Plasticity of the proteasome-targeting signal Fat10 enhances substrate degradation

  1. Hitendra Negi
  2. Aravind Ravichandran
  3. Pritha Dasgupta
  4. Shridivya Reddy
  5. Ranabir Das  Is a corresponding author
  1. Tata Institute of Fundamental Research, India

Abstract

The proteasome controls levels of most cellular proteins, and its activity is regulated under stress, quiescence, and inflammation. However, factors determining the proteasomal degradation rate remain poorly understood. Proteasome substrates are conjugated with small proteins (tags) like ubiquitin and Fat10 to target them to the proteasome. It is unclear if the structural plasticity of proteasome-targeting tags can influence substrate degradation. Fat10 is upregulated during inflammation, and its substrates undergo rapid proteasomal degradation. We report that the degradation rate of Fat10 substrates critically depends on the structural plasticity of Fat10. While the ubiquitin tag is recycled at the proteasome, Fat10 is degraded with the substrate. Our results suggest significantly lower thermodynamic stability and faster mechanical unfolding in Fat10 compared to ubiquitin. Long-range salt bridges are absent in the Fat10 structure, creating a plastic protein with partially unstructured regions suitable for proteasome engagement. Fat10 plasticity destabilizes substrates significantly and creates partially unstructured regions in the substrate to enhance degradation. NMR-relaxation-derived order parameters and temperature dependence of chemical shifts identify the Fat10-induced partially unstructured regions in the substrate, which correlated excellently to Fat10-substrate contacts, suggesting that the tag-substrate collision destabilizes the substrate. These results highlight a strong dependence of proteasomal degradation on the structural plasticity and thermodynamic properties of the proteasome-targeting tags.

Data availability

The manuscript and supporting file include all data generated or analyzed during this study.

Article and author information

Author details

  1. Hitendra Negi

    National Center for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  2. Aravind Ravichandran

    National Center for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  3. Pritha Dasgupta

    National Center for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  4. Shridivya Reddy

    National Center for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  5. Ranabir Das

    National Center for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India
    For correspondence
    rana@ncbs.res.in
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5114-6817

Funding

Tata Institute of Fundamental Research (RTI4006)

  • Ranabir Das

Science and Engineering Research Board (CRG/2021/006032)

  • Ranabir Das

Department of Biotechnology, Ministry of Science and Technology, India (dbt/pr12422/med/31/287/2014)

  • Ranabir Das

Council of Scientific and Industrial Research, India

  • Hitendra Negi

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Volker Dötsch, Goethe University, Germany

Version history

  1. Received: July 20, 2023
  2. Accepted: July 9, 2024
  3. Accepted Manuscript published: July 10, 2024 (version 1)

Copyright

© 2024, Negi et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 108
    views
  • 40
    downloads
  • 0
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Hitendra Negi
  2. Aravind Ravichandran
  3. Pritha Dasgupta
  4. Shridivya Reddy
  5. Ranabir Das
(2024)
Plasticity of the proteasome-targeting signal Fat10 enhances substrate degradation
eLife 13:e91122.
https://doi.org/10.7554/eLife.91122

Share this article

https://doi.org/10.7554/eLife.91122

Further reading

    1. Developmental Biology
    2. Structural Biology and Molecular Biophysics
    Samuel C Griffiths, Jia Tan ... Hsin-Yi Henry Ho
    Research Article Updated

    The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception. Functionally, we showed that the ROR2 CRD, but not other domains, is required and minimally sufficient to promote WNT5A signaling, and Robinow mutations in the CRD and the adjacent Kr impair ROR2 secretion and function. Moreover, using function-activating and -perturbing antibodies against the Frizzled (FZ) family of WNT receptors, we demonstrate the involvement of FZ in WNT5A-ROR signaling. Thus, ROR2 acts via its CRD to potentiate the function of a receptor super-complex that includes FZ to transduce WNT5A signals.

    1. Microbiology and Infectious Disease
    2. Structural Biology and Molecular Biophysics
    Ai Nguyen, Huaying Zhao ... Peter Schuck
    Research Article

    Genetic diversity is a hallmark of RNA viruses and the basis for their evolutionary success. Taking advantage of the uniquely large genomic database of SARS-CoV-2, we examine the impact of mutations across the spectrum of viable amino acid sequences on the biophysical phenotypes of the highly expressed and multifunctional nucleocapsid protein. We find variation in the physicochemical parameters of its extended intrinsically disordered regions (IDRs) sufficient to allow local plasticity, but also observe functional constraints that similarly occur in related coronaviruses. In biophysical experiments with several N-protein species carrying mutations associated with major variants, we find that point mutations in the IDRs can have nonlocal impact and modulate thermodynamic stability, secondary structure, protein oligomeric state, particle formation, and liquid-liquid phase separation. In the Omicron variant, distant mutations in different IDRs have compensatory effects in shifting a delicate balance of interactions controlling protein assembly properties, and include the creation of a new protein-protein interaction interface in the N-terminal IDR through the defining P13L mutation. A picture emerges where genetic diversity is accompanied by significant variation in biophysical characteristics of functional N-protein species, in particular in the IDRs.