Diverse polyubiquitin chains accumulate following 26S proteasomal dysfunction in mammalian neurones

doi:10.1016/j.neulet.2010.12.064

Neuroscience Letters

Volume 491, Issue 1, 10 March 2011, Pages 44-47

https://doi.org/10.1016/j.neulet.2010.12.064 Get rights and content

Abstract

A generality has been that polyubiquitin chain linkage can differentially address proteins for various physiological processes. 26S proteasomal degradation is the most established function of ubiquitin signalling, classically linked to Lys48 polyubiquitin chains. The other well-characterised polyubiquitin linkage, via Lys63, mediates nonproteolytic functions. However, there are five other lysine residues and ubiquitin's amino terminus which can participate in polyubiquitination. Our 26S proteasome knockout mouse provides a unique opportunity to comprehensively investigate the ubiquitin signals in their physiological context in neurones following genetic inhibition of the proteasome, using quantitative mass spectrometry of ubiquitin linkage-specific signature peptides. We provide the first evidence for diverse polyubiquitin chains in mammalian neurones in vivo and show that polyubiquitin linked via Lys6, Lys11, Lys29 and Lys48, but not Lys63, accumulates upon 26S proteasome dysfunction. This adaptable nature of ubiquitin signals for proteasomal targeting could reflect the extensive cellular processes which are regulated by proteasome proteolysis and/or may involve specific ubiquitin linkage preferences for subsets of proteins in mammalian neurones. Our molecular pathological findings make a significant contribution to the understanding of ubiquitin signalling in ubiquitin–proteasome function.

Research highlights

▶ An extensive repertoire of non-Lys63-linked polyubiquitin chains function in 26S proteasomal targeting in mammalian neurones. ▶ Mass spectrometry approaches can be used to investigate ubiquitin–ubiquitin isopeptide linkages in their physiological context in vivo.

Section snippets

Acknowledgements

This work was supported by a Parkinson's UK Senior Research Fellowship (F-0702) and a Research Scholar Grant from the American Cancer Society (RSG-09-181).

References (34)

G. Alexandru et al.
UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover
Cell
(2008)
B. Crosas et al.
Ubiquitin chains are remodeled at the proteasome by opposing ubiquitin ligase and deubiquitinating activities
Cell
(2006)
L. Deng et al.
Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain
Cell
(2000)
A.D. Jacobson et al.
The lysine 48 and lysine 63 ubiquitin conjugates are processed differently by the 26s proteasome
The Journal of Biological Chemistry
(2009)
L. Jin et al.
Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex
Cell
(2008)
E.S. Johnson et al.
A proteolytic pathway that recognizes ubiquitin as a degradation signal
The Journal of Biological Chemistry
(1995)
H.T. Kim et al.
Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin–protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages
The Journal of Biological Chemistry
(2007)
L. Kundrat et al.
Identification of residues on Hsp70 and Hsp90 ubiquitinated by the cochaperone CHIP
Journal of Molecular Biology
(2010)
S. Paine et al.
Immunoreactivity to Lys63-linked polyubiquitin is a feature of neurodegeneration
Neuroscience Letters
(2009)
C.M. Pickart et al.
Polyubiquitin chains: polymeric protein signals
Current Opinion in Chemical Biology
(2004)

H. Richly et al.

A series of ubiquitin binding factors connects CDC48/p97 to substrate multiubiquitylation and proteasomal targeting

Cell

(2005)

Y. Saeki et al.

Definitive evidence for Ufd2-catalyzed elongation of the ubiquitin chain through Lys48 linkage

Biochemical and Biophysical Research Communications

(2004)

H.D. Ulrich

Regulating post-translational modifications of the eukaryotic replication clamp PCNA

DNA Repair

(2009)

P. Xu et al.

Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation

Cell

(2009)

W. Zeng et al.

Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity

Cell

(2010)

L. Bedford et al.

Depletion of 26S proteasomes in mouse brain neurons causes neurodegeneration and Lewy-like inclusions resembling human pale bodies

The Journal of Neuroscience

(2008)

E.J. Bennett et al.

Global changes to the ubiquitin system in Huntington's disease

Nature

(2007)

Cited by (19)

Major histocompatibility complex (MHC) class i processing of the NY-ESO-1 antigen is regulated by Rpn10 and Rpn13 Proteins and immunoproteasomes following non-lysine ubiquitination
2016, Journal of Biological Chemistry
Citation Excerpt :
Noteworthy is that such NY-ESO-1 Ub species were detected in the presence of MG-132, confirming their potency as signals for targeting NY-ESO-1 to proteasome-mediated degradation. The notion that proteasomes do not necessarily recognize canonical linkages is in agreement with previous studies showing that atypical poly-Ub may actively participate in the regulation of protein breakdown (25–27). Importantly, our analysis of the NY-ESO-1 ubiquitination profile was carried out following enrichment of the full-length protein using a C-terminal epitope tagging strategy.
The supply of MHC class I-restricted peptides is primarily ensured by the degradation of intracellular proteins via the ubiquitin-proteasome system. Depending on the target and the enzymes involved, ubiquitination is a process that may dramatically vary in terms of linkages, length, and attachment sites. Here we identified the unique lysine residue at position 124 of the NY-ESO-1 cancer/testis antigen as the acceptor site for the formation of canonical Lys-48-linkages. Interestingly, a lysine-less form of NY-ESO-1 was as efficient as its wild-type counterpart in supplying the HLA-A*0201-restricted NY-ESO-1_157–165 antigenic peptide. In fact, we show that the regulation of NY-ESO-1 processing by the ubiquitin receptors Rpn10 and Rpn13 as a well as by the standard and immunoproteasome is governed by non-canonical ubiquitination on non-lysine sites. In summary, our data underscore the significance of atypical ubiquitination in the modulation of MHC class I antigen processing.
The complexity of recognition of ubiquitinated substrates by the 26S proteasome
2014, Biochimica et Biophysica Acta - Molecular Cell Research
Citation Excerpt :
These chains, like the ‘canonical’ Lys48-based chains, appear also to bind to the Rpn10/S5a subunit of 19S sub-complex [21], probably via a TEK box motif that is found on both the substrate and the Ub moiety [22]. Other studies have shown that Lys33- [23,24] and Lys63-based chains [25], are also recognized by the proteasome. It should be noted that some of these results were obtained in studies using cell free reconstituted systems.
The Ubiquitin Proteasome System (UPS) was discovered in two steps. Initially, APF-1 (ATP-dependent proteolytic Factor 1) later identified as ubiquitin (Ub), a hitherto known protein of unknown function, was found to covalently modify proteins. This modification led to degradation of the tagged protein by – at that time – an unknown protease. This was followed later by the identification of the 26S proteasome complex which is composed of a previously identified Multi Catalytic Protease (MCP) and an additional regulatory complex, as the protease that degrades Ub-tagged proteins. While Ub conjugation and proteasomal degradation are viewed as a continued process responsible for most of the regulated proteolysis in the cell, the two processes have also independent roles. In parallel and in the years that followed, the hallmark signal that links the substrate to the proteasome was identified as an internal Lys48-based polyUb chain. However, since these initial findings were described, our understanding of both ends of the process (i.e. Ub-conjugation to proteins, and their recognition and degradation), have advanced significantly. This enabled us to start bridging the ends of this continuous process which suffered until lately from limited structural data regarding the 26S proteasomal architecture and the structure and diversity of the Ub chains. These missing pieces are of great importance because the link between ubiquitination and proteasomal processing is subject to numerous regulatory steps and are found to function improperly in several pathologies. Recently, the molecular architecture of the 26S proteasome was resolved in great detail, enabling us to address mechanistic questions regarding the various molecular events that polyubiquitinated (polyUb) substrates undergo during binding and processing by the 26S proteasome. In addition, advancement in analytical and synthetic methods enables us to better understand the structure and diversity of the degradation signal. The review summarizes these recent findings and addresses the extrapolated meanings in light of previous reports. Finally, it addresses some of the still remaining questions to be solved in order to obtain a continuous mechanistic view of the events that a substrate undergoes from its initial ubiquitination to proteasomal degradation. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.
Identification of ubiquitin-modified lysine residues and novel phosphorylation sites on eukaryotic initiation factor 2B epsilon
2013, Biochemical and Biophysical Research Communications
Citation Excerpt :
Ubiquitin itself was modified at several residues in the present investigation. Beyond the canonical Lys-48 linked polyubiquitin chains, polyubiquitin chains linked via Lys-11 and Lys-29 of ubiquitin are also implicated in mediating proteasomal degradation [27,28]. Polyubiquitin linkage via Lys-63 has been reported to lead to both lysosomal [27] and proteasomal degradation [29] although others report that Lys-63 linked polyubiquitin chains do not accumulate upon conditional knockout of the 26S proteasome [28], suggesting that Lys-63 may not be involved in proteasomal degradation.
Eukaryotic initiation factor 2Bε (eIF2Bε) plays a critical role in the initiation of mRNA translation and its expression and guanine nucleotide exchange activity are major determinants of the rate of protein synthesis. In this work we provide evidence that the catalytic epsilon subunit of eIF2B is subject to ubiquitination and proteasome-mediated degradation. Lysates of C2C12 myoblasts treated with proteasome inhibitor were subjected to sequential immunoprecipitations for eIF2Bε followed by ubiquitin. Tandem mass spectrometry (LC–MS/MS) analysis of immunoprecipitated proteins resulted in the identification of five peptides containing ubiquitin (diglycine) modifications on eIF2Bε. The specific lysine residues containing the ubiquitin modifications were localized as Lys-56, Lys-98, Lys-136, Lys-212 and Lys-500 (corresponding to the rat protein sequence). In addition three novel phosphorylation sites were identified including Ser-22, Ser-125, and Thr-317. Moreover, peptides corresponding to the amino acid sequence of the E3 ligase NEDD4 were also detected in the LC–MS/MS analysis, and an interaction between endogenous eIF2Bε with NEDD4 was confirmed by co-immunoprecipitation.
Selectivity of the ubiquitin-binding modules
2012, FEBS Letters
Citation Excerpt :
Among various polyubiquitin chains, Lys48- and Lys63-linked types have been the focuses of initial ubiquitin-signaling investigations [14,15]. However, extensive functional and structural studies in recent years have manifested larger variety of ubiquitin chains such as Lys11- and Met1-linked polyubiquitins to be synthesized in vivo and participate in the biological processes [10,16–19]. So far, there are several factors proposed to determine the specificity of ubiquitin-binding modules for different ubiquitin species.
Ubiquitin-binding modules are constituents of cellular proteins that mediate the effects of ubiquitylation by making transient, non-covalent interactions with ubiquitin molecules. While some ubiquitin-binding modules bind single ubiquitin moieties, others are selective for specific ubiquitin chains of different linkage types and lengths. In recent years, functions of ubiquitin chains that are polymerized through their Lys or N-terminal Met (i.e. linear chains) residues have been linked to a variety of cellular processes. Selectivity of ubiquitin-binding modules for different ubiquitin chain types appears as a key to the distinct regulatory consequences during protein quality control pathways, receptor endocytosis, gene transcription, signaling via the NF-κB pathway, and autophagy.
The ubiquitinated axon: Local control of axon development and function by ubiquitin
2021, Journal of Neuroscience
RNF4—A Paradigm for SUMOylation-Mediated Ubiquitination
2019, Proteomics

View all citing articles on Scopus

View full text

Diverse polyubiquitin chains accumulate following 26S proteasomal dysfunction in mammalian neurones

Abstract

Research highlights

Section snippets

Acknowledgements

Cell

Cell

Cell

The Journal of Biological Chemistry

Cell

The Journal of Biological Chemistry

The Journal of Biological Chemistry

Journal of Molecular Biology

Neuroscience Letters

Current Opinion in Chemical Biology

Cell

Biochemical and Biophysical Research Communications

DNA Repair

Cell

Cell

Depletion of 26S proteasomes in mouse brain neurons causes neurodegeneration and Lewy-like inclusions resembling human pale bodies

The Journal of Neuroscience

Global changes to the ubiquitin system in Huntington's disease

Nature