Date of Award

12-2024

Access Control

Open Access

Degree Name

Biology, M.A.

Department

Biology

Advisor

Xiang-Dong Zhang, Ph.D.

Department Home page

https://biology.buffalostate.edu/faculty

First Reader

Xiang-Dong Zhang, Ph.D.

Second Reader

Gregory J. Wadsworth, Ph.D.

Third Reader

Sandra M. Borbor-Sawyer, Ph.D.

Abstract

Polymeric SUMO-2/3 chain modification is critical for chromosome segregation, DNA damage repair, genome stability, stress responses, and protein degradation. However, how these signals are regulated remains poorly understood. Both the SUMO-targeted ubiquitin E3 ligase (STUbL) RNF4 and the SUMO-specific isopeptidase SENP6 recognize poly-SUMO-2/3 chains through multiple SUMO-interacting motifs (SIMs). RNF4 contains four tandem SIMs within a short (~80 amino acids) region, whereas SENP6 possesses eight SIMs spanning a much larger (~500 amino acids) domain.

Our preliminary results show that overexpressing an RNF4 N-terminal fragment containing four tandem SIMs (wild type, WT), but not an inactive SIMs mutant (Mut), increases levels of poly-SUMO-2/3 chain conjugates in human cells. To test this model, we first performed in vitro SUMO-deconjugation assays by incubating poly-SUMO-2 chains with YFP-tagged SENP6 in the presence of His-tagged RNF4 fragment containing either SIMs WT or SIMs Mut followed by immunoblot analysis. We found that His-RNF4 SIMs WT, but not SIMs Mut, effectively inhibited SENP6-mediated disassembly of poly-SUMO-2 chains in vitro.

Furthermore, we performed both in vitro and in vivo binding assays using the linear fusion proteins containing multiple SUMO-2 moieties, demonstrated the RNF4 fragment containing SIMs WT, but not SIMs Mut, competitively reduced the interaction of SENP6 with poly-SUMO-2 chain signals. Therefore, our results have elucidated that poly-SUMO-2/3 chain signals are antagonistically controlled by two SIMs-containing proteins, RNF4 and SENP6, for degrading and stabilizing poly-SUMOylated proteins, respectively. A better understanding of poly-SUMO-2/3 chain modification signals may help us develop novel therapeutic treatments for human diseases, including cancer.

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