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Unfolding or aggregation, that is the question

  • Marcel Bolten
    Affiliations
    Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V5T 1Z4, Canada
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  • Jonathan P. Bernardini
    Affiliations
    Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V5T 1Z4, Canada
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  • Thibault Mayor
    Correspondence
    To whom correspondence should be addressed:Dept. of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, 2125 East Mall, Vancouver, British Columbia V5T 1Z4, Canada
    Affiliations
    Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V5T 1Z4, Canada
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  • Author Footnotes
    2 The abbreviations used are:PNproteostasis networkDDdestabilizing domainS1Shield-1Prupleckstrin-like receptor for ubiquitin.
Open AccessPublished:November 08, 2019DOI:https://doi.org/10.1074/jbc.H119.011242
      Cellular processes accompanying protein aggregation are diverse and entangled, making it difficult to investigate the underlying molecular processes in a time-resolved way. Gottlieb, Thompson, and colleagues address this shortcoming using a chemical biology approach to monitor ubiquitination within the first 10 min after the initiation of protein aggregation. Intriguingly, unfolding rather than aggregation seems to trigger the observed events. This work might provide a method to answer open questions regarding the regulation of the proteostasis network upon protein misfolding.

      Introduction

      Protein homeostasis is fundamental to maintain a functional proteome. Malfunction of the proteostasis network (PN)
      The abbreviations used are: PN
      proteostasis network
      DD
      destabilizing domain
      S1
      Shield-1
      Pru
      pleckstrin-like receptor for ubiquitin.
      can result in protein misfolding and eventually the accumulation of the intracellular protein aggregates that are hallmarks of various proteopathies including Alzheimer’s disease and ALS. Untangling the precise relations between the different elements of the PN has been challenging due to the myriad of events that are triggered simultaneously by protein misfolding and aggregation but might provide necessary insights for drug development against these malfunctions. The PN prevents the accumulation of misfolded proteins in part through the ubiquitin proteasome system, which targets proteins for proteasomal destruction (
      • Galves M.
      • Rathi R.
      • Prag G.
      • Ashkenazi A.
      Ubiquitin Signaling and Degradation of Aggregate-Prone Proteins.
      ). However, the kinetic link between consecutive events along the trajectory of the targeted proteins and the interplay between the different elements of the PN at the molecular level are poorly defined. Gottlieb et al. (
      • Gottlieb C.D.
      • Thompson A.C.S.
      • Ordureau A.
      • Harper J.W.
      • Kopito R.R.
      Acute unfolding of a single protein immediately stimulates recruitment of ubiquitin protein ligase E3C (UBE3C) to 26S proteasomes.
      ) now show in their recent work that several proteins—including the proteasomal subunit RPN13—are ubiquitinated within 10 min following acute unfolding of engineered proteins, mediated in part by the recruitment of the E3 ubiquitin ligase UBE3C to the 26S proteasome. These results indicate that the proteasome is responsive to protein unfolding, not just aggregation, and provide new strategies to gain further insights in this area.
      To untangle the events occurring immediately after protein unfolding, Gottlieb et al. (
      • Gottlieb C.D.
      • Thompson A.C.S.
      • Ordureau A.
      • Harper J.W.
      • Kopito R.R.
      Acute unfolding of a single protein immediately stimulates recruitment of ubiquitin protein ligase E3C (UBE3C) to 26S proteasomes.
      ) elegantly combined protein destabilizing domains (DDs) and proteomic approaches. To this end, they used a protein termed AgDD engineered by Wandless and colleagues (
      • Miyazaki Y.
      • Mizumoto K.
      • Dey G.
      • Kudo T.
      • Perrino J.
      • Chen L.-C.
      • Meyer T.
      • Wandless T.J.
      A method to rapidly create protein aggregates in living cells.
      ). AgDD contains the FK506-binding protein (FKBP1A) mutant that typifies the DDs but is extended on the N terminus with an aggregation-prone peptide and on the C terminus with superfolder GFP (Fig. 1A). The fusion protein is stable when bound to the high-affinity synthetic ligand Shield-1 (S1) but readily aggregates and is ubiquitinated upon S1 removal (
      • Miyazaki Y.
      • Mizumoto K.
      • Dey G.
      • Kudo T.
      • Perrino J.
      • Chen L.-C.
      • Meyer T.
      • Wandless T.J.
      A method to rapidly create protein aggregates in living cells.
      ).
      Figure thumbnail gr1
      Figure 1A, AgDD remains soluble in the presence of S1 but readily aggregates upon S1 washout. B, AgDD unfolding stimulates UBE3C recruitment to the 26S proteasome, leading to increased ubiquitination of AgDD, perhaps to increase substrate processivity, and increased RPN13 ubiquitination, to potentially prevent the recruitment of other proteasome substrates to the engaged particle. When the cell is not overloaded with misfolded proteins, these other substrates would then be degraded by other—nonengaged—proteasome particles. NES, nuclear export signal; sfGFP, superfolder GFP.
      With these constructs in hand, the authors were able to identify the first proteins that undergo changes in ubiquitination upon S1 removal and consequently AgDD aggregation in a time-resolved manner using ubiquitin remnant immunoaffinity profiling and a quantitative MS approach with tandem mass tagging. Among the 48 identified proteins, including AgDD, members of the proteostasis network were highly enriched and showed increased ubiquitination levels within 2.5–10 min. Interestingly, many ribosomal proteins and ribosome biogenesis factors were identified next to proteasomal subunits, indicating a rapid targeting of key players of the protein synthesis and degradation machineries. Of particular interest is the strong increase in ubiquitination of RPN13 (also known as ADRM1) on residues Lys-21 and Lys-34. Ubiquitination of AgDD and RPN13 upon S1 washout was confirmed by pulldown analysis. Importantly, the authors did not observe any changes in the degradation kinetics of another model substrate targeted by the ubiquitin proteasome system upon aggregation of AgDD, indicating that the observed RPN13 ubiquitination was unlikely to be leading to a general impairment of the 26S proteasome activity. Interestingly, the proteolytic activity itself was increased in these conditions, when measured with a small proteasomal peptide substrate. The E3 ligase UBE3C was previously shown to ubiquitinate a nonaggregating DD reporter and RPN13 (
      • Chu B.W.
      • Kovary K.M.
      • Guillaume J.
      • Chen L.-C.
      • Teruel M.N.
      • Wandless T.J.
      The E3 ubiquitin ligase UBE3C enhances proteasome processivity by ubiquitinating partially proteolyzed substrates.
      ,
      • Besche H.C.
      • Sha Z.
      • Kukushkin N.V.
      • Peth A.
      • Hock E.M.
      • Kim W.
      • Gygi S.
      • Gutierrez J.A.
      • Liao H.
      • Dick L.
      • Goldberg A.L.
      Autoubiquitination of the 26S Proteasome on Rpn13 regulates breakdown of ubiquitin conjugates.
      ), and Gottlieb et al. confirmed that UBE3C was responsible for AgDD polyubiquitination (i.e. attachment of subsequent ubiquitin moieties to form a chain) and RPN13 multiubiquitination (i.e. conjugation of ubiquitin to different substrate lysine residues) using an siRNA knockdown approach (
      • Gottlieb C.D.
      • Thompson A.C.S.
      • Ordureau A.
      • Harper J.W.
      • Kopito R.R.
      Acute unfolding of a single protein immediately stimulates recruitment of ubiquitin protein ligase E3C (UBE3C) to 26S proteasomes.
      ). Importantly, these events occurred rapidly, within minutes of the induced aggregation of AgDD.
      As a result of the high aggregation rate, it was not easily possible to distinguish whether the observed events were due to AgDD unfolding or aggregation. Therefore, to discriminate between these two possibilities, the authors returned to a DD variant lacking the N-terminal aggregation-prone peptide, which converts to an unfolded yet soluble molten globule-like state in the absence of S1. The observed results were highly congruent to AgDD within the first 10 min, indicating that indeed unfolding but not aggregation of the model substrate was triggering UBE3C recruitment, and RPN13 and AgDD ubiquitination, although aggregation seems to accelerate the response (
      • Gottlieb C.D.
      • Thompson A.C.S.
      • Ordureau A.
      • Harper J.W.
      • Kopito R.R.
      Acute unfolding of a single protein immediately stimulates recruitment of ubiquitin protein ligase E3C (UBE3C) to 26S proteasomes.
      ). Notably, the observed time resolution that goes along with the chemical biology approach presented by Gottlieb et al. offers a unique view of the chain of events that take place when the PN is challenged.
      So, what is the role of RPN13 ubiquitination in the context of protein misfolding? Previous work from Goldberg and colleagues (
      • Chu B.W.
      • Kovary K.M.
      • Guillaume J.
      • Chen L.-C.
      • Teruel M.N.
      • Wandless T.J.
      The E3 ubiquitin ligase UBE3C enhances proteasome processivity by ubiquitinating partially proteolyzed substrates.
      ) showed that RPN13 ubiquitination increases upon proteasome inhibition as well as other conditions that lead to a general increase of the cellular ubiquitination levels, such as heat shock and arsenite treatment—conditions that all lead to an accumulation of misfolded proteins. Importantly, UBE3C-mediated ubiquitination of RPN13 reduces the capacity of the proteasome to bind to polyubiquitinated substrates (
      • Chu B.W.
      • Kovary K.M.
      • Guillaume J.
      • Chen L.-C.
      • Teruel M.N.
      • Wandless T.J.
      The E3 ubiquitin ligase UBE3C enhances proteasome processivity by ubiquitinating partially proteolyzed substrates.
      ), consistent with the fact that the RPN13 ubiquitination sites (Lys-21 and Lys-34) are adjacent and within the RPN13-Pru domain that binds ubiquitin (
      • Husnjak K.
      • Elsasser S.
      • Zhang N.
      • Chen X.
      • Randles L.
      • Shi Y.
      • Hofmann K.
      • Walters K.J.
      • Finley D.
      • Dikic I.
      Proteasome subunit Rpn13 is a novel ubiquitin receptor.
      ). UBE3C was previously shown to ubiquitinate the nonaggregating DD reporter to promote its degradation by increasing proteasomal processivity (
      • Besche H.C.
      • Sha Z.
      • Kukushkin N.V.
      • Peth A.
      • Hock E.M.
      • Kim W.
      • Gygi S.
      • Gutierrez J.A.
      • Liao H.
      • Dick L.
      • Goldberg A.L.
      Autoubiquitination of the 26S Proteasome on Rpn13 regulates breakdown of ubiquitin conjugates.
      ). Yet recruitment of UBE3C to the proteasome is also enhanced in the presence of ubiquitinated substrates (
      • Kuo C.-L.
      • Goldberg A.L.
      Ubiquitinated proteins promote the association of proteasomes with the deubiquitinating enzyme Usp14 and the ubiquitin ligase Ube3c.
      ). One model that could reconcile these findings is that UBE3C is recruited to the proteasome to promote the complete degradation of misfolded proteins by conjugating additional ubiquitin moieties, increasing their affinity to the proteasome. The concomitant ubiquitination of RPN13 would then prevent the recruitment of additional proteasome substrates, because RPN13-conjugated complexes would appear to be substrate-engaged particles with potentially “more difficult” proteins to process (Fig. 1B), similar to what Goldberg and colleagues proposed (
      • Chu B.W.
      • Kovary K.M.
      • Guillaume J.
      • Chen L.-C.
      • Teruel M.N.
      • Wandless T.J.
      The E3 ubiquitin ligase UBE3C enhances proteasome processivity by ubiquitinating partially proteolyzed substrates.
      ,
      • Kuo C.-L.
      • Goldberg A.L.
      Ubiquitinated proteins promote the association of proteasomes with the deubiquitinating enzyme Usp14 and the ubiquitin ligase Ube3c.
      ). The molecular mechanism that mediates UBE3C proteasomal recruitment remains to be determined in future investigations. More generally, the present study demonstrates that changes at the proteasomal level rapidly occur in the cell in the presence of a single pool of unfolded proteins (i.e. AgDD or DD), in conditions where the overall activity of the ubiquitin proteasome system does not appear to be impaired. The future challenges will be to determine whether this mechanism is specific to proteasome substrates that are potentially more difficult to process (e.g. due to misfolding) and whether it plays a role in proteopathies more broadly.

      References

        • Galves M.
        • Rathi R.
        • Prag G.
        • Ashkenazi A.
        Ubiquitin Signaling and Degradation of Aggregate-Prone Proteins.
        Trends Biochem. Sci. 2019; 44 (31079890): 872-884
        • Gottlieb C.D.
        • Thompson A.C.S.
        • Ordureau A.
        • Harper J.W.
        • Kopito R.R.
        Acute unfolding of a single protein immediately stimulates recruitment of ubiquitin protein ligase E3C (UBE3C) to 26S proteasomes.
        J. Biol. Chem. 2019; 294 (31375563): 16511-16524
        • Miyazaki Y.
        • Mizumoto K.
        • Dey G.
        • Kudo T.
        • Perrino J.
        • Chen L.-C.
        • Meyer T.
        • Wandless T.J.
        A method to rapidly create protein aggregates in living cells.
        Nat. Commun. 2016; 7 (27229621): 11689
        • Chu B.W.
        • Kovary K.M.
        • Guillaume J.
        • Chen L.-C.
        • Teruel M.N.
        • Wandless T.J.
        The E3 ubiquitin ligase UBE3C enhances proteasome processivity by ubiquitinating partially proteolyzed substrates.
        J. Biol. Chem. 2013; 288 (24158444): 34575-34587
        • Besche H.C.
        • Sha Z.
        • Kukushkin N.V.
        • Peth A.
        • Hock E.M.
        • Kim W.
        • Gygi S.
        • Gutierrez J.A.
        • Liao H.
        • Dick L.
        • Goldberg A.L.
        Autoubiquitination of the 26S Proteasome on Rpn13 regulates breakdown of ubiquitin conjugates.
        EMBO J. 2014; 33 (24811749): 1159-1176
        • Husnjak K.
        • Elsasser S.
        • Zhang N.
        • Chen X.
        • Randles L.
        • Shi Y.
        • Hofmann K.
        • Walters K.J.
        • Finley D.
        • Dikic I.
        Proteasome subunit Rpn13 is a novel ubiquitin receptor.
        Nature. 2008; 453 (18497817): 481-488
        • Kuo C.-L.
        • Goldberg A.L.
        Ubiquitinated proteins promote the association of proteasomes with the deubiquitinating enzyme Usp14 and the ubiquitin ligase Ube3c.
        Proc. Natl. Acad. Sci. U.S.A. 2017; 114 (28396413): E3404-E3413

      Linked Article

      • Acute unfolding of a single protein immediately stimulates recruitment of ubiquitin protein ligase E3C (UBE3C) to 26S proteasomes
        Journal of Biological ChemistryVol. 294Issue 45
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          The intracellular accumulation of aggregated misfolded proteins is a cytopathological hallmark of neurodegenerative diseases. However, the functional relationship between protein misfolding or aggregation and the cellular proteostasis network that monitors and maintains proteome health is poorly understood. Previous studies have associated translational suppression and transcriptional remodeling with the appearance of protein aggregates, but whether these responses are induced by aggregates or their misfolded monomeric or oligomeric precursors remains unclear.
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