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  • Review Article
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Proteasomes and their kin: proteases in the machine age

Nature Reviews Molecular Cell Biology volume 5, pages 177–187 (2004)Cite this article

Key Points

  • The 26S proteasome is the most famous member of a family of 'chambered proteases' — multi-subunit enzymes that share a barrel-shaped structure and interior active sites that can only be accessed through a gated pore.

  • The architecture of these enzymes promotes processive substrate degradation and makes substrate unfolding a prerequisite for proteolysis. Chambered proteases therefore have dedicated chaperone (regulatory) complexes that confer the ability to recognize and unfold cognate substrates.

  • The substrates of each protease display a signal(s) that is recognized by its regulatory complex. Chambered proteases are exquisitely specific because the signals that they recognize are independent of the proteolytic cleavage sites

  • Unlike prokaryotic and archaebacterial family members, which usually recognize primary sequence motifs in the substrate in a direct manner, the eukaryotic 26S proteasome typically recognizes substrates that are tagged with a 'polyubiquitin chain' — a polymer assembled from the small, conserved protein ubiquitin. Eukaryotes have a vast array of enzymes that mediate the highly regulated process of polyubiquitin tagging.

  • Protease regulatory complexes carry out numerous functions, including recognition of substrate-based signals, unfolding of the substrate polypeptide chain, and gating of the protease pore. The regulatory (19S) complex of the 26S proteasome must also remove the polyubiquitin-chain signal, the constituent ubiquitins of which are then released and re-used.

  • Recent studies of the protein unfolding that is catalysed by protease regulatory complexes indicate that signal recognition, unfolding and translocation are intimately coupled and intrinsically energy dependent. Substrates of prokaryotic regulatory complexes seem to start unfolding at the degradation signal; this is not necessarily the case for the 26S proteasome.

  • In eukaryotes, the processes of signal (polyubiquitin) recognition and removal are surprisingly complex. Polyubiquitin removal must be precisely coordinated with downstream events in order to ensure that tagged substrates are, in fact, degraded.

  • Understanding the precise mechanisms of substrate unfolding by protease regulatory complexes is a goal of many laboratories at present, as is elucidating the specific functions of the subunits of the 19S regulatory complex. The answers to these and other questions could open new avenues for the design of proteasome-directed therapeutics.

Abstract

'Chambered proteases', including the eukaryotic 26S proteasome, use the energy of ATP to drive the unfolding and translocation of a polypeptide substrate into a chamber of sequestered proteolytic active sites. These proteases have diverse functions and are found in all three kingdoms of life. Understanding chambered proteases requires answers to two questions — how do these remarkable machines select the correct target proteins and how do they bring about the processive degradation of these molecules?

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Figure 1: A gallery of chambered proteases.
Figure 2: Electron-microscopy images of chambered-protease complexes.
Figure 3: The subunit composition of the 19S complex of Saccharomyces cerevisiae.
Figure 4: Steps in substrate proteolysis by 26S proteasomes.

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Acknowledgements

We apologize to the many individuals whose work could not be cited due to space limitations. We thank M. Hochstrasser for comments on the manuscript and T. Yao, M. Bewley and A. Cohen for help with the figures. Work in our laboratories is funded by grants from the National Institutes of Health.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, 21205, Maryland, USA

    Cecile M. Pickart

  2. Department of Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa, USA

    Robert E. Cohen

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  1. Cecile M. Pickart
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  2. Robert E. Cohen
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Correspondence to Cecile M. Pickart.

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Related links

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DATABASES

Entrez

ClpA

ClpX

HslU

HslV

Saccharomyces genome database

Hul5

Rpn10

Rpn11

RPT1

Rpt2

RPT6

ubiquitin-processing protease 6

Swiss-Prot

Az

ODC

POH1

S4

S5a

ubiquitin-fusion degradation 4

ubiquitin-specific protease 14

UCH37

Glossary

CHAMBERED PROTEASE

A multi-subunit proteolytic enzyme that has a barrel-shaped structure and sequestered active sites. It is sometimes called a compartmentalized or self-compartmentalized protease.

26S PROTEASOME

The main chambered protease of eukaryotes. Named for its approximate sedimentation coefficient, it is assembled from proteolytic (20S) and regulatory (19S) complexes. We use the term '26S' for hybrid 20S–19S and 19S–20S–19S complexes.

UBIQUITIN

A conserved, 76-amino-acid protein in eukaryotes that is conjugated through its carboxyl terminus to amino groups (usually on lysine residues) of cellular proteins.

HslV

(heat-shock-locus protease). A chambered protease of E. coli, the proteolytic and regulatory complexes of which consist of HslV dodecamers and HslU hexamers (sometimes called ClpQ and ClpY), respectively. These subcomplexes associate to form the holoenzyme that is known as HslUV.

ClpP

(caseinolytic protease). A chambered protease of E. coli in which the proteolytic complex consists of 14 ClpP subunits (arranged as two rings). Hexameric rings of ClpA or ClpX subunits (two distinct regulatory complexes) associate with ClpP to form ClpAP or ClpXP holoenzymes.

20S PROTEASOME

The proteolytic complex of proteasomes has 28 subunits arranged as four rings. It is named for its approximate sedimentation coefficient and has orthologues in some eubacteria and archaebacteria.

AAA+ ATPASES

ATPases that are associated with a variety of cellular activities. A superfamily of proteins with one or two nucleotide-binding domains ('AAA modules'), which often form ring-like oligomers and function as chaperones in diverse cellular processes.

19S REGULATORY COMPLEX

Named for its approximate sedimentation coefficient and also known as PA700, one or two copies of this ∼18-subunit complex associate with the 20S proteasome to form a 26S proteasome.

COP9 SIGNALOSOME

A multi-protein complex that has numerous regulatory roles. Its subunits are homologous to a subset of the Rpn subunits in the 19S regulatory complex.

Ubp/USP

(ubiquitin-processing proteases/ubiquitin-specific proteases). Yeast and mammalian members, respectively, of a family of cysteine proteases that hydrolyse peptide or isopeptide bonds that involve the carboxyl terminus of ubiquitin.

POLYUBIQUITIN CHAIN

A polymer that results from the conjugation of ubiquitin to itself. Chains that are linked through lysine 48 of each ubiquitin target substrates for degradation by 26S proteasomes.

E3 UBIQUITIN LIGASES

Enzymes that are responsible for the conjugation of ubiquitin to substrate proteins.

Ufd4

A ubiquitin ligase that is part of the 'ubiquitin-fusion degradation' pathway, which consists of enzymes that facilitate the polyubiquitylation of ubiquitin-fusion proteins.

UBL–UBA PROTEINS

A family of proteins in which UBL (ubiquitin-like) and UBA (ubiquitin-associated) domains are present in a single polypeptide chain.

SSRA

A peptide tag that is encoded by a small stable RNA (10Sa RNA) and is added to the carboxyl termini of stalled translation products in E. coli in order to target these aberrant polypeptides for degradation by chambered proteases.

IκBα–NF-κB

The nuclear factor-κB transcription factor is processed (from a p105 form to a p50 form) by 26S proteasomes. NF-κB is sequestered in the cytoplasm through associations with members of the inhibitor of κB family, which includes IκBα. IκBα must be degraded for NF-κB to activate the expression of inflammatory genes in the nucleus.

PA28

(proteasome activator-28). A family of regulatory complexes that have a role in the production of antigenic peptides. They consist of heptamers of 28-kDa non-ATPase subunits.

MPN+

The protein domain MPN+ ('Mpr1, Pad1, amino-terminal'; also known as JAMM (Jab1, MPN-domain metalloprotease)) is thought to coordinate zinc and to catalyse proteolysis.

Zn2+-METALLOPROTEASE

A protease that has an essential zinc atom in its active site.

CYSTEINE PROTEASE

A protease that has a catalytic cysteine residue in its active site.

UCH

Ubiquitin-carboxy-terminal hydrolases are ubiquitin-specific cysteine proteases that are similar to Ubp/USPs, but that belong to a different protein family.

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Pickart, C., Cohen, R. Proteasomes and their kin: proteases in the machine age. Nat Rev Mol Cell Biol 5, 177–187 (2004). https://doi.org/10.1038/nrm1336

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