The ATP-dependent proteases and proteolytic complexes involved into intracellular protein degradation

The review characterizes the main enzymatic systems of selective proteolysis responsible for maintenance of intracellular proteome in prokaryotes, eukaryotes and archaea. The features of proteolytic components of the ATP-dependent proteases as well as similarity and diversity of their regulatory components belonging to AAA⁺ ATPases are discussed.     

Allophanate hydrolase of Oleomonas sagaranensis involved in an ATP-dependent degradation pathway specific to urea

The first prokaryotic urea carboxylase has previously been purified and characterized from Oleomonas sagaranensis. As the results indicated the presence of an ATP-dependent urea degradation pathway in Bacteria, the characterization of the second component of this pathway, allophanate hydrolase, was carried out. The gene encoding allophanate hydrolase was found adjacent to the urea carboxylase gene. The purified, recombinant enzyme exhibited ammonia-generating activity towards allophanate, and, together with urea carboxylase, efficiently produced ammonia from urea in an ATP-dependent manner. The substrate specificity of the enzyme was strict, and analogs of allophanate were not hydrolyzed. Moreover, although the urea carboxylase exhibited carboxylase activity towards urea, acetamide, and formamide, ammonia-releasing activity of the two enzymes combined was detected only towards urea, indicating that the pathway was specific for urea degradation.     

ATP-dependent steps in the binding of ubiquitin conjugates to the 26S proteasome that commit to degradation

Eukaryotic cells target proteins for degradation by the 26S proteasome by attaching a ubiquitin chain. Using a rapid assay, we analyzed the initial binding of ubiquitinated proteins to purified 26S particles as an isolated process at 4°C. Subunits Rpn10 and Rpn13 contribute equally to the high-affinity binding of ubiquitin chains, but in their absence, ubiquitin conjugates bind to another site with 4-fold lower affinity. Conjugate binding is stimulated 2- to 4-fold by binding of ATP or the nonhydrolyzable analog, ATPγS (but not ADP), to the 19S ATPases. Following this initial, reversible association, ubiquitin conjugates at 37°C become more tightly bound through a step that requires ATP hydrolysis and a loosely folded domain on the protein, but appears independent of ubiquitin. Unfolded or loosely folded polypeptides can inhibit this tighter binding. This commitment step precedes substrate deubiquitination and allows for selection of ubiquitinated proteins capable of being unfolded and efficiently degraded.     

Ubiquitin-independent proteasomal degradation driven by C-degron pathways

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Ubiquitin-independent proteasomal degradation during oncogenic viral infections

Most eukaryotic proteins destined for imminent destruction are first tagged with a chain of ubiquitin molecules and are subsequently dismantled by the proteasome. Ubiquitin-independent degradation of substrates by the proteasome, however, also occurs. The number of documented proteasome-dependent, ubiquitin-independent degradation events remains relatively small but continues to grow. Proteins involved in oncogenesis and tumor suppression make up the majority of the known cases for this type of protein destruction. Provocatively, viruses with confirmed or suspected oncogenic properties are also prominent participants in the pantheon of ubiquitin-independent proteasomal degradation events. In this review, we identify and describe examples of proteasome-dependent, ubiquitin-independent protein degradation that occur during tumor virus infections, speculate why this type of protein destruction may be preferred during oncogenesis, and argue that this uncommon type of protein turnover represents a prime target for antiviral and anticancer therapeutics.     

Pathway choice between proteasomal and autophagic degradation

Efficient degradation of abnormal or aggregated proteins is crucial to protect the cell against proteotoxic stress. Selective targeting and disposal of such proteins usually occurs in a ubiquitin-dependent manner by proteasomes and macroautophagy/autophagy. Whereas proteasomes are efficient in degrading abnormal soluble proteins, protein aggregates are typically targeted for degradation by autophagic vesicles. Both processes require ubiquitin-binding receptors, which are targeted to proteasomes via ubiquitin-like domains or to phagophores (the precursors to autophagosomes) via Atg8/LC3 binding motifs, respectively. The use of substrate modification by ubiquitin in both pathways raised the question of how degradative pathway choice is achieved. In contrast to previous models, proposing different types of ubiquitin linkages for substrate targeting, we find that pathway choice is a late event largely determined by the oligomeric state of the receptors. Monomeric proteasome receptors bind soluble substrates more efficiently due to their higher affinity for ubiquitin. Upon substrate aggregation, autophagy receptors with lower ubiquitin binding affinity gain the upper hand due to higher avidity achieved by receptor bundling. Thus, our work suggests that ubiquitination is a shared signal of an adaptive protein quality control system, which targets substrates for the optimal proteolytic pathway.     

Pupylation as a signal for proteasomal degradation in bacteria

Posttranslational modifications in the form of covalently attached proteins like ubiquitin (Ub), were long considered an exclusive feature of eukaryotic organisms. The discovery of pupylation, the modification of lysine residues with a prokaryotic, ubiquitin-like protein (Pup), demonstrated that certain bacteria use a tagging pathway functionally related to ubiquitination in order to target proteins for proteasomal degradation. However, functional analogies do not translate into structural or mechanistic relatedness. Bacterial Pup, unlike eukaryotic Ub, does not adopt a β-grasp fold, but is intrinsically disordered. Furthermore, isopeptide bond formation in the pupylation process is carried out by enzymes evolutionary descendent from glutamine synthetases. While in eukaryotes, the proteasome is the main energy-dependent protein degradation machine, bacterial proteasomes exist in addition to other architecturally related degradation complexes, and their specific role along with the role of pupylation is still poorly understood. In Mycobacterium tuberculosis (Mtb), the Pup–proteasome system contributes to pathogenicity by supporting the bacterium's persistence within host macrophages. Here, we describe the mechanism and structural framework of pupylation and the targeting of pupylated proteins to the proteasome complex. Particular attention is given to the comparison of the bacterial Pup–proteasome system and the eukaryotic ubiquitin–proteasome system. Furthermore, the involvement of pupylation and proteasomal degradation in Mtb pathogenesis is discussed together with efforts to establish the Pup–proteasome system as a drug target. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

Antizyme, a mediator of ubiquitin-independent proteasomal degradation

Ornithine decarboxylase (ODC) is among the small set of proteasome substrates that is not ubiquitinated. It is instead degraded in conjunction with the protein antizyme (AZ). ODC and AZ are participants in a regulatory circuit that restricts pools of polyamines, the downstream products of ODC enzymatic activity. Functional studies using directed mutagenesis have identified regions of ODC and AZ required for the process of ODC degradation. Within ODC, there is a region that is required for AZ binding which lies on the surface of an alpha-beta barrel forming one domain of the ODC monomer. A carboxy-terminal ODC domain is needed for both AZ-dependent and AZ-independent degradation. Within AZ, the carboxy-terminal half molecule is sufficient for binding to ODC, but an additional domain found within the AZ amino terminus must be present for stimulation of ODC degradation by the proteasome. Recently, the AZs have been found to consist of an ancient gene family. Within vertebrate species, multiple isoforms are found, with distinct functions that remain to be sorted out. Although AZ homologs have been found in some yeast species, homology searches have failed to identify an AZ homolog in Saccharomyces cerevisiae. Nevertheless, the close parallel between polyamine-induced ODC degradation in S. cerevisiae and in animal cells suggests that this organism will also be found to harbor an AZ-like protein.     

Accelerated proteasomal degradation of membrane Ig heavy chains

Membrane IgG H chains turn over considerably more rapidly than secretory Ig H chains in the 18-81 A2 pre-B cell line. This rapid degradation occurs in proteasomes. N-Glycosylated membrane Ig H chains accumulate in the endoplasmic reticulum in the presence of proteasomal inhibitors, suggesting that retrotranslocation and proteasomal degradation of membrane Ig H chains may be closely coupled processes. Accelerated proteasomal degradation of membrane Ig H chains was also observed in transfected nonlymphoid cells. At steady state, the membrane form of the H chain associates more readily with Bip and calnexin than its secretory counterpart. The preferential recognition of membrane, as opposed to secretory, Ig H chains by some endoplasmic reticulum chaperones, may provide an explanation for the accelerated proteasomal degradation of the former.     

Ubiquitin proteasomal pathway mediated degradation of p53 in melanoma

Ubiquitin proteasomal pathway (UPP) is the principle mechanism for protein catabolism and affects cellular processes critical for survival and proliferation. Levels of tumor suppressor protein p53 are very low in cells due to its rapid turnover by UPP-mediated degradation. While p53 is mutated in human cancers, most human melanomas maintain wild-type conformation. In this study, to investigate the effects of UPP inhibitor invitro and in vivo, we used a genetically-engineered mouse model (GEMM) that has the same genetic alterations as those of human melanomas. Melanoma cells were established from mouse tumors and named 8B20 cells. Treatment of 8B20 cells with the UPP inhibitors, MG132 and clasto-lactacystin-β-lactone, led to an increase in levels of p53 while treatment with non-proteasomal inhibitors did not alter p53 levels. UPP inhibitors induced formation of heavy molecular weight ubiquitinated proteins, a hallmark of UPP inhibition, and p53-specific poly-ubiquitinated products in 8B20 cells. To further decipher the mechanism of p53 stabilization, we investigated half-life of p53 in cells treated with cycloheximide to block de novo protein synthesis. Treatment of 8B20 cells with MG132 led to an increase in the half-life of p53. Further analysis revealed that p53 stabilization was not mediated by phosphorylation of Ser-15 and Ser-20 residues. In vivo studies showed that MG132 induced p53 overexpression and reduced tumor growth, suggesting an important role of p53 stabilization in controlling melanoma. Taken together, our studies provide a proof of principle for using a GEMM to address the mechanisms of action and efficacy of melanoma treatment.     

Ferritin oxidation and proteasomal degradation: protection by antioxidants

The accumulation of oxidatively damaged proteins is a well-known hallmark of aging and several neurodegenerative diseases including Alzheimer's, Parkinson's and Huntigton's diseases. These highly oxidized protein aggregates are in general not degradable by the main intracellular proteolytic machinery, the proteasomal system. One possible strategy to reduce the accumulation of such oxidized protein aggregates is the prevention of the formation of oxidized protein derivatives or to reduce the protein oxidation to a degree that can be handled by the proteasome. To do so an antioxidative strategy might be successful. Therefore, we undertook the present study to test whether antioxidants are able to prevent the protein oxidation and to influence the proteasomal degradation of moderate oxidized proteins. As a model protein we choose ferritin. H₂O₂ induced a concentration dependent increase of protein oxidation accompanied by an increased proteolytic susceptibility. This increase of proteolytic susceptibility is limited to moderate hydrogen peroxide concentrations, whereas higher concentrations are accompanied by protein aggregate formation.

Protective effects of the vitamin E derivative Trolox, the pyridoindole derivative Stobadine and of the standardized extracts of flavonoids from bark of Pinus Pinaster Pycnogenol^® and from leaves of Ginkgo biloba (EGb 761) were studied on moderate damaged ferritin.