Protein degradation through the Ubiquitin (Ub)-26S Proteasome System (UPS) is a major gene expression regulatory pathway in plants. In this pathway, the 76-amino acid Ub proteins are covalently linked onto a large array of UPS substrates with the help of three enzymes (E1 activating, E2 conjugating, and E3 ligating enzymes) and direct them for turnover in the 26S proteasome complex. The S-phase Kinase-associated Protein 1 (Skp1), CUL1, F-box (FBX) protein (SCF) complexes have been identified as the largest E3 ligase group in plants due to the dramatic number expansion of the FBX genes in plant genomes. Since it is the FBX proteins that recognize and determine the specificity of SCF substrates, much effort has been done to characterize their genomic, physiological, and biochemical roles in the past two decades of functional genomic studies. However, the sheer size and high sequence diversity of the FBX gene family demands new approaches to uncover unknown functions. In this work, we first identified 82 known FBX members that have been functionally characterized up to date in Arabidopsis thaliana . Through comparing the genomic structure, evolutionary selection, expression patterns, domain compositions, and functional activities between known and unknown FBX gene members, we developed a neuralmore »
This content will become publicly available on December 1, 2023
Predicting the structural basis of targeted protein degradation by integrating molecular dynamics simulations with structural mass spectrometry
Abstract Targeted protein degradation (TPD) is a promising approach in drug discovery for degrading proteins implicated in diseases. A key step in this process is the formation of a ternary complex where a heterobifunctional molecule induces proximity of an E3 ligase to a protein of interest (POI), thus facilitating ubiquitin transfer to the POI. In this work, we characterize 3 steps in the TPD process. (1) We simulate the ternary complex formation of SMARCA2 bromodomain and VHL E3 ligase by combining hydrogen-deuterium exchange mass spectrometry with weighted ensemble molecular dynamics (MD). (2) We characterize the conformational heterogeneity of the ternary complex using Hamiltonian replica exchange simulations and small-angle X-ray scattering. (3) We assess the ubiquitination of the POI in the context of the full Cullin-RING Ligase, confirming experimental ubiquitinomics results. Differences in degradation efficiency can be explained by the proximity of lysine residues on the POI relative to ubiquitin.
- Authors:
- ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more »
- Award ID(s):
- 1761320
- Publication Date:
- NSF-PAR ID:
- 10370111
- Journal Name:
- Nature Communications
- Volume:
- 13
- Issue:
- 1
- ISSN:
- 2041-1723
- Sponsoring Org:
- National Science Foundation
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Abstract Motivation Ubiquitination is widely involved in protein homeostasis and cell signaling. Ubiquitin E3 ligases are critical regulators of ubiquitination that recognize and recruit specific ubiquitination targets for the final rate-limiting step of ubiquitin transfer reactions. Understanding the ubiquitin E3 ligase activities will provide knowledge in the upstream regulator of the ubiquitination pathway and reveal potential mechanisms in biological processes and disease progression. Recent advances in mass spectrometry-based proteomics have enabled deep profiling of ubiquitylome in a quantitative manner. Yet, functional analysis of ubiquitylome dynamics and pathway activity remains challenging.
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Availability and<a href='#' onclick='$(this).hide().next().show().next().show();return false;' style='margin-left:10px;'>more »</a><span style='display:none;'>implementation Source code and binaries are freely available for download at URL: https://github.com/Chenlab-UMN/Ub-E3-ligase-Activity-Profiling-Analysis, implemented in python and supported on Linux and MS Windows.
Supplementary information Supplementary data are available at Bioinformatics online.
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