Paradigms of protein degradation by the proteasome

Inobe, Tomonao; Matouschek, Andreas T

doi:10.1016/j.sbi.2014.02.002

Cited by 105 publications

(90 citation statements)

References 82 publications

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“…In vivo half-lives of intracellular proteins range from less than a minute to many days (1)(2)(3)(4)(5)(6). The term "half-life" is, at best, an approximate descriptor of a protein's degradation curve.…”

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confidence: 99%

A reference-based protein degradation assay without global translation inhibitors

Oh¹,

Chen²,

Varshavsky

2017

Journal of Biological Chemistry

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confidence: 99%

A reference-based protein degradation assay without global translation inhibitors

Oh¹,

Chen²,

Varshavsky

2017

Journal of Biological Chemistry

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“…Another determinant of an N-degron is an internal Lys residue(s). It functions as a site of protein polyubiquitylation, is often engaged stochastically (in competition with other "eligible" lysines), and tends to be located in a conformationally disordered region (2,9,19,20). Bacteria also contain the N-end rule pathway, but Ub-independent versions of it (21-26).…”

mentioning

confidence: 99%

Analyzing N-terminal Arginylation through the Use of Peptide Arrays and Degradation Assays

Wadas

Piatkov

Brower

et al. 2016

Journal of Biological Chemistry

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N␣ -terminal arginylation (Nt-arginylation) of proteins is mediated by the Ate1 arginyltransferase (R-transferase), a component of the Arg/N-end rule pathway. This proteolytic system recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. The definitively identified ("canonical") residues that are Nt-arginylated by R-transferase are N-terminal Asp, Glu, and (oxidized) Cys. Over the last decade, several publications have suggested (i) that Ate1 can also arginylate non-canonical N-terminal residues; (ii) that Ate1 is capable of arginylating not only ␣-amino groups of N-terminal residues but also ␥-carboxyl groups of internal (non-N-terminal) Asp and Glu; and (iii) that some isoforms of Ate1 are specific for substrates bearing N-terminal Cys residues. In the present study, we employed arrays of immobilized 11-residue peptides and pulse-chase assays to examine the substrate specificity of mouse R-transferase. We show that amino acid sequences immediately downstream of a substrate's canonical (Nt-arginylatable) N-terminal residue, particularly a residue at position 2, can affect the rate of Nt-arginylation by R-transferase and thereby the rate of degradation of a substrate protein. We also show that the four major isoforms of mouse R-transferase have similar Nt-arginylation specificities in vitro, contrary to the claim about the specificity of some Ate1 isoforms for N-terminal Cys. In addition, we found no evidence for a significant activity of the Ate1 R-transferase toward previously invoked non-canonical N-terminal or internal amino acid residues. Together, our results raise technical concerns about earlier studies that invoked non-canonical arginylation specificities of Ate1.The N-end rule pathway is a set of intracellular proteolytic systems whose unifying feature is the ability to recognize and polyubiquitylate proteins containing N-terminal (Nt) 2 degradation signals called N-degrons, thereby causing the processive degradation of these proteins by the proteasome (Fig. 1, A and B) (1-13). Recognition components of the N-end rule pathway are called N-recognins. In eukaryotes, N-recognins are E3 ubiquitin (Ub) ligases that can target N-degrons. Some N-recognins contain several substrate-binding sites and thereby can recognize (bind to) not only N-degrons but also specific internal (non-N-terminal) degradation signals (Fig. 1A) (14 -18). The main determinant of a protein's N-degron is either an unmodified or chemically modified N-terminal residue. Another determinant of an N-degron is an internal Lys residue(s). It functions as a site of protein polyubiquitylation, is often engaged stochastically (in competition with other "eligible" lysines), and tends to be located in a conformationally disordered region (2,9,19,20). Bacteria also contain the N-end rule pathway, but Ub-independent versions of it (21-26).Regulated degradation of proteins and their natural fragments by the N-end rule pathway has been s...

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“…Translocation of this initiation region of the substrate serves to break interactions in the ternary complex, allowing degradation to begin and the adaptor to be recycled. An unstructured initiation region is also required for unfolding and degradation by the proteasome (41)(42)(43). Proteins are targeted to the proteasome by a two-part degradation signal consisting of a disordered region within the substrate and a polyubiquitin tag.…”

Section: Discussionmentioning

confidence: 99%

Remodeling of a delivery complex allows ClpS-mediated degradation of N-degron substrates

Rivera-Rivera¹,

Roman-Hernandez²,

Sauer³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The ClpS adaptor collaborates with the AAA+ ClpAP protease to recognize and degrade N-degron substrates. ClpS binds the substrate N-degron and assembles into a high-affinity ClpS-substrateClpA complex, but how the N-degron is transferred from ClpS to the axial pore of the AAA+ ClpA unfoldase to initiate degradation is not known. Here we demonstrate that the unstructured N-terminal extension (NTE) of ClpS enters the ClpA processing pore in the active ternary complex. We establish that ClpS promotes delivery only in cis, as demonstrated by mixing ClpS variants with distinct substrate specificity and either active or inactive NTE truncations. Importantly, we find that ClpA engagement of the ClpS NTE is crucial for ClpS-mediated substrate delivery by using ClpS variants carrying "blocking" elements that prevent the NTE from entering the pore. These results support models in which enzymatic activity of ClpA actively remodels ClpS to promote substrate transfer, and highlight how ATPase/motor activities of AAA+ proteases can be critical for substrate selection as well as protein degradation.AAA+ ATPase adaptor | N-degron substrate selection | adaptor remodeling | AAA+ unfoldase/translocase A AA+ molecular machines power cellular processes as diverse as protein degradation, microtubule severing, membrane fusion, and initiation of DNA replication, with the common theme that macromolecules are actively remodeled (1-3). Furthermore, protein quality control in all organisms involves deployment of ATPdependent proteases, consisting of hexameric AAA+ rings that unfold and translocate specific substrates into an associated peptidase barrel (3, 4). Adaptor proteins are known to aid recognition and degradation of certain substrates (5-8), but how enzyme-adaptor pairs ensure proper substrate selection is poorly understood.In prokaryotes and eukaryotes, the N-end rule pathway governs degradation of proteins with specific N-terminal amino acids (9, 10). In Escherichia coli, the primary destabilizing N-degron amino acids are Phe, Tyr, Trp, and Leu (11,12). ClpS, a widespread bacterial adaptor, recognizes and delivers N-degron substrates to the ClpAP or ClpCP AAA+ proteases (6,11,13). These enzymes consist of the AAA+ ClpA or ClpC unfoldases coaxially stacked with the ClpP peptidase (14-16). In eukaryotes, a family of E3 ligases shares homology with the substrate-binding region of ClpS (17, 18). These ligases recognize N-degron substrates and promote ubiquitination, which then targets the modified protein to the 26S proteasome (17,18).Multiple crystal structures reveal the regions of ClpS that bind to the N-degron, as well as a patch that binds the N-terminal domain of . This bivalent binding to the substrate and the enzyme tethers N-degron substrates to ClpAP. Tethering alone is insufficient for ClpS to promote substrate delivery however, given that deletion of 12 amino acids of the ClpS unstructured N-terminal extension (NTE; residues 1-25 in E. coli ClpS) (Fig. 1A) prevents N-degron substrate degradation, but does not block format...

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Paradigms of protein degradation by the proteasome

Cited by 105 publications

References 82 publications

A reference-based protein degradation assay without global translation inhibitors

A reference-based protein degradation assay without global translation inhibitors

Analyzing N-terminal Arginylation through the Use of Peptide Arrays and Degradation Assays

Remodeling of a delivery complex allows ClpS-mediated degradation of N-degron substrates

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