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Biomedical subjects

Michael H Glickman

Publications and source records attributed to Michael H Glickman.

12 recordsLinked to original sources
PubMed2005

Proteasome plasticity.

The 26S proteasome is responsible for regulated proteolysis of most intracellular proteins yet the focus of intense regulatory action itself. Proteasome abundance is responsive to cell needs or stress conditions, and dynamically localized to concentrations of substrates. Proteasomes are continually assembled and disassembled, and their subunits subject to a variety of posttranslational modifications. Furthermore, as robust and multi-tasking as this complex is, it does not function alone. A spattering of closely associating proteins enhances complex stability, fine-tunes activity, assists in substrate-binding, recycling of ubiquitin, and more. HEAT repeat caps activate proteasomes, yet share remarkable features with nuclear importins. Fascinating cross talk even occurs with ribosomes through common maturation factors. The dynamics of proteasome configurations and how they relate to diverse activities is the topic of this review.

Animals↗
PubMed2005

A window of opportunity: timing protein degradation by trimming of sugars and ubiquitins.

Of the many post-translational modifications of proteins, ubiquitination and N-glycosylation stand out because they are polymeric additions. In contrast to single-unit modifications, the fate of the modified protein is determined by the dynamic equilibrium of polymerization versus depolymerization, rather than by the initial addition itself. Notably, it is the trimming of sugar chains and elongation of polyubiquitin that target the protein to degradation. Recent research suggests that, for each process, special receptors recognize chains that reach an appropriate length and commit the conjugated substrate for proteasomal disposal. We propose that the 'magic numbers' are loss of at least three mannose residues from the initial chain, or extension to at least four ubiquitins. Although these processes are compartmentalized to either side of the endoplasmic reticulum (ER) membrane, some proteins are sequentially subjected to both because they transverse this membrane for ER-associated degradation.

Calnexin↗
PubMed2005

Purification of proteasomes, proteasome subcomplexes, and proteasome-associated proteins from budding yeast.

The proteasome is a highly complex, ATP-dependent protease, consisting of over 30 subunits, and dedicated mainly to the degradation of ubiquitin-protein conjugates. Proteasomes are evolutionarily conserved in the eukaryotic kingdom, and those of yeast are well suited to serve as a general model. We describe techniques for the purification of proteasomes from budding yeast in milligram amounts via conventional and affinity-based strategies. While both approaches yield highly purified material, the affinity method is faster and easier. In addition, the affinity method is more suitable for identifying proteasome-associated proteins. We also describe methods for purifying the major subassemblies of the proteasome, such as the CP, the RP, the lid, and the base. A variety of activity assays and native gel procedures are available to evaluate purified proteasomes functionally. When coupled with the genetic methods available in yeast, these biochemical procedures allow for detailed functional analysis of this unique protein complex.

Chromatography, Affinity↗
PubMed2004

Participation of the proteasomal lid subunit Rpn11 in mitochondrial morphology and function is mapped to a distinct C-terminal domain.

Substrates destined for degradation by the 26 S proteasome are labelled with polyubiquitin chains. Rpn11/Mpr1, situated in the lid subcomplex, partakes in the processing of these chains or in their removal from substrates bound to the proteasome. Rpn11 also plays a role in maintaining mitochondrial integrity, tubular structure and proper function. The recent finding that Rpn11 participates in proteasome-associated deubiquitination focuses interest on the MPN+ (Mpr1, Pad1, N-terminal)/JAMM (JAB1/MPN/Mov34) metalloprotease site in its N-terminal domain. However, Rpn11 damaged at its C-terminus (the mpr1-1 mutant) causes pleiotropic effects, including proteasome instability and mitochondrial morphology defects, resulting in both proteolysis and respiratory malfunctions. We find that overexpression of WT (wild-type) RPN8, encoding a paralogous subunit that does not contain the catalytic MPN+ motif, corrects proteasome conformations and rescues cell cycle phenotypes, but is unable to correct defects in the mitochondrial tubular system or respiratory malfunctions associated with the mpr1-1 mutation. Transforming mpr1-1 with various RPN8-RPN11 chimaeras or with other rpn11 mutants reveals that a WT C-terminal region of Rpn11 is necessary, and more surprisingly sufficient, to rescue the mpr1-1 mitochondrial phenotype. Interestingly, single-site mutants in the catalytic MPN+ motif at the N-terminus of Rpn11 lead to reduced proteasome-dependent deubiquitination connected with proteolysis defects. Nevertheless, these rpn11 mutants suppress the mitochondrial phenotypes associated with mpr1-1 by intragene complementation. Together, these results point to a unique role for the C-terminal region of Rpn11 in mitochondrial maintenance that may be independent of its role in proteasome-associated deubiquitination.

Acetyltransferases↗
PubMed2004

Deubiquitinating enzymes are IN/(trinsic to proteasome function).

Covalent conjugation of the ubiquitin tag to cellular proteins plays a central role in a number of processes, the most notable among them being degradation by the 26S proteasome. A fundamental property of this process is that ubiquitination, in contrast to subsequent degradation, is reversible due to a number of deubiquitinating enzymes that mediate the disassembly of ubiquitin-protein conjugates. The uniqueness of ubiquitin as a reversible tag necessitates mechanisms to guarantee its efficiency. Interestingly, some deubiquitinating enzymes are associated with the 26S proteasome itself. We include a brief overview of the key proteasome-associated deubiquitinating enzymes such as Rpn11/POH1, UCH37/Uch2, Ubp6/Usp14 and Doa4/Ubp4. We go on to discuss how these enzymes may contribute to, or possibly counteract, proteolysis by the proteasome. For example, cumulative evidence points to a partitioning of proteasome action between proteolysis and deubiquitination. On the one hand, inhibition of proteolysis promotes deubiquitination, while on the other hand, inhibition of deubiquitination can promote proteolysis. The plethora of deubiquitinating enzymes may serve as proof reading devices altering the equilibrium between these two processes and allowing for reversal of fortune at various stages of the process. To promote degradation over deubiquitination, certain polyubiquitin conformations could be stabilized or protected from deubiquitinating enzymes in order that they can serve as efficient targeting signals leading to the proteasome. We hypothesize that polvubiquitin chains could also serve as "timers": by slowing down chain disassembly, longer chains allow ample time for unfolding and proteolysis of the substrate.

Animals↗
PubMed2003

Complementary roles for Rpn11 and Ubp6 in deubiquitination and proteolysis by the proteasome.

Substrates destined for degradation by the 26 S proteasome are labeled with polyubiquitin chains. These chains can be dismantled by deubiquitinating enzymes (DUBs). A number of reports have identified different DUBs that can hydrolyze ubiquitin from substrates bound to the proteasome. We measured deubiquitination by both isolated lid and base-core particle subcomplexes, suggesting that at least two different DUBs are intrinsic components of 26 S proteasome holoenzymes. In agreement, we find that highly purified proteasomes contain both Rpn11 and Ubp6, situated within the lid and base subcomplexes, respectively. To study their relative contributions, we purified proteasomes from a mutant in the putative metalloprotease domain of Rpn11 and from a ubp6 null. Interestingly, in both preparations we observed slower deubiquitination rates, suggesting that Rpn11 and Ubp6 serve complementary roles. In accord, the double mutant is synthetically lethal. In contrast to WT proteasomes, proteasomes lacking the lid subcomplex or those purified from the rpn11 mutant are less sensitive to metal chelators, supporting the prediction that Rpn11 may be a metalloprotein. Treatment of proteasomes with ubiquitin-aldehyde or with cysteine modifiers also inhibited deubiquitination but simultaneously promoted degradation of a monoubiquitinated substrate along with the ubiquitin tag. Degradation is unique to 26 S proteasome holoenzymes; we could not detect degradation of a ubiquitinated protein by "lidless" proteasomes, although they were competent for deubiquitination. The fascinating observation that a single ubiquitin moiety is sufficient for targeting an otherwise stable substrate to proteasomes exposes how rapid deubiquitination of poorly ubiquitinated substrates may counteract degradation.

Cysteine Endopeptidases↗
PubMed2003

Proteasome disassembly and downregulation is correlated with viability during stationary phase.

During prolonged starvation, yeast cells enter a stationary phase (SP) during which the synthesis of many proteins is dramatically decreased. We show that a parallel decrease in proteasome-dependent proteolysis also occurs. The reduction in proteolysis is correlated with disassembly of 26S proteasome holoenzymes into their 20S core particle (CP) and 19S regulatory particle (RP) components. Proteasomes are reassembled, and proteolysis resumes prior to cell cycle reentry. Free 20S CPs are found in an autoinhibited state in which the N-terminal tails from neighboring alpha subunits are anchored by an intricate lattice of interactions blocking the channel that leads into the 20S CPs. By deleting channel gating residues of CP alpha subunits, we generated an "open channel" proteasome that exhibits faster rates of protein degradation both in vivo and in vitro, indicating that gating contributes to regulation of proteasome activity. This open channel mutant is delayed in outgrowth from SP and cannot survive following prolonged starvation. In summary, we have found that the ubiquitin-proteasome pathway can be subjected to global downregulation, that the proteasome is a target of this regulation, and that proteasome downregulation is linked to survival of SP cells. Maintaining high viability during SP is essential for evolutionary fitness, which may explain the extreme conservation of channel gating residues in eukaryotic proteasomes.

Caseins↗
PubMed2003

The COP9 signalosome-like complex in S. cerevisiae and links to other PCI complexes.

The COP9 signalosome (CSN), the lid subcomplex of the proteasome and translational initiation factor 3 (eIF3) share structural similarities and are often referred to as the PCI family of complexes. In multicellular eukaryotes, the CSN is highly conserved as an 8-subunit complex but in Saccharomyces cerevisiae the complex is rather divergent. We further characterize the composition and properties of the CSN in budding yeast and its interactions with these related complexes. Using the generalized profile method we identified CSN candidates, four with PCI domains: Csn9, Csn10, Pci8/Csn11, and Csn12, and one with an MPN domain, Csn5/Rri1. These proteins and an additional interactor, Csi1, were tested for pairwise interactions by yeast two-hybrid and were found to form a cluster surrounding Csn12. Csn5 and Csn12 cofractionate in a complexed form with an apparent molecular weight of roughly 250kDa. However, Csn5 migrates as a monomer in Deltacsn12 supporting the pivotal role of Csn12 in stabilizing the complex. Confocal fluorescence microscopy detects GFP-tagged Csn5 preferentially in the nucleus, whereas in absence of Csn12, Csn10, Pci8/Csn11, or Csi1, Csn5 is delocalized throughout the cell, indicating that multiple subunits are required for nuclear localization of Csn5. Two CSN subunits, Csn9 and Csi1, interact with the proteasome lid subunit Rpn5. Pci8/Csn11 has previously been shown to interact with eIF3. Together, these results point to a network of interactions between these three structurally similar, yet functionally diverse, complexes.

Amino Acid Sequence↗
PubMed2002

COP9 signalosome components play a role in the mating pheromone response of S. cerevisiae.

A family of genetically and structurally homologous complexes, the proteasome lid, Cop9 signalosome (CSN) and eukaryotic translation initiation factor 3, mediate different regulatory pathways. The CSN functions in numerous eukaryotes as a regulator of development and signaling, yet until now no evidence for a complex has been found in Saccharomyces cerevisiae. We identified a group of proteins, including a homolog of Csn5/Jab1 and four uncharacterized PCI components, that interact in a manner suggesting they form a complex analogous to the CSN in S. cerevisiae. These newly identified subunits play a role in adaptation to pheromone signaling. Deletants for individual subunits enhance pheromone response and increase mating efficiency. Overexpression of individual subunits or a human homolog mitigates sst2-induced pheromone sensitivity. Csi1, a novel CSN interactor, exhibits opposite phenotypes. Deletants also accumulate Cdc53/cullin in a Rub1-modified form; however, this role of the CSN appears to be distinct from that in the mating pathway.

COP9 Signalosome Complex↗
PubMed2002

MPN+, a putative catalytic motif found in a subset of MPN domain proteins from eukaryotes and prokaryotes, is critical for Rpn11 function.

BACKGROUND: Three macromolecular assemblages, the lid complex of the proteasome, the COP9-Signalosome (CSN) and the eIF3 complex, all consist of multiple proteins harboring MPN and PCI domains. Up to now, no specific function for any of these proteins has been defined, nor has the importance of these motifs been elucidated. In particular Rpn11, a lid subunit, serves as the paradigm for MPN-containing proteins as it is highly conserved and important for proteasome function. RESULTS: We have identified a sequence motif, termed the MPN+ motif, which is highly conserved in a subset of MPN domain proteins such as Rpn11 and Csn5/Jab1, but is not present outside of this subfamily. The MPN+ motif consists of five polar residues that resemble the active site residues of hydrolytic enzyme classes, particularly that of metalloproteases. By using site-directed mutagenesis, we show that the MPN+ residues are important for the function of Rpn11, while a highly conserved Cys residue outside of the MPN+ motif is not essential. Single amino acid substitutions in MPN+ residues all show similar phenotypes, including slow growth, sensitivity to temperature and amino acid analogs, and general proteasome-dependent proteolysis defects. CONCLUSIONS: The MPN+ motif is abundant in certain MPN-domain proteins, including newly identified proteins of eukaryotes, bacteria and archaea thought to act outside of the traditional large PCI/MPN complexes. The putative catalytic nature of the MPN+ motif makes it a good candidate for a pivotal enzymatic function, possibly a proteasome-associated deubiquitinating activity and a CSN-associated Nedd8/Rub1-removing activity.

Amino Acid Sequence↗
PubMed2002

The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction.

Between the 1960s and 1980s, most life scientists focused their attention on studies of nucleic acids and the translation of the coded information. Protein degradation was a neglected area, considered to be a nonspecific, dead-end process. Although it was known that proteins do turn over, the large extent and high specificity of the process, whereby distinct proteins have half-lives that range from a few minutes to several days, was not appreciated. The discovery of the lysosome by Christian de Duve did not significantly change this view, because it became clear that this organelle is involved mostly in the degradation of extracellular proteins, and their proteases cannot be substrate specific. The discovery of the complex cascade of the ubiquitin pathway revolutionized the field. It is clear now that degradation of cellular proteins is a highly complex, temporally controlled, and tightly regulated process that plays major roles in a variety of basic pathways during cell life and death as well as in health and disease. With the multitude of substrates targeted and the myriad processes involved, it is not surprising that aberrations in the pathway are implicated in the pathogenesis of many diseases, certain malignancies, and neurodegeneration among them. Degradation of a protein via the ubiquitin/proteasome pathway involves two successive steps: 1) conjugation of multiple ubiquitin moieties to the substrate and 2) degradation of the tagged protein by the downstream 26S proteasome complex. Despite intensive research, the unknown still exceeds what we currently know on intracellular protein degradation, and major key questions have remained unsolved. Among these are the modes of specific and timed recognition for the degradation of the many substrates and the mechanisms that underlie aberrations in the system that lead to pathogenesis of diseases.

Animals↗