Author ORCID Identifier

https://orcid.org/0000-0003-1701-5535

Date of Award

6-1-2025

Document Type

Thesis

School

School of Chemical & Biotechnology

Programme

Ph.D.-Doctoral of Philosophy

First Advisor

Dr Ranabir Das

Second Advisor

Dr Dipita Guha

Keywords

Computational Structural biology, Protein Thermodynamics, Cellular Homeostasis, Molecular Dynamics Simulations, Fat10ylation, Conformational Entropy, Substrate Destabilization, Proteasome

Abstract

Degradation of proteins by the proteasome is crucial in regulating protein levels in the cell. Post-translational modifications, such as ubiquitylation and Fat10ylation, trigger proteasomal degradation of the substrate proteins. While ubiquitylation orchestrates multiple cellular processes, Fat10ylation is primarily involved in the inflammatory response. Unlike ubiquitin, recycled upon substrate degradation, Fat10 is degraded along with its substrate. Although the thermodynamic properties of the substrate are critical for effective proteasomal degradation, they remain poorly understood for the Fat10-proteasome pathway.

Here, we demonstrate that Fat10 exhibits markedly lower thermodynamic stability and faster unfolding kinetics compared to ubiquitin. This is due to the absence of long-range electrostatic interactions within Fat10, resulting in a flexible structure with partially unstructured regions. By investigating the Fat10∼substrate conjugate, we reveal that the mechanical unfolding pathway and energy are influenced by the site of Fat10 modification. Our findings suggest that the entry of Fat10 into the proteasome, followed by the substrate, is the energetically preferred pathway. Furthermore, we explored the impact of Fat10 on the thermodynamic properties of substrates, considering their size, flexibility, and surface characteristics.

Fat10ylation induces significant entropic destabilisation, especially in smaller substrates. For larger substrates, multi-monoFat10ylation is necessary to achieve similar destabilization. Notably, Fat10 modification at negatively charged patches on the substrate surface is crucial for optimal destabilization and subsequent degradation. These insights provide a detailed mechanistic understanding of the Fat10-proteasome degradation pathway, with potential implications for therapeutic strategies targeting protein homeostasis.

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