Proteolytic Machine Research Articles

Mechanisms of protein aggregation in the retinal pigment epithelial cells

The pathogenesis of age-related macular degeneration (AMD) essentially involves chronic oxidative stress, increased accumulation of lipofuscin in retinal pigment epithelial (RPE) cells and extracellular drusen formation, as well as the presence of chronic inflammation. The capacity to prevent the accumulation of cellular cytotoxic protein aggregates is decreased in senescent cells which may evoke lipofuscin accumulation into lysosomes in postmitotic RPE cells. This presence of lipofuscin decreases lysosomal enzyme activity and impairs autophagic clearance of damaged proteins which should be removed from cells. Proteasomes are another crucial proteolytic machine which degrade especially cellular proteins damaged by oxidative stress. This review examines the cross-talk between lysosomes, autophagy and proteasomes in RPE cell protein aggregation, their role as a possible therapeutic target and their involvement in the pathogenesis of AMD.

Degrons in protein substrates program the speed and operating efficiency of the AAA+ Lon proteolytic machine

AAA+ proteases are ATP-fueled machines that bind protein substrates via a degradation tag, unfold the molecule if necessary, and then translocate the polypeptide into a chamber for proteolysis. Tag recognition is normally viewed as a passive reaction. By contrast, for the AAA+ Lon protease, we show that degron tags are also regulatory elements that determine protease activity levels. Indeed, different tags fused to the same protein change degradation speeds and energetic efficiencies by 10-fold or more. Degron binding to multiple sites in the Lon hexamer appears to differentially stabilize specific enzyme conformations, including one with high protease and low ATPase activity, and results in positively cooperative degradation. These allosteric mechanisms allow Lon to operate in either a fast or slow proteolysis mode, according to specific physiological needs, and may help maximize degradation of misfolded proteins following stress-induced denaturation.

Autocatalytic Processing of m-AAA Protease Subunits in Mitochondria

m-AAA proteases are ATP-dependent proteolytic machines in the inner membrane of mitochondria which are crucial for the maintenance of mitochondrial activities. Conserved nuclear-encoded subunits, termed paraplegin, Afg3l1, and Afg3l2, form various isoenzymes differing in their subunit composition in mammalian mitochondria. Mutations in different m-AAA protease subunits are associated with distinct neuronal disorders in human. However, the biogenesis of m-AAA protease complexes or of individual subunits is only poorly understood. Here, we have examined the processing of nuclear-encoded m-AAA protease subunits upon import into mitochondria and demonstrate autocatalytic processing of Afg3l1 and Afg3l2. The mitochondrial processing peptidase MPP generates an intermediate form of Afg3l2 that is matured autocatalytically. Afg3l1 or Afg3l2 are also required for maturation of newly imported paraplegin subunits after their cleavage by MPP. Our results establish that mammalian m-AAA proteases can act as processing enzymes in vivo and reveal overlapping activities of Afg3l1 and Afg3l2. These findings might be of relevance for the pathogenesis of neurodegenerative disorders associated with mutations in different m-AAA protease subunits.

Adapting the machine: adaptor proteins for Hsp100/Clp and AAA+ proteases

Members of the AAA+ protein superfamily contribute to many diverse aspects of protein homeostasis in prokaryotic cells. As a fundamental component of numerous proteolytic machines in bacteria, AAA+ proteins play a crucial part not only in general protein quality control but also in the regulation of developmental programmes, through the controlled turnover of key proteins such as transcription factors. To manage these many, varied tasks, Hsp100/Clp and AAA+ proteases use specific adaptor proteins to enhance or expand the substrate recognition abilities of their cognate protease. Here, we review our current knowledge of the modulation of bacterial AAA+ proteases by these cellular arbitrators.

Thioredoxin Txnl1/TRP32 Is a Redox-active Cofactor of the 26 S Proteasome

The 26 S proteasome is a large proteolytic machine, which degrades most intracellular proteins. We found that thioredoxin, Txnl1/TRP32, binds to Rpn11, a subunit of the regulatory complex of the human 26 S proteasome. Txnl1 is abundant, metabolically stable, and widely expressed and is present in the cytoplasm and nucleus. Txnl1 has thioredoxin activity with a redox potential of about -250 mV. Mutant Txnl1 with one active site cysteine replaced by serine formed disulfide bonds to eEF1A1, a substrate-recruiting factor of the 26 S proteasome. eEF1A1 is therefore a likely physiological substrate. In response to knockdown of Txnl1, ubiquitin-protein conjugates were moderately stabilized. Hence, Txnl1 is the first example of a direct connection between protein reduction and proteolysis, two major intracellular protein quality control mechanisms.

The N-Terminal Penultimate Residue of 20S Proteasome α1 Influences its N α Acetylation and Protein Levels as Well as Growth Rate and Stress Responses of Haloferax volcanii

Proteasomes are energy-dependent proteolytic machines. We elaborate here on the previously observed N(alpha) acetylation of the initiator methionine of the alpha1 protein of 20S core particles (CPs) of Haloferax volcanii proteasomes. Quantitative mass spectrometry revealed this was the dominant N-terminal form of alpha1 in H. volcanii cells. To further examine this, alpha1 proteins with substitutions in the N-terminal penultimate residue as well as deletion of the CP "gate" formed by the alpha1 N terminus were examined for their N(alpha) acetylation. Both the "gate" deletion and Q2A substitution completely altered the N(alpha)-acetylation pattern of alpha1, with the deletion rendering alpha1 unavailable for N(alpha) acetylation and the Q2A modification apparently enhancing cleavage of alpha1 by methionine aminopeptidase (MAP), resulting in acetylation of the N-terminal alanine. Cells expressing these two alpha1 variants were less tolerant of hypoosmotic stress than the wild type and produced CPs with enhanced peptidase activity. Although alpha1 proteins with Q2D, Q2P, and Q2T substitutions were N(alpha) acetylated in CPs similar to the wild type, cells expressing these variants accumulated unusually high levels of alpha1 as rings in N(alpha)-acetylated, unmodified, and/or MAP-cleaved forms. More detailed examination of this group revealed that while CP peptidase activity was not impaired, cells expressing these alpha1 variants displayed higher growth rates and were more tolerant of hypoosmotic and high-temperature stress than the wild type. Overall, these results suggest that N(alpha) acetylation of alpha1 is important in CP assembly and activity, high levels of alpha1 rings enhance cell proliferation and stress tolerance, and unregulated opening of the CP "gate" impairs the ability of cells to overcome salt stress.

Activation mechanisms of coagulation factor IX

Blood haemostasis is accomplished by a complex network of coagulatory and fibrinolytic processes. These processes have to be delicately balanced, as clinically manifested by bleeding disorders, such as haemophilia A and B. These disorders are caused by defects in coagulation factor VIII and factor IX, respectively. Following a dual strategy, we emphasise on the one hand principles conserved in most coagulation enzymes, thus mirroring much of the underlying complexity in haemostasis; on the other hand, we identify enzymatic properties of the factor IXa-factor VIIIa system (Xase) that distinguish this proteolytic machine from other components of the coagulation system. While the exact mechanisms of its activity modulation remain baffling until today, superactive factor IX mutants significantly improve our current understanding and serve as a specific and testable model of Xase action.

A degradation signal recognition in prokaryotes

The degradation of ssrA-tagged substrates in prokaryotes is conducted by a subset of ATP-dependent proteases, including ClpXP complex. More than 630 sequences of ssrA have been identified from 514 species, and are conserved in a wide range of prokaryotes. SspB protein markedly stimulates the degradation of these ssrA-tagged substrates by the ClpXP proteolytic machine. The dimeric SspB protein is composed of a compact ssrA-binding domain, which has a dimerization surface and a flexible C-terminal tail with a ClpX-binding motif at its very end. Since SspB is an adaptor protein for the ClpXP complex, designed mutagenesis, fluorescence spectroscopy, biochemistry and X-ray crystallography have been used to investigate the mechanism of delivery of ssrA-tagged proteins. In this paper the structural basis of ssrA-tag recognition by ClpX and SspB, as well as SspB-tail recognition by ZBD, is described.

Resolving Individual Steps in the Operation of ATP-Dependent Proteolytic Molecular Machines: From Conformational Changes to Substrate Translocation and Processivity

Clp, Lon, and FtsH proteases are proteolytic molecular machines that use the free energy of ATP hydrolysis to unfold protein substrates and processively present them to protease active sites. Here we review recent biochemical and structural studies relevant to the mechanism of ATP-dependent processive proteolysis. Despite the significant structural differences among the Clp, Lon, and FtsH proteases, these enzymes share important mechanistic features. In these systems, mechanistic studies have provided evidence for ATP binding and hydrolysis-driven conformational changes that drive translocation of substrates, which has significant implications for the processive mechanism of proteolysis. These studies indicate that the nucleotide (ATP, ADP, or nonhydrolyzable ATP analogues) occupancy of the ATPase binding sites can influence the binding mode and/or binding affinity for protein substrates. A general mechanism is proposed in which the communication between ATPase active sites and protein substrate binding regions coordinates a processive cycle of substrate binding, translocation, proteolysis, and product release.

MT4-(MMP17) and MT6-MMP (MMP25), A unique set of membrane-anchored matrix metalloproteinases: properties and expression in cancer

The process of cancer progression involves the action of multiple proteolytic systems, among which the family of matrix metalloproteinases (MMPs) play a pivotal role. The MMPs evolved to accomplish their proteolytic tasks in multiple cellular and tissue microenvironments including lipid rafts by incorporation and deletions of specific structural domains. The membrane type-MMPs (MT-MMPs) incorporated membrane anchoring domains that display these proteases at the cell surface, and thus they are optimal pericellular proteolytic machines. Two members of the MT-MMP subfamily, MMP-17 (MT4-MMP) and MMP-25 (MT6-MMP), are anchored to the plasma membrane via a glycosyl-phosphatidyl inositol (GPI) anchor, which confers these enzymes a unique set of regulatory and functional mechanisms that separates them from the rest of the MMP family. Discovered almost a decade ago, the body of work on GPI-MT-MMPs today is still surprisingly limited when compared to other MT-MMPs. However, new evidence shows that the GPI-MT-MMPs are highly expressed in human cancer, where they are associated with progression. Accumulating biochemical and functional evidence also highlights their distinct properties. In this review, we summarize the structural, biochemical, and biological properties of GPI-MT-MMPs and present an overview of their expression and role in cancer. We further discuss the potential implications of GPI-anchoring for enzyme function. Finally, we comment on the new scientific challenges that lie ahead to better understand the function and role in cancer of these intriguing but yet unique MMPs.

Conserved residues in the N‐domain of the AAA+ chaperone ClpA regulate substrate recognition and unfolding

Protein degradation in the cytosol of Escherichia coli is carried out by a variety of different proteolytic machines, including ClpAP. The ClpA component is a hexameric AAA+ (ATPase associated with various cellular activities) chaperone that utilizes the energy of ATP to control substrate recognition and unfolding. The precise role of the N-domains of ClpA in this process, however, remains elusive. Here, we have analysed the role of five highly conserved basic residues in the N-domain of ClpA by monitoring the binding, unfolding and degradation of several different substrates, including short unstructured peptides, tagged and untagged proteins. Interestingly, mutation of three of these basic residues within the N-domain of ClpA (H94, R86 and R100) did not alter substrate degradation. In contrast mutation of two conserved arginine residues (R90 and R131), flanking a putative peptide-binding groove within the N-domain of ClpA, specifically compromised the ability of ClpA to unfold and degrade selected substrates but did not prevent substrate recognition, ClpS-mediated substrate delivery or ClpP binding. In contrast, a highly conserved tyrosine residue lining the central pore of the ClpA hexamer was essential for the degradation of all substrate types analysed, including both folded and unstructured proteins. Taken together, these data suggest that ClpA utilizes two structural elements, one in the N-domain and the other in the pore of the hexamer, both of which are required for efficient unfolding of some protein substrates.

Distinct Static and Dynamic Interactions Control ATPase-Peptidase Communication in a AAA+ Protease

In the ClpXP proteolytic machine, ClpX uses the energy of ATP hydrolysis to unfold protein substrates and translocate them through a central pore and into the degradation chamber of ClpP. Here, we demonstrate a bipartite system of ClpX-ClpP interactions that serves multiple functional roles. High-affinity contacts between six loops near the periphery of the hexameric ClpX ring and a ClpP ring establish correct positioning and increase degradation activity but are insensitive to nucleotide state. These static peripheral interactions maintain a stable ClpXP complex, while other parts of this machine change conformation hundreds of times per minute. By contrast, relatively weak axial contacts between loops at the bottom of the ClpX central channel and N-terminal loops of ClpP vary dynamically with the nucleotide state of individual ClpX subunits, control ATP-hydrolysis rates, and facilitate efficient protein unfolding. Thus, discrete static and dynamic interactions mediate binding and communication between ClpX and ClpP.

Roles for the Human ATP-dependent Lon Protease in Mitochondrial DNA Maintenance

Human mitochondrial Lon is an ATP-powered proteolytic machine that specifically binds to single-stranded G-rich DNA and RNA in vitro. However, it is unknown whether Lon binds mitochondrial DNA (mtDNA) in living cells or functions in mtDNA integrity. Here, we demonstrate that Lon interacts with the mitochondrial genome in cultured cells using mtDNA immunoprecipitation (mIP). Lon associates with sites distributed primarily within one-half of the genome and preferentially with the control region for mtDNA replication and transcription. Bioinformatic analysis of mIP data revealed a G-rich consensus sequence. Consistent with these findings, in vitro experiments showed that the affinity of Lon for single-stranded DNA oligonucleotides correlates with conformity to this consensus. To examine the role of Lon in mtDNA maintenance, cells carrying an inducible short hairpin RNA for Lon depletion were used. In control and Lon-depleted cells, mtDNA copy number was essentially the same in the presence or absence of oxidative stress. However when oxidatively stressed, control cells exhibited an increased frequency of mtDNA lesions, whereas Lon-depleted cells showed little if any mtDNA damage. This suggests that oxidative mtDNA damage is permitted when Lon is present and prevented when Lon is substantially depleted. Upon oxidative stress, mIP showed reduced Lon binding to mtDNA; however binding to the control region was unaffected. It is unlikely that oxidative modification of Lon blocks its ability to bind DNA in vivo as results show that oxidized purified Lon retains sequence-specific DNA binding. Taken together, these results demonstrate that mtDNA binding is a physiological function of Lon and that cellular levels of Lon influence sensitivity to mtDNA damage. These findings suggest roles for Lon in linking protein and mtDNA quality control.

Localization of proteasomes and proteasomal proteolysis in the mammalian interphase cell nucleus by systematic application of immunocytochemistry

Proteasomes are ATP-driven, multisubunit proteolytic machines that degrade endogenous proteins into peptides and play a crucial role in cellular events such as the cell cycle, signal transduction, maintenance of proper protein folding and gene expression. Recent evidence indicates that the ubiquitin-proteasome system is an active component of the cell nucleus. A characteristic feature of the nucleus is its organization into distinct domains that have a unique composition of macromolecules and dynamically form as a response to the requirements of nuclear function. Here, we show by systematic application of different immunocytochemical procedures and comparison with signature proteins of nuclear domains that during interphase endogenous proteasomes are localized diffusely throughout the nucleoplasm, in speckles, in nuclear bodies, and in nucleoplasmic foci. Proteasomes do not occur in the nuclear envelope region or the nucleolus, unless nucleoplasmic invaginations expand into this nuclear body. Confirmedly, proteasomal proteolysis is detected in nucleoplasmic foci, but is absent from the nuclear envelope or nucleolus. The results underpin the idea that the ubiquitin-proteasome system is not only located, but also proteolytically active in distinct nuclear domains and thus may be directly involved in gene expression, and nuclear quality control.

Protein Degradation within Mitochondria: Versatile Activities of AAA Proteases and Other Peptidases

ABSTRACTCell survival depends on essential processes in mitochondria. Various proteases within these organelles regulate mitochondrial biogenesis and ensure the complete degradation of excess or damaged proteins. Many of these proteases are highly conserved and ubiquitous in eukaryotic cells. They can be assigned to three functional classes: processing peptidases, which cleave off mitochondrial targeting sequences of nuclearly encoded proteins and process mitochondrial proteins with regulatory functions; ATP-dependent proteases, which either act as processing peptidases with regulatory functions or as quality-control enzymes degrading non-native polypeptides to peptides; and oligopeptidases, which degrade these peptides and mitochondrial targeting sequences to amino acids. Disturbances of protein degradation within mitochondria cause severe phenotypes in various organisms and can lead to the induction of apoptotic programmes and cell-specific neurodegeneration in mammals. After an overview of the proteolytic system of mitochondria, we will focus on versatile functions of ATP-dependent AAA proteases in the inner membrane. These conserved proteolytic machines conduct protein quality surveillance of mitochondrial inner membrane proteins, mediate vectorial protein dislocation from membranes, and, acting as processing enzymes, control ribosome assembly, mitochondrial protein synthesis, and mitochondrial fusion. Implications of these functions for cell-specific axonal degeneration in hereditary spastic paraplegia will be discussed.

Hsp70 Molecular Chaperones: Emerging Roles in Human Disease and Identification of Small Molecule Modulators

Molecular chaperones are best known for their ability to aid in the solubilization of mis-folded proteins, and as a result play essential roles in protein folding, degradation, and transport. However, many molecular chaperones also play essential roles in signal transduction cascades. For example, Hsp70 molecular chaperones are a highly conserved, abundant class of chaperones that are found in every species and in nearly every cellular compartment in eukaryotes. In addition to their well-established roles in facilitating protein folding and in the targeting of proteins to organelles and to proteolytic machines, Hsp70s are anti-apoptotic and inhibition of Hsp70 function in some cases is sufficient to induce tumor cell death. Hsp70 function is also vital for the replication of viruses. Based on these data, small molecule Hsp70 modulators might, in principle, be used for the treatment of specific cancers, infections, and protein conformational diseases. In this review, we summarize the structural and functional characteristics of Hsp70 chaperones, and then discuss their roles in cellular physiology. Finally, we will review the recent discovery of small molecules that alter Hsp70 expression and function.

The 1.9 Å Structure of a Proteasome-11S Activator Complex and Implications for Proteasome-PAN/PA700 Interactions

Proteasomes are cylindrical structures that function in multiple cellular processes by degrading a wide variety of cytosolic and nuclear proteins. Substrate access and product release from the enclosed catalytic chamber occurs through axial pores that are opened by activator complexes. Here, we report high-resolution structures of wild-type and mutant archaeal proteasomes bound to the activator PA26. These structures support the proposal that an ordered open conformation is required for proteolysis and that its formation can be triggered by outward displacement of surrounding residues. The structures and associated biochemical assays reveal the mechanism of binding, which involves an interaction between the PA26 C terminus and a conserved lysine. Surprisingly, biochemical observations implicate an equivalent interaction for the unrelated ATP-dependent activators PAN and PA700.

Nuclear Waste Disposal

CELL BIOLOGY Quality-control systems within the cytosol are important for the overall health of the cell; aberrant proteins (incorrectly assembled or damaged during use) may not function properly, and the cell has mechanisms for disposing of such waste (and recycling the components) if they cannot be repaired. Gardner et al. find that a similar quality-control system operates within the cell nucleus of the yeast Saccharomyces cerevisiae. Mutant nuclear proteins are targeted for destruction by the proteasome: a proteolytic machine of the cytosol. A set of nuclear quality-control proteins—San1, which is a ubiquitin-protein ligase, and two ubiquitin-conjugating enzymes, Cdc34 and Ubc1—act together to tag mutant proteins with ubiquitin, the molecular label for proteasomal degradation. San1 possesses a nuclear localization signal that is required for its function, and cells lacking San1 suffer from chronic stress, presumably due to the accumulation of aberrant proteins within the nucleus. Thus, the eukaryotic cell has surveillance and quality-control strategies to protect each of its compartments from the harmful consequences of dysfunctional proteins. — SMH Cell 120 , 803 (2005).

Proteasome-associated proteins: regulation of a proteolytic machine

The proteasome is a compartmentalized, ATP-dependent protease composed of more than 30 subunits that recognizes and degrades polyubiquitinated substrates. Despite its physiological importance, many aspects of the proteasome's structural organization and regulation remain poorly understood. In addition to the proteins that form the proteasome holocomplex, there is increasing evidence that proteasomal function is affected by a wide variety of associating proteins. A group of ubiquitin-binding proteins assist in delivery of substrates to the proteasome, whereas proteasome-associated ubiquitin ligases and deubiquitinating enzymes may alter the dynamics of ubiquitin chains already associated with the proteasome. Some proteins appear to influence the overall stability of the complex, and still others have the capacity to activate or inhibit the hydrolytic activity of the core particle. The increasing number of interacting proteins identified suggests that proteasomes, as they exist in the cell, are larger and more diverse in composition than previously assumed. Thus, the study of proteasome-associated proteins will lead to new perspectives on the dynamics of this uniquely complex proteolytic machine.

Protein degradation and protection against misfolded or damaged proteins.

The ultimate mechanism that cells use to ensure the quality of intracellular proteins is the selective destruction of misfolded or damaged polypeptides. In eukaryotic cells, the large ATP-dependent proteolytic machine, the 26S proteasome, prevents the accumulation of non-functional, potentially toxic proteins. This process is of particular importance in protecting cells against harsh conditions (for example, heat shock or oxidative stress) and in a variety of diseases (for example, cystic fibrosis and the major neurodegenerative diseases). A full understanding of the pathogenesis of the protein-folding diseases will require greater knowledge of how misfolded proteins are recognized and selectively degraded.