A Conserved Unfoldase Activity for the p97 AAA-ATPase in Proteasomal Degradation

The multifunctional AAA-ATPase p97 is one of the most abundant and conserved proteins in eukaryotic cells. The p97/Npl4/Ufd1 complex dislocates proteins that fail the protein quality control in the endoplasmic reticulum to the cytosol where they are subject to degradation by the ubiquitin/proteasome system. Substrate dislocation depends on the unfoldase activity of p97. Interestingly, p97 is also involved in the degradation of specific soluble proteasome substrates but the exact mode of action of p97 in this process is unclear. Here, we show that both the central pore and ATPase activity of p97 are necessary for the degradation of cytosolic ubiquitin-fusion substrates. Addition of a flexible extended C-terminal peptide to the substrate relieves the requirement for p97. Deletion mapping reveals a conserved length dependency of 20 residues for the peptide, which allows p97-independent degradation to occur. Our results suggest that initiation of unfolding may be more complex than previously anticipated and that the 19S regulatory complex of the proteasome can require preprocessing of highly folded, ubiquitylated substrates by the p97^Ufd1/Npl4 complex. Our data provide an explanation for the observation that p97 is only essential for a subpopulation of soluble substrates and predict that a common characteristic of soluble p97-dependent substrates is the lack of an initiation site to facilitate unfolding by the 26S proteasome.     

The FBXL family of F-box proteins: variations on a theme

The ubiquitin–proteasome system (UPS) is responsible for the rapid targeting of proteins for degradation at 26S proteasomes and requires the orchestrated action of E1, E2 and E3 enzymes in a well-defined cascade. F-box proteins (FBPs) are substrate-recruiting subunits of Skp1-cullin1-FBP (SCF)-type E3 ubiquitin ligases that determine which proteins are ubiquitinated. To date, around 70 FBPs have been identified in humans and can be subdivided into distinct families, based on the protein-recruiting domains they possess. The FBXL subfamily is defined by the presence of multiple leucine-rich repeat (LRR) protein-binding domains. But how the 22 FBPs of the FBXL family achieve their individual specificities, despite having highly similar structural domains to recruit their substrates, is not clear. Here, we review and explore the FBXL family members in detail highlighting their structural and functional similarities and differences and how they engage their substrates through their LRRs to adopt unique interactomes.     

Ubiquitin‐like domains can target to the proteasome but proteolysis requires a disordered region

Ubiquitin and some of its homologues target proteins to the proteasome for degradation. Other ubiquitin‐like domains are involved in cellular processes unrelated to the proteasome, and proteins containing these domains remain stable in the cell. We find that the 10 yeast ubiquitin‐like domains tested bind to the proteasome, and that all 11 identified domains can target proteins for degradation. Their apparent proteasome affinities are not directly related to their stabilities or functions. That is, ubiquitin‐like domains in proteins not part of the ubiquitin proteasome system may bind the proteasome more tightly than domains in proteins that are bona fide components. We propose that proteins with ubiquitin‐like domains have properties other than proteasome binding that confer stability. We show that one of these properties is the absence of accessible disordered regions that allow the proteasome to initiate degradation. In support of this model, we find that Mdy2 is degraded in yeast when a disordered region in the protein becomes exposed and that the attachment of a disordered region to Ubp6 leads to its degradation.     

The shaping of invasive glioma phenotype by the ubiquitin–proteasome system

Abstract

Protein degradation is an indispensable process for cells which is often deregulated in various diseases, including malignant conditions. Depending on the specific cell type and functions of expressed proteins, this aberration may have different effects on the determination of malignant phenotypes. A discrete, inherent feature of malignant glioma is its profound invasive and migratory potential, regulated by the expression of signaling and effector proteins, many of which are also subjected to post-translational regulation by the ubiquitin–proteasome system (UPS). Here we provide an overview of this connection, focusing on important pro-invasive protein signals targeted by the UPS.     

Biochemical evidence for glucose-independent induction of HXT expression in Saccharomyces cerevisiae

The yeast glucose sensors Rgt2 and Snf3 generate a signal in response to glucose that leads to degradation of Mth1 and Std1, thereby relieving repression of Rgt1-repressed genes such as the glucose transporter genes (HXT). Mth1 and Std1 are degraded via the Yck1/2 kinase-SCF(Grr1)-26S proteasome pathway triggered by the glucose sensors. Here, we show that RGT2-1 promotes ubiquitination and subsequent degradation of Mth1 and Std1 regardless of the presence of glucose. Site-specific mutagenesis reveals that the conserved lysine residues of Mth1 and Std1 might serve as attachment sites for ubiquitin, and that the potential casein kinase (Yck1/2) sites of serine phosphorylation might control their ubiquitination. Finally, we show that active Snf1 protein kinase in high glucose prevents degradation of Mth1 and Std1.     

The role of ubiquitin linkages on α-synuclein induced-toxicity in a Drosophila model of Parkinson's disease

Parkinson's disease (PD) is a common movement disorder marked by the loss of dopaminergic (DA) neurons in the brain stem and the presence of intraneuronal inclusions designated as Lewy bodies (LB). The cause of neurodegeneration in PD is not clear, but it has been suggested that protein misfolding and aggregation contribute significantly to the development of the disease. Misfolded and aggregated proteins are cleared by ubiquitin proteasomal system (UPS) and autophagy lysosomal pathway (ALP). Recent studies suggested that different types of ubiquitin linkages can modulate these two pathways in the process of protein degradation. In this study, we found that co-expression of ubiquitin can rescue neurons from α-syn-induced neurotoxicity in a Drosophila model of PD. This neuroprotection is dependent on the formation of lysine 48 polyubiquitin linkage which is known to target protein degradation via the proteasome. Consistent with our results that we observed in vivo , we found that ubiquitin co-expression in the cell can facilitate cellular protein degradation by the proteasome in a lysine 48 polyubiquitin-dependent manner. Taken together, these results suggest that facilitation of proteasomal protein degradation can be a potential therapeutic approach for PD.     

UBL/UBA ubiquitin receptor proteins bind a common tetraubiquitin chain

The ubiquitin-proteasome pathway is essential throughout the life cycle of a cell. This system employs an astounding number of proteins to ubiquitylate and to deliver protein substrates to the proteasome for their degradation. At the heart of this process is the large and growing family of ubiquitin receptor proteins. Within this family is an intensely studied group that contains both ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains: Rad23, Ddi1 and Dsk2. Although UBL/UBA family members are reported to regulate the degradation of other proteins, their individual roles in ubiquitin-mediated protein degradation has proven difficult to resolve due to their overlapping functional roles and interaction with each other and other ubiquitin family members. Here, we use a combination of NMR spectroscopy and molecular biology to reveal that Rad23 and Ddi1 interact with each other by using UBL/UBA domain interactions in a manner that does not preclude their interaction with ubiquitin. We demonstrate that UBL/UBA proteins can bind a common tetraubiquitin molecule and thereby provide strong evidence for a model in which chains adopt an opened structure to bind multiple receptor proteins. Altogether our results suggest a mechanism through which UBL/UBA proteins could protect chains from premature de-ubiquitylation and unnecessary elongation during their transit to the proteasome.     

The ubiquitin-proteasome system

The 2004 Nobel Prize in chemistry for the discovery of protein ubiquitination has led to the recognition of cellular proteolysis as a central area of research in biology. Eukaryotic proteins targeted for degradation by this pathway are first ‘tagged’ by multimers of a protein known as ubiquitin and are later proteolyzed by a giant enzyme known as the proteasome. This article recounts the key observations that led to the discovery of ubiquitin-proteasome system (UPS). In addition, different aspects of proteasome biology are highlighted. Finally, some key roles of the UPS in different areas of biology and the use of inhibitors of this pathway as possible drug targets are discussed.     

Substrate selection by the proteasome through initiation regions

Proteins in the cell have to be eliminated once their function is no longer desired or they become damaged. Most regulated protein degradation is achieved by a large enzymatic complex called the proteasome. Many proteasome substrates are targeted for degradation by the covalent attachment of ubiquitin molecules. Ubiquitinated proteins can be bound by the proteasome, but for proteolysis to occur the proteasome needs to find a disordered tail somewhere in the target at which it initiates degradation. The initiation step contributes to the specificity of proteasomal degradation. Here, we review how the proteasome selects initiation sites within its substrates and discuss how the initiation step affects physiological processes.     

Recruitment of APPL1 to ubiquitin-rich aggresomes in response to proteasomal impairment

Inhibitors of proteasomes have been shown to affect endocytosis of multiple membrane receptors, in particular at the step of cargo sorting for lysosomal degradation. Here we demonstrate that the inhibition of proteasomes causes specific redistribution of an endosomal adaptor APPL1, which undergoes initial solubilization from APPL endosomes followed by clustering in the perinuclear region. MG132 treatment decreases APPL1 labeling of endosomes while the staining of the canonical early endosomes with EEA1 remains unaffected. Upon prolonged treatment with proteasome inhibitors, endogenous APPL1 localizes to the site of aggresome formation, with perinuclear APPL1 clusters encapsulated within a vimentin cage and co-localizing with aggregates positive for ubiquitin. The clustering of APPL1 is concomitant with increased ubiquitination and decreased solubility of this protein. We determined that the ubiquitin ligase Nedd4 enhances polyubiquitination of APPL1, and the ubiquitin molecules attached to APPL1 are linked through lysine-63. Taken together, these results add APPL1 to only a handful of endogenous cellular proteins known to be recruited to aggresomes induced by proteasomal stress. Moreover, our studies suggest that the proteasome inhibitors that are already in clinical use affect the localization, ubiquitination and solubility of APPL1.     

Receptors make the pathway choice for protein degradation

Damaged or aggregated proteins and organelles accumulate with age and contribute to various age-related pathologies including Alzheimer, Parkinson or Huntington diseases. In eukaryotic cells, there are 2 major pathways for degradation of the cytoplasm: The ubiquitin–proteasome system (UPS) and macroautophagy/autophagy. Both pathways can share the characteristic of initiating the process by ubiquitination of the substrate, but they utilize different ubiquitin receptors. In a paper described in a punctum in this issue, Lu et al. used the yeast Saccharomyces cerevisiae to demonstrate that the decision to use a particular pathway is made through a mechanism that depends on the receptors rather than the specific type of substrate ubiquitination.     

Regulation of apoptosis by E3 ubiquitin ligases in ubiquitin proteasome system

Apoptosis is an organised ATP‐dependent programmed cell death that organisms have evolved to maintain homoeostatic cell numbers and eliminate unnecessary or unhealthy cells from the system. Dysregulation of apoptosis can have serious manifestations culminating into various diseases, especially cancer. Accurate control of apoptosis requires regulation of a wide range of growth enhancing as well as anti‐oncogenic factors. Appropriate regulation of magnitude and temporal expression of key proteins is vital to maintain functional apoptotic signalling. Controlled protein turnover is thus critical to the unhindered operation of the apoptotic machinery, disruption of which can have severe consequences, foremost being oncogenic transformation of cells. The ubiquitin proteasome system (UPS) is one such major cellular pathway that maintains homoeostatic protein levels. Recent studies have found interesting links between these two fundamental cellular processes, wherein UPS depending on the cue can either inhibit or promote apoptosis. A diverse range of E3 ligases are involved in regulating the turnover of key proteins of the apoptotic pathway. This review summarises an overview of key E3 ubiquitin ligases involved in the regulation of the fundamental proteins involved in apoptosis, linking UPS to apoptosis and attempts to emphasize the significance of this relationship in context of cancer.     

The effects of proteasomal inhibition on synaptic proteostasis

Synaptic function crucially depends on uninterrupted synthesis and degradation of synaptic proteins. While much has been learned on synaptic protein synthesis, little is known on the routes by which synaptic proteins are degraded. Here we systematically studied how inhibition of the ubiquitin‐proteasome system (UPS) affects the degradation rates of thousands of neuronal and synaptic proteins. We identified a group of proteins, including several proteins related to glutamate receptor trafficking, whose degradation rates were significantly slowed by UPS inhibition. Unexpectedly, however, degradation rates of most synaptic proteins were not significantly affected. Interestingly, many of the differential effects of UPS inhibition were readily explained by a quantitative framework that considered known metabolic turnover rates for the same proteins. In contrast to the limited effects on protein degradation, UPS inhibition profoundly and preferentially suppressed the synthesis of a large number of synaptic proteins. Our findings point to the importance of the UPS in the degradation of certain synaptic proteins, yet indicate that under basal conditions most synaptic proteins might be degraded through alternative pathways.     

Ubiquitin Signaling Regulates RNA Biogenesis,Processing, and Metabolism

The fate of eukaryotic proteins, from their synthesis to destruction, is supervised by the ubiquitin–proteasome system (UPS). The UPS is the primary pathway responsible for selective proteolysis of intracellular proteins, which is guided by covalent attachment of ubiquitin to target proteins by E1 (activating), E2 (conjugating), and E3 (ligating) enzymes in a process known as ubiquitylation. The UPS can also regulate protein synthesis by influencing multiple steps of RNA (ribonucleic acid) metabolism. Here, recent publications concerning the interplay between the UPS and different types of RNA are reviewed. This interplay mainly involves specific RNA-binding E3 ligases that link RNA-dependent processes with protein ubiquitylation. The emerging understanding of their modes of RNA binding, their RNA targets, and their molecular and cellular functions are primarily focused on. It is discussed how the UPS adapted to interact with different types of RNA and how RNA molecules influence the ubiquitin signaling components.     

Sulforaphane enhances proteasomal and autophagic activities in mice and is a potential therapeutic reagent for Huntington's disease

The ubiquitin proteasome system (UPS) is impaired in Huntington's disease, a devastating neurodegenerative disorder. Sulforaphane, a naturally occurring compound, has been shown to stimulate UPS activity in cell cultures. To test whether sulforaphane enhances UPS function in vivo, we treated UPS function reporter mice ubiquitously expressing the green fluorescence protein (GFP) fused to a constitutive degradation signal that promotes its rapid degradation in the conditions of a healthy UPS. The modified GFP is termed GFP UPS reporter (GFPu). We found that both GFPu and ubiquitinated protein levels were significantly reduced and the three peptidase activities of the proteasome were increased in the brain and peripheral tissues of the mice. Interestingly, sulforaphane treatment also enhanced autophagy activity in the brain and the liver. To further examine whether sulforaphane promotes mutant huntingtin (mHtt) degradation, we treated Huntington's disease cells with sulforaphane and found that sulforaphane not only enhanced mHtt degradation but also reduced mHtt cytotoxicity. Sulforaphane‐mediated mHtt degradation was mainly through the UPS pathway as the presence of a proteasome inhibitor abolished this effect. Taken together, these data indicate that sulforaphane activates protein degradation machineries in both the brain and peripheral tissues and may be a therapeutic reagent for Huntington's disease and other intractable disorders.