Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions

UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently it was reported that UCH37 activity is stimulated by branched ubiquitin chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and NMR structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal ubiquitins that emanate from a branched ubiquitin. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear ubiquitin chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique ubiquitin chain architecture is aided by a DUB.

Branched ubiquitin chain binding and deubiquitination by UCH37 facilitate proteasome clearance of stress-induced inclusions

UCH37, also known as UCHL5, is a highly conserved deubiquitinating enzyme (DUB) that associates with the 26S proteasome. Recently it was reported that UCH37 activity is stimulated by branched ubiquitin chain architectures. To understand how UCH37 achieves its unique debranching specificity, we performed biochemical and NMR structural analyses and found that UCH37 is activated by contacts with the hydrophobic patches of both distal ubiquitins that emanate from a branched ubiquitin. In addition, RPN13, which recruits UCH37 to the proteasome, further enhances branched-chain specificity by restricting linear ubiquitin chains from having access to the UCH37 active site. In cultured human cells under conditions of proteolytic stress, we show that substrate clearance by the proteasome is promoted by both binding and deubiquitination of branched polyubiquitin by UCH37. Proteasomes containing UCH37(C88A), which is catalytically inactive, aberrantly retain polyubiquitinated species as well as the RAD23B substrate shuttle factor, suggesting a defect in recycling of the proteasome. These findings provide a foundation to understand how proteasome degradation of substrates modified by a unique ubiquitin chain architecture is aided by a DUB.

Adaptability of the ubiquitin-proteasome system to proteolytic and folding stressors

Aging, disease, and environmental stressors are associated with failures in the ubiquitin-proteasome system (UPS), yet a quantitative understanding of how stressors affect the proteome and how the UPS responds is lacking. Here we assessed UPS performance and adaptability in yeast under stressors using quantitative measurements of misfolded substrate stability and stress-dependent UPS regulation by the transcription factor Rpn4. We found that impairing degradation rates (proteolytic stress) and generating misfolded proteins (folding stress) elicited distinct effects on the proteome and on UPS adaptation. Folding stressors stabilized proteins via aggregation rather than overburdening the proteasome, as occurred under proteolytic stress. Still, the UPS productively adapted to both stressors using separate mechanisms: proteolytic stressors caused Rpn4 stabilization while folding stressors increased RPN4 transcription. In some cases, adaptation completely prevented loss of UPS substrate degradation. Our work reveals the distinct effects of proteotoxic stressors and the versatility of cells in adapting the UPS.

Quantitative analysis of the ubiquitin-proteasome system under proteolytic and folding stressors

Aging, disease, and environmental stressors are associated with failures in the ubiquitin-proteasome system (UPS), yet a quantitative understanding of how stressors affect the proteome and how the UPS responds is lacking. Here we assessed UPS performance and adaptability in yeast under stressors using quantitative measurements of misfolded substrate stability and stress-dependent UPS regulation by the transcription factor Rpn4. We found that impairing degradation rates (proteolytic stress) and generating misfolded proteins (folding stress) elicited distinct effects on the proteome and on UPS adaptation. Folding stressors stabilized proteins via aggregation rather than overburdening the proteasome, as occurred under proteolytic stress. Still, the UPS productively adapted to both stressors using separate mechanisms: proteolytic stressors caused Rpn4 stabilization while folding stressors increased RPN4 transcription. In some cases, adaptation completely prevented loss of UPS substrate degradation. Our work reveals the distinct effects of proteotoxic stressors and the versatility of cells in adapting the UPS.

Marcks Marks Resistance to Proteasome Inhibitors: Exocytosis of Polyubiquitinated Proteins in Bortezomib-Resistant Leukemia Cells

Despite the proven efficacy of proteasome inhibitors like bortezomib (BTZ) in multiple myeloma, mantle cell lymphoma, and in an experimental setting in pediatric acute leukemia, development of drug resistance remains a primary hindrance. To further understand the molecular basis underlying this chemoresistance phenomenon, various leukemia cell line models with acquired resistance to BTZ were developed and characterized. One common characteristic was that acquisition of point mutations in PSMB5 and upregulation of the β5-subunit of the proteasome were key determinants of BTZ-resistance in vitro. However, it remains unclear how these drug resistance modalities translate to the overcoming of proteolytic stress imposed by proteasome inhibition. From this perspective, we here undertook a multi-modality (DNA, mRNA, miRNA) array-based analyses of human CCRF-CEM acute leukemia cells and two BTZ-resistant subclones [one with a low resistance level [(10-fold, CEM/BTZ7) and another subline with a high resistance level (140-fold, CEM/BTZ200)] to determine whether or not complementary mechanisms contribute to BTZ resistance. Gene expression profiling studies revealed markedly reduced proteolytic stress induction in drug resistant cells over a broad BTZ concentration range. Moreover, several genes involved in cytoskeleton regulation and vesicle migration were increased in resistant cells. Of all genes, myristoylated alanine-rich C-kinase substrate (MARCKS) was the most differentially overexpressed gene with 25- to 42-fold upregulation in CEM/BTZ7 and CEM/BTZ200 cells, respectively. These observations were corroborated at the protein level and solely included unphosphorylated MARCKS rather than phosphorylated MARCKS, which was marginally expressed in CEM/BTZ cells. Interestingly, MARCKS upregulation was also observed in other BTZ-resistant and leukemia cells (CEM and THP1) with acquired resistance to the proteasome inhibitor salinosporamide A and the immunoproteasome inhibitor PR924. Given the overexpression of MARCKS in proteasome inhibitor-resistant leukemia cells, we further explored whether or not MARCKS overexpression may serve as a predictive marker of BTZ resistance in clinical samples of acute leukemia patients. To this end, we examined primary patient specimens from a phase II childhood refractory/relapsed ALL trial in which BTZ is administered in two intensive re-induction regimens containing vincristine, prednisone, PEG-asparaginase, doxorubicin or cyclophosphamide and etoposide followed by methotrexate treatment. MARCKS expression was demonstrated in 64% of therapy-refractory pediatric leukemia specimens (n=44) wherein higher MARCKS expression trended (p=0.09) towards a dismal response to BTZ-containing chemotherapy. Finally, from a mechanistic perspective, we showed a concentration-dependent association of MARCKS protein with the emergence of ubiquitin-containing vesicles in the resistant cells. This association with MARCKS protein was lost upon exocytosis of these vesicles, which were found to be extruded and taken up in co-cultures with recipient HeLa cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance through vesicular exocytosis of ubiquitinated proteins in BTZ-resistant cells to overcome proteolytic stress over a broad range of cytotoxic BTZ concentrations. Disclosures Kaspers: Janssen-Cilag: Research Funding. Smeets:Novartis: Employment. Zweegman:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Cloos:Takeda: Honoraria.

Polyubiquitin Linkage Profiles in Three Models of Proteolytic Stress Suggest the Etiology of Alzheimer Disease

Polyubiquitin chains on substrates are assembled through any of seven lysine residues or the N terminus of ubiquitin (Ub), generating diverse linkages in the chain structure. PolyUb linkages regulate the fate of modified substrates, but their abundance and function in mammalian cells are not well studied. We present a mass spectrometry-based method to measure polyUb linkages directly from total lysate of mammalian cells. In HEK293 cells, the level of polyUb linkages was found to be 52% (Lys48), 38% (Lys63), 8% (Lys29), 2% (Lys11), and 0.5% or less for linear, Lys6, Lys27, and Lys33 linkages. Tissue specificity of these linkages was examined in mice fully labeled by heavy stable isotopes (i.e. SILAC mice). Moreover, we profiled the Ub linkages in brain tissues from patients of Alzheimer disease with or without concurrent Lewy body disease as well as three cellular models of proteolytic stress: proteasome deficiency, lysosome deficiency, and heat shock. The data support that polyUb chains linked through Lys6, Lys11, Lys27, Lys29, and Lys48 mediate proteasomal degradation, whereas Lys63 chains are preferentially involved in the lysosomal pathway. Mixed linkages, including Lys48, may also contribute to lysosomal targeting, as both Lys63 and Lys48 linkages are colocalized in LC3-labeled autophagosomes. Interestingly, heat shock treatment augments Lys11, Lys48, and Lys63 but not Lys29 linkages, and this unique pattern is similar to that in the profiled neurodegenerative cases. We conclude that different polyUb linkages play distinct roles under the three proteolytic stress conditions, and protein folding capacity in the heat shock responsive pathway might be more affected in Alzheimer disease.