Allosteric regulation of proteins continues to be an engaging research question for the scientific community. Models describing allosteric communication have evolved from focusing on conformation-based descriptors of protein structural changes to appreciating the role of internal protein dynamics as a mediator of allostery. Here we explain a "Violin Model" for allostery as a contemporary method for approaching the Cooper-Dryden model based on redistribution of protein thermal fluctuations. Based on graph theory, the violin model makes use of community network analysis to functionally cluster correlated protein motions obtained from molecular dynamics simulations. This review provides the theory and workflow of the methodology and explains the application of violin model to unraveling the workings of Protein Kinase A.
Dynamics-driven allostery provides important insights into the working mechanics of proteins, especially enzymes. In this study we employ this paradigm to answer a basic question: in enzyme superfamilies where the catalytic mechanism, active sites and protein fold are conserved, what accounts for the difference in the catalytic prowess of the individual members? We show that when subtle changes in sequence do not translate to changes in structure, they do translate to changes in dynamics. We use sequentially diverse PTP1B, TbPTP1, and YopH as the representatives of the conserved Protein Tyrosine Phosphatase (PTP) superfamily. Using amino acid network analysis of group behavior (community analysis) and influential node dominance on networks (eigenvector centrality), we explain the dynamic basis of catalytic variations seen between the three proteins. Importantly, we explain how a dynamics-based blueprint makes PTP1B amenable to allosteric control and how the same is abstracted in TbPTP1 and YopH.
The ubiquitin proteasome system regulates the degradation of proteins in order to maintain cellular homeostasis, which is often disrupted in cancer cells. The UBR5 (Ubiquitin Protein Ligase E3 Component N-Recognin 5) protein is an E3 ubiquitin ligase that catalyzes the direct ubiquitination of protein substrates, most often resulting in substrate protein degradation. UBR5 is the human homolog of the Drosophilia hyperplastic discs (hyd) tumor suppressor gene. However, ubr5 is amplified and over-expressed in human ovarian and breast cancers, among others, and thought to play more of a tumor-promoting role in cancer. We previously demonstrated that targeting UBR5 expression in human ovarian cancer xenografts in mice with liposomal delivery of UBR5 siRNA inhibited tumor growth and sensitized tumors to the DNA-damaging agent cisplatin. To identify novel substrates of UBR5-stimulated degradation, we transduced OVCAR5 cells with doxycycline-inducible shRNA targeting UBR5 or a control shRNA and metabolically labelled proteins with heavy or light lysine and arginine using Stable Isotope Labeling with Amino acids in Cell culture (SILAC). Doxycycline was added for the last 48 hr and cisplatin for the last 12 hrs before cell harvest. Equal amounts of protein from both cultures were mixed, digested with trypsin and analyzed by LC-MS/MS (Orbitrap Elite MS) in three independent experiments including a label swap control. Peptides and proteins were identified and quantified using MaxQuant (FDR<0.01). To identify proteins differentially expressed after UBR5 knockdown, relative protein abundance was compared by two-sided t-test with permutation-based correction of the p-value. We identified multiple proteins with increased expression following UBR5 knockdown, suggesting these proteins are regulated by ubiquitin-mediated degradation by UBR5. We validated UBR5-mediated regulation of several proteins by Western blotting, including an oncogene involved in breast and ovarian cancer progression, which we further characterized. In summary, we were able to identify several novel UBR5 candidate substrates whose expression is increased following UBR5 knockdown. Further characterization of these substrates will allow us to identify novel mechanisms of UBR5 regulation of tumor progression. Citation Format: Scott T. Eblen, Jennifer R. Bethard, D Ralph Rogers, Abdelkader Daoud, Lalima K. Madan, Lauren E. Ball. Identification of novel substrates of the UBR5 E3 ubiquitin ligase in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 326.
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