Feeding the machine: mechanisms of proteasome-catalyzed degradation of ubiquitinated proteins
Introduction
The proteasome comprises two types of large multiprotein complexes, the 20S and two 19S regulatory complexes (Figure 1). The 19S complex is a 17-subunit machine, itself composed of two subcomplexes: the base, thought to be responsible for the denaturation of the substrate via the action of six ATPase subunits; and the lid, which sits over the base and is believed to recognize polyubiquitinated proteasome substrates and remove the ubiquitin tag although the action of an inherent isopeptidase activity. The cylindrical 20S proteolytic complex comprises four rings, each containing seven subunits. The two inner rings possess three catalytic sites each, which are characterized by their individual preference for cleavage after specific amino acids. This inner proteolytic chamber is accessible only through one openings at each end of the 20S cylinder, each of which is gated by one of the 19S complexes.
Proteins are targeted for degradation by the proteasome via the concerted effort of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2) and a ubiquitin ligase (E3). Whereas there is one E1 enzyme in eukaryotic cells, multiple E2 and E3 enzymes pair up to provide the specificity needed to direct selective protein degradation. Subsequent multiple rounds of ubiquitination result in polyubiquitinated proteins that are recognized by the 19S regulatory particle (RP) of the 26S proteasome. Once ubiquitinated, protein substrates are recognized and degraded by the 26S proteasome into short peptides. Each step of this process has been the focus of several recent studies, which have advanced our knowledge of the mechanisms involved (Figure 1).
Section snippets
Substrate recognition: E3 ubiquitin ligase specificity
Given the constitutive nature of ubiquitinated protein proteolysis by the proteasome, researchers interested in the regulation of this degradation cascade have focused on the mechanisms of substrate selection. E3 ubiquitin ligases provide the specificity required for regulated protein degradation. Whereas budding yeast have only one E1 ubiquitin activating enzyme, 13 E2 ubiquitin conjugating enzymes and at least 60 putative E3 ubiquitin ligases have been reported in the yeast genome. The great
Sugar attraction
E3 ubiquitin ligases recognize their corresponding protein substrates via a variety of structural determinants including primary sequence, post-translational modifications and protein folding state (Figure 2). In recent years, several new ubiquitin ligase recognition motifs have been reported. For example, an F-box-containing protein, Fbx2, with no known binding specificity was recently reported to recognize N-linked high-mannose oligosaccharides bound to proteins [1]. Recognition of N-glycan
Sensing oxygen
Cells sense and respond to varying levels of oxygen and oxidative stress using several intracellular mechanisms. For example, the iron regulatory proteins IRP1 and IRP2 are post-transcriptional regulators of cellular iron metabolism that bind to an iron-responsive element in the untranslated region of mRNAs involved in iron homeostasis and can serve as a link between iron homeostasis and oxidative stress sensing 2., 3.. IRP2 is regulated by iron in a heme-dependent manner via its iron-dependent
Catching unfolded proteins
The molecular chaperones heat shock protein (Hsp)70 and Hsp90 bind misfolded or unfolded proteins to prevent protein aggregation and catalyze the proper folding through an ATP-dependent mechanism. If, however, chaperone-mediated renaturation is not feasible, misfolded proteins then become substrates for ubiquitination and proteasomal degradation. Links between these two pathways have recently been described. Molecular chaperones work in concert with co-chaperones such as CHIP (carboxyl terminus
Recruitment mechanisms
Once ubiquitinated, targeted proteins are bound by the 19S regulatory complex of the 26S proteasome for subsequent translocation and degradation. How proteins find and bind the 26S proteasome has been the focus of several studies [21]. Recent studies identified a direct binding of polyubiquitin chains by a specific ATPase subunit S6′ (also known as Rpt5) in the 19S RP [22]. Although this interaction is readily detectable in in vitro experiments, recent studies suggest that polyubiquitinated
Deubiquitination of substrates: an isopeptidase revealed
Once bound to the 26S proteasome, the isopeptide bond between the protein substrate and the polyubiquitin chain must be cleaved before subsequent substrate translocation through the axial pore of the proteolytic chamber.
Several deubiquitinating enzymes (DUBs) belonging to the cysteine protease family have been reported [32]. Kessler and colleagues [33••] have recently employed a creative approach to identify all cysteine protease DUBs within a cell using ubiquitin-modified electrophilic
Channel access mechanisms
It has long been known that small peptides can serve as substrates for the purified 20S proteasome but that, in general, full-length proteins must be polyubiquitinated, recognized by the 19S regulatory complex, deubiquitinated and unfolded in an ATP-dependent manner before translocation into the proteolytic 20S chamber. Experiments have shown that access to the 20S cylinder is gated and thus regulated by a key protein within the 19S complex, namely the rpt2 subunit [36••]. X-ray
Conclusions
In the past two years, several studies have advanced our understanding of the mechanisms by which the ubiquitin-proteasome pathway degrades proteins. These include an ever expanding repertoire of E3 structural determinants such as hydroxyproline, oligosaccharide moieties and protein denaturation. A clearer role has also emerged for molecular chaperones as triage agents to shunt non-native proteins down the degradative pathway if renaturation is not a viable option. In addition, many of the
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
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of special interest
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of outstanding interest
Acknowledgements
I thank the NIH (GM62120) for its support of our work, and members of the lab for helpful comments.
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