TY - JOUR A1 - Tolay, Nazife A1 - Buchberger, Alexander T1 - Role of the ubiquitin system in stress granule metabolism JF - International Journal of Molecular Sciences N2 - Eukaryotic cells react to various stress conditions with the rapid formation of membrane-less organelles called stress granules (SGs). SGs form by multivalent interactions between RNAs and RNA-binding proteins and are believed to protect stalled translation initiation complexes from stress-induced degradation. SGs contain hundreds of different mRNAs and proteins, and their assembly and disassembly are tightly controlled by post-translational modifications. The ubiquitin system, which mediates the covalent modification of target proteins with the small protein ubiquitin (‘ubiquitylation’), has been implicated in different aspects of SG metabolism, but specific functions in SG turnover have only recently emerged. Here, we summarize the evidence for the presence of ubiquitylated proteins at SGs, review the functions of different components of the ubiquitin system in SG formation and clearance, and discuss the link between perturbed SG clearance and the pathogenesis of neurodegenerative disorders. We conclude that the ubiquitin system plays an important, medically relevant role in SG biology. KW - 26S proteasome KW - p97/VCP KW - Cdc48 KW - DUB KW - G3BP KW - granulostasis KW - granulophagy KW - ALS Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-284061 SN - 1422-0067 VL - 23 IS - 7 ER - TY - JOUR A1 - Tolay, Nazife A1 - Buchberger, Alexander T1 - Comparative profiling of stress granule clearance reveals differential contributions of the ubiquitin system JF - Life Science Alliance N2 - Stress granules (SGs) are cytoplasmic condensates containing untranslated mRNP complexes. They are induced by various proteotoxic conditions such as heat, oxidative, and osmotic stress. SGs are believed to protect mRNPs from degradation and to enable cells to rapidly resume translation when stress conditions subside. SG dynamics are controlled by various posttranslationalmodifications, but the role of the ubiquitin system has remained controversial. Here, we present a comparative analysis addressing the involvement of the ubiquitin system in SG clearance. Using high-resolution immuno-fluorescence microscopy, we found that ubiquitin associated to varying extent with SGs induced by heat, arsenite, H2O2, sorbitol, or combined puromycin and Hsp70 inhibitor treatment. SG-associated ubiquitin species included K48- and K63-linked conjugates, whereas free ubiquitin was not significantly enriched. Inhibition of the ubiquitin activating enzyme, deubiquitylating enzymes, the 26S proteasome and p97/VCP impaired the clearance of arsenite- and heat-induced SGs, whereas SGs induced by other stress conditions were little affected. Our data underline the differential involvement of the ubiquitin system in SG clearance, a process important to prevent the formation of disease-linked aberrant SGs. KW - phase transition KW - quality control KW - protein KW - inhibition KW - complexity KW - separation KW - diversity KW - autophagy KW - ALS KW - P97 Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259810 VL - 4 IS - 5 ER - TY - JOUR A1 - Prieto-Garcia, Cristian A1 - Hartmann, Oliver A1 - Reissland, Michaela A1 - Braun, Fabian A1 - Bozkurt, Süleyman A1 - Pahor, Nikolett A1 - Fuss, Carmina A1 - Schirbel, Andreas A1 - Schülein-Völk, Christina A1 - Buchberger, Alexander A1 - Calzado Canale, Marco A. A1 - Rosenfeldt, Mathias A1 - Dikic, Ivan A1 - Münch, Christian A1 - Diefenbacher, Markus E. T1 - USP28 enables oncogenic transformation of respiratory cells, and its inhibition potentiates molecular therapy targeting mutant EGFR, BRAF and PI3K JF - Molecular Oncology N2 - Oncogenic transformation of lung epithelial cells is a multistep process, frequently starting with the inactivation of tumour suppressors and subsequent development of activating mutations in proto-oncogenes, such as members of the PI3K or MAPK families. Cells undergoing transformation have to adjust to changes, including altered metabolic requirements. This is achieved, in part, by modulating the protein abundance of transcription factors. Here, we report that the ubiquitin carboxyl-terminal hydrolase 28 (USP28) enables oncogenic reprogramming by regulating the protein abundance of proto-oncogenes such as c-JUN, c-MYC, NOTCH and ∆NP63 at early stages of malignant transformation. USP28 levels are increased in cancer compared with in normal cells due to a feed-forward loop, driven by increased amounts of oncogenic transcription factors such as c-MYC and c-JUN. Irrespective of oncogenic driver, interference with USP28 abundance or activity suppresses growth and survival of transformed lung cells. Furthermore, inhibition of USP28 via a small-molecule inhibitor resets the proteome of transformed cells towards a ‘premalignant’ state, and its inhibition synergizes with clinically established compounds used to target EGFR\(^{L858R}\)-, BRAF\(^{V600E}\)- or PI3K\(^{H1047R}\)-driven tumour cells. Targeting USP28 protein abundance at an early stage via inhibition of its activity is therefore a feasible strategy for the treatment of early-stage lung tumours, and the observed synergism with current standard-of-care inhibitors holds the potential for improved targeting of established tumours. KW - buparlisib KW - c-MYC KW - gefitinib KW - lung cancer KW - USP28 KW - vemurafenib Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-312777 VL - 16 IS - 17 ER - TY - JOUR A1 - Körner, Maria A1 - Meyer, Susanne R. A1 - Marincola, Gabriella A1 - Kern, Maximilian J. A1 - Grimm, Clemens A1 - Schuelein-Voelk, Christina A1 - Fischer, Utz A1 - Hofmann, Kay A1 - Buchberger, Alexander T1 - The FAM104 proteins VCF1/2 promote the nuclear localization of p97/VCP JF - eLife N2 - The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and DNA damage repair, and its de-regulation is linked to neuromuscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97–cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members VCP nuclear cofactor family member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth, and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions. KW - p97 VCP Cdc48 KW - ubiquitin proteasome system KW - nuclear import KW - DNA damage repair KW - FAM104A KW - FLJ14775 KW - FAM104B KW - FLJ20434 KW - CXorf44 KW - cell biology Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-350222 VL - 12 ER - TY - JOUR A1 - Buchberger, Alexander A1 - Böhm, Stephanie T1 - The Budding Yeast Cdc48Shp1 Complex Promotes Cell Cycle Progression by Positive Regulation of Protein Phosphatase 1 (Glc7) JF - PLoS One N2 - The conserved, ubiquitin-selective AAA ATPase Cdc48 regulates numerous cellular processes including protein quality control, DNA repair and the cell cycle. Cdc48 function is tightly controlled by a multitude of cofactors mediating substrate specificity and processing. The UBX domain protein Shp1 is a bona fide substrate-recruiting cofactor of Cdc48 in the budding yeast S. cerevisiae. Even though Shp1 has been proposed to be a positive regulator of Glc7, the catalytic subunit of protein phosphatase 1 in S. cerevisiae, its cellular functions in complex with Cdc48 remain largely unknown. Here we show that deletion of the SHP1 gene results in severe growth defects and a cell cycle delay at the metaphase to anaphase transition caused by reduced Glc7 activity. Using an engineered Cdc48 binding-deficient variant of Shp1, we establish the Cdc48Shp1 complex as a critical regulator of mitotic Glc7 activity. We demonstrate that shp1 mutants possess a perturbed balance of Glc7 phosphatase and Ipl1 (Aurora B) kinase activities and show that hyper-phosphorylation of the kinetochore protein Dam1, a key mitotic substrate of Glc7 and Ipl1, is a critical defect in shp1. We also show for the first time a physical interaction between Glc7 and Shp1 in vivo. Whereas loss of Shp1 does not significantly affect Glc7 protein levels or localization, it causes reduced binding of the activator protein Glc8 to Glc7. Our data suggest that the Cdc48Shp1 complex controls Glc7 activity by regulating its interaction with Glc8 and possibly further regulatory subunits. KW - alleles KW - cell cycle KW - immunoprecipitation KW - phosphatases KW - genetic interactions Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-96073 ER -