62. 2. Parge HE, Hallewell RA, Tainer JA (1992) Atomic structures of wild type and thermostable mutant recombinant human Cu,Zn superoxide dismutase Proc Natl Acad Sci USA 89 : 6109 6113. 3. Ogihara NL, Parge HE, Hart PJ, Weiss MS, Goto JJ, et al. (1996) Unusual trigonal planar copper configuration revealed in the atomic structure of yeast copper zinc superoxide dismutase Biochemistry 35 : 2316 2321. 4. Borchelt DR, Lee MK, Slunt HH, Guarnieri M, Xu Z S, et al. (1994) Superoxide dismutase 1 with mutations linked to familial amyotrophic lateral sclerosis possesses significant activity Proc Natl Acad Sci USA 91 : 8292 8296. 5. Nishida CR, Butler Gralla E, Selverstone Valentine J (1994) Characterization of three yeast copper zinc superoxide dismutase mutants analogous to those coded for in familial amyotrophic lateral sclerosis Proc Natl Acad Sci USA 91 : 9906 9910. 6. Ratovitski T, Corson LB, Strain J, Wong P, Cleveland DW, et al. (1999) Variation in the biochemical/biophysical properties of mutant superoxide dismutase 1 en zymes and the rate of disease progression in familial amyotrophic lateral sclerosis kindreds Hum Mol Genet 8 : 1451 1460. 7. Wiedau Pazos M, Goto JJ, Rabizadeh S, Gralla EB, Roe JA, et al. (1996) Altered reactivity of superoxide dismutase in familial amyotrop hic lateral sclerosis Science 271 : 515 518. 8. Hayward LJ, Rodriguez JA, Kim JW, Tiwari A, Goto JJ, et al. (2002) Decreased Metallation and Activity in Subsets of Mutant Superoxide Dismutases Associated with Familial Amyotrophic Lateral Sclerosis J Biol Ch em 277 : 15923 15931. 9. Jonsson PA, Ernhill K, Andersen PM, Bergemalm D, Brannstrom T, et al. (2004) Minute quantities of misfolded mutant superoxide dismutase 1 cause amyotrophic lateral sclerosis Brain 127 : 73 88. 10. Wang J, Xu G, Li H, Gonzales V, Fromholt D, et al. (2005) Somatodendritic accumulation of misfolded SOD1 L126Z in motor neurons mediates degeneration: {alpha}B crystallin modulates aggregation Hum Mol Genet 14 : 2335 2347. 11. Wang J, Caruano Yzermans A, Rodriguez A, Scheurmann JP, Slunt HH, et al. ( 2007) Disease associated mutations at copper ligand histidine residues of superoxide dismutase 1 diminish the binding of copper and compromise dimer stability J Biol Chem 282 : 345 352. 12. Gurney ME, Pu H, Chiu AY, Dal Canto MC, Polchow CY, et al. (1994) Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation Science 264 : 1772 1775. 13. Wong PC, Pardo CA, Borchelt DR, Lee MK, Copeland NG, et al. (1995) An adverse property of a familial ALS linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria Neuron 14 : 1105 1116. 14. Jonsson PA, Graffmo KS, Brannstrom T, Nilsson P, Andersen PM, et al. (2006) Motor neuron disease in mice expressing the wild type like D90A mutant superoxide dism utase 1 J Neuropathol Exp Neuro l 65 : 1126 1136.

PAGE 14

15. Reaume AG, Elliott JL, Hoffman EK, Kowall NW, Ferrante RJ, et al. (1996) Motor neurons in Cu/Zn superoxide dismutase deficient mice develop normally but exhibit enhanced cell death after axonal injury Nature Genetics 13 : 43 47. 16. Johnston JA, Dalton MJ, Gurney ME, Kopito RR (2000) Formation of high molecular weight complexes of mutant Cu, Zn superoxide dismutase in a mouse model for familial amyotrophic lateral sclerosis Proc Natl Acad Sci USA 97 : 12571 12576. 17. Shinder GA, Lacourse MC, Minotti S, Durham HD (2001) Mutant Cu/Zn superoxide dismutase proteins have altered solubility and interact with heat shock/stress proteins in models of amyotrophic lateral sclerosis J Biol Chem 276 : 12791 12796. 18. Wang J, Xu G, Borchelt DR (2002) High molecular weight complexes of mutant superoxide dismutase 1: age dependent and tissue specific accumulation Neurobiol Dis 9 : 139 148. 19. Wang J, Xu G, Gonzales V, Coonfield M, Fromholt D, et al. (2002) Fibrillar inclusions and motor neuron degeneration in transgenic mice expressing superoxide dismutase 1 with a disrupted copper binding site Neurobiol Dis 10 : 128 138. 20. Prudencio M, Hart PJ, Borchelt DR, Andersen PM (2009) Variation in aggregation propensities among ALS assoc iated variants of SOD1: correlation to human disease Hum Mol Genet 18 : 3217 3226. 21. Prudencio M, Borchelt DR (2011) Superoxide dismutase 1 encoding mutations linked to ALS adopts a spectrum of misfolded states Mol Neurodegener 6 : 77. 22. Matsumoto G, Stojanovic A, Holmberg CI, Kim S, Morimoto RI (2005) Structural properties and neuronal toxicity of amyotrophic lateral sclerosis associated Cu/Zn superoxide dismutase 1 aggregates J Cell Biol 171 : 75 85. 23. Prudencio M, Durazo A, Whitelegge JP, Borchelt DR (2009) Modulation of mutant superoxide dismutase 1 aggregation by co expression of wild type enzyme J Neurochem 108 : 1009 1018. 24. Chiti F, Massimo S, Taddei N, Ramponi G, Dobson CM (2003) Rationalization of the effects of mutations on peptide and protein ag gregation rates. Nature 424: 805 808. 25. Monsellier E, Chiti F (2007) Prevention of amyloid like aggregation as a driving force of protein evolution. EMBO reports 8: 737 742. 26. Corcoran LJ, Mitchison TJ, Liu Q (2004) A novel action of histone deacetylase inhibitors in a protein aggresome disease model Curr Biol 14: 488 492. 27. Turner BJ, Atkin JD, Farg MA, Zang DW, Rembach A, et al. (2005) Impaired extracellular secretion of mutant superoxide dismutase 1 associates with neurotoxicity in familial amyotrophic lateral sclerosis. J Neurosci 25: 108 117. 28. Matsumoto G, Stojanovic A, Holmberg CI, Kim S, Morimoto RI (2005) Structural properties and neuronal toxicity of amyotrophic lateral sclerosis associated Cu/Zn superoxide dismutase 1 aggre gates J Cell Biol 171: 75 85. 29. Zhang F, Zhu H (2006) Intracellular conformational alterations of mutant SOD1 and the implications for fALS associated SOD1 mutant induced motor neuron cell death. Biochim Biophys Acta 1760: 404 414. 30. Fei E, Jia N, Yan M, Ying Z, Sun Q, et al. (20 06) SUMO 1 modification increases human SOD1 stability and aggregation. Biochem Biophys Res Commun 347: 406 412. 31. Urushitani M, Ezzi SA, Matsuo A, Tooyama I, Julien JP (2008) The endoplasmic reticulum Golgi pathway is a target for translocation and aggrega tion of mutant superoxide dismutase linked to ALS. FASEB J 22: 2476 2487.

PAGE 15

32. Witan H, Gorlovoy P, Kaya AM, Koziollek Drechsler I, Neumann H, et al. (2009) Wild type Cu/Zn superoxide dismutase (SOD1) does not facilitate, but impedes the formation of protein a ggregates of amyotrophic lateral sclerosis causing mutant SOD1. Neurobiol Dis 36: 331 342. 33. Prudencio M, Durazo A, Whitelegge JP, Borchelt DR (2010) An examination of wild type SOD1 in modulating the toxicity and aggregation of ALS associated mutant SOD1. H um Mol Genet 19: 4774 4789. 34. Roberts BLT, Patel K, Brown HH, Borchelt DR (2012) Role of Disulfide Cross Linking of Mutant SOD1 in the Formation of Inclusion Body Like Structures. PLoS ONE 7(10): e47838. doi:10.1371/journal.pone.0047838