Citation
Characterization of archaeal ubiquitin-like SAMP2 modification on lysine 53 and 63 of TATA binding protein (TBP2)

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Title:
Characterization of archaeal ubiquitin-like SAMP2 modification on lysine 53 and 63 of TATA binding protein (TBP2)
Series Title:
Annual UF Undergraduate Research Symposium
Creator:
Sanchez, Iona
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Language:
English

Subjects

Subjects / Keywords:
Archaea, proteasome
proteasome
ubiquitin-like protein
TBP2

Notes

Abstract:
The ubiquitin-proteasome system (UPS) constitutes the primary pathway for protein degradation in eukaryotes. UPS mediated proteolysis plays a pivotal role in a large number of cellular processes, including intracellular homeostasis, signaling, transcription regulation, and protein quality control (1). However, the regulated and targeted proteolysis of archaea, one of three major evolutionary lineages of life, still remains largely unknown. Interestingly, UPS gene homologs are widespread in archaeal genome sequences.  Although evidence suggests the recent disclosed ubiquitin-like small archaeal modifier proteins (SAMPs) are associated with targeting of proteins for degradation by proteasomes, a direct link between sampylation and proteasome mediated protein degradation is still missing. Previous work shows TATA binding protein (TBP2) level is accumulated by deletion of UPS gene homologs in archaea, suggesting TBP2 is targeted for degradation by archaeal UPS. This study aims to further characterize archaeal UPS mediated TBP2 degradation by mutating SAMP2 modification sites on TBP2.
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Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Iona Sanchez.
General Note:
Center for Undergraduate Research
Exhibitions:
Poster presented at the 2015 UF Undergraduate Research Symposium

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University of Florida Institutional Repository
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University of Florida
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Copyright Iona Sanchez. Permission granted to University of Florida to digitize and display 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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Characterization of archaeal ubiquitin like SAMP2 modification on lysine 53 and 63 of TATA binding protein (TBP2) Iona A. Sanchez, Xian Fu, Julie A. Maupin Furlow University of Florida, Department of Microbiology and Cell Science, Gainesville, FL. 32608 Abstract The ubiquitin proteasome system (UPS) constitutes the primary pathway for protein degradation in eukaryotes UPS mediated proteolysis plays a pivotal role in a large number of cellular processes, including intracellular homeostasis, signaling, transcription regulation, and protein quality control ( 1 ) However, the regulated and targeted proteolysis of archaea, one of three major evolutionary lineages of life, still remains largely unknown Interestingly, UPS gene homologs are widespread in archaeal genome sequences Although evidence suggests the recent disclosed ubiquitin like small archaeal modifier proteins (SAMPs) are associated with targeting of proteins for degradation by proteasomes, a direct link between sampylation and proteasome mediated protein degradation is still missing Previous work shows TATA binding protein (TBP 2 ) level is accumulated by deletion of UPS gene homologs in archaea, suggesting TBP 2 is targeted for degradation by archaeal UPS This study aims to further characterize archaeal UPS mediated TBP 2 degradation by mutating SAMP 2 modification sites on TBP 2 Introduction Proteasomes are highly specialized proteases with compartmentalized protein structures They are energy dependent and function to degrade poorly synthesized, misfolded, and damaged proteins In a word, proteasome mediated degradation is essential in a large number of cellular processes ( 3 ) In eukaryotic cells, the process used to target proteins for proteasome mediated degradation is called ubiquitylation which is the conjugation of ubiquitin ( Ub ) to protein targets by formation of covalent attachment This is mediated by ATP dependent systems including E 1 ubiquitin activating enzyme, E 2 Ub conjugating enzyme, and E 3 Ub protein ligase enzyme as shown in Figure 1 ( 2 ) Archaea conduct a form of post translational modification named sampylation that resembles the ubiquitylation in eukaryotes ( 3 ) Our lab uses halophilic archaeon Haloferax volcanii as a model organism to study UPS in archaea Hfx volcanii encodes ubiquitin proteasome pathway homologs, for example, UbaA is E 1 homologue and SAMP 2 is Ub like protein as shown in Figure 1 Results Discussion & Future Work Acknowledgements 1. Proteasome System as a Prospective Molecular Target for Cancer Treatment and Current protein & peptide science 11.6 (2010): 459 470. Print. 2. M.A. Humbard H.V. Miranda, J. M. Lim, D.J. Krause, J.R. Pritz G. Zhou, et al. Ubiquitin like small archaeal modifier proteins (SAMPs) in Haloferax volcanii Nature, 463 (2010), pp. 54 60 3. Maupin Furlow J. Proteasomes and protein conjugation across domains of life. Nat Rev Microbiol (2012) 10:100 11. doi:10.1038/nrmicro2696 WT TBP 2 level is not increased by site directed mutagenesis (SDM) of TBP 2 lysine 53 and 63 which may be because WT TBP 2 lacks the signal for sampylation and then for degradation TBP 2 S 2 E variant mimics the TBP 2 serine 2 phosphorylation that may serve as a signal for SAMP 2 modification of TBP 2 K 53 and K 63 (data not shown) TBP 2 S 2 E stationary level is not increased by single site directed mutagenesis of lysine 53 and 63 The next step is to construct a TBP 2 S 2 E site direct mutation with lysine 53 and 63 to arginine (K 53 / 63 R) double variant W e hypothesize that the SDM of both K 53 and K 63 to arginine may stabilized TBP 2 S 2 E by preventing it from being modified by SAMP 2 We also hypothesize that other lysine residues like K 146 and K 156 (mirror image of K 53 and K 63 ) can also serve as additional sites for SAMP 2 modification if K 53 and K 63 are inaccessible for sampylation References This work was funded in part of the National Institute of Health (R01 GN057498) and the Department of Energy (DE FG02 05ER15650) to J.A. Maupin Furlow Figure 1 To initiate ubiquitylation, ATP adenylation of Ub /Ub 1 is catalyzed by E 1 The activated Ub /Ub 1 form transfers to a conserved E 1 cysteine residue The E 1 Ub thioester intermediate transfers Ub to a conserved E 2 cysteine residue forming a second thioester intermediate E 2 Ub conjugating enzyme transfers Ub /Ub 1 to a lysine residue within the target protein with the assistance of E 3 Ub protein ligase enzyme Ultimately, a covalent bond occurs between Ub and the lysine residue on the target protein Addition of more Ub on the lysine residue of the previous Ub results in polyubiquitin chains This is the signal for degradation by the proteasome ( 2 ) As shown in Figure 4 the stationary level of TBP 2 is significantly higher in UPS mutants compared with WT, suggesting TBP 2 is degraded by a UPS like pathway We hypothesize SAMP 2 modification on lysine 53 and lysine 63 on TBP 2 serve as the signal for TBP 2 degradation by UPS like pathway in Haloferax volcanii Figure 3 Structural model of HVO_ 1727 (T bp 2 ) in complex with DNA promoter elements and transcription factor B (TFB) Figure 2 C terminal diglycine of SAMP 2 is isopeptide linked to the K 53 and K 63 of Tbp 2 There are dozens of proteins found to be modified by SAMPs in Hfx Volcanii For example TBP 2 lysine 53 and 63 is covalent linked to SAMP 2 based on mass spectrometry (Figure 2 ) + + + + + + + wt wt ubaA jamm2 jamm1/2 panB panAB cdc48c Genome Flag SAMP2 TBP2 StrepII 2 5 2 0 M r (kDa) 50 TBP2 IB : StrepII Loading control Figure 4 Steady level of TBP 2 is increased by deletion of UPS gene homologs in Hfx volcanii TBP 2 StrepII was co expressed with Flag SAMP 2 in wild type ( wt ) and UPS deletion mutants (Xian Fu et al unpublished data) + + + + + + + + + K53R K63R K53/63R + K53R K63R K53/63R wt wt wt wt ubaA ubaA ubaA ubaA TBP2 StrepII Flag SAMP2 In trans Genome TBP2 IB : StrepII Loading control Figure 5 Effect of K 53 / 63 R site directed mutagenesis on TBP 2 WT TBP 2 and TBP 2 variant was co expressed with Flag SAMP 2 in wild type ( wt ) Stationary phase cells were harvested, boiled ( 10 min) in reducing SDS PAGE buffer, and separated by 12 % SDS PAGE TBP 2 were detected by StrepII immunoblotting ( IB) Equal loading was confirmed by Coomassie Blue staining + + + + + + + K53R K63R + K53R K63R wt wt wt ubaA ubaA ubaA TBP2 S2E StrepII Flag SAMP2 In trans Genome TBP2 IB : StrepII Loading control Figure 6 Effect of K 53 / 63 R site directed mutagenesis on TBP 2 S 2 E TBP 2 S 2 E and TBP 2 variant was co expressed with Flag SAMP 2 in wild type ( wt ) Stationary phase cells were harvested, boiled ( 10 min) in reducing SDS PAGE buffer, and separated by 12 % SDS PAGE TBP 2 were detected by StrepII immunoblotting ( IB) Equal loading was confirmed by Coomassie Blue staining