bims-proteo Biomed News
on Proteostasis
Issue of 2023–01–22
25 papers selected by
Eric Chevet, INSERM
  1. From seeds to trees: how E2 enzymes grow ubiquitin chains.
  2. Distinct role of ERp57 and ERdj5 as a disulfide isomerase and reductase during ER protein folding.
  3. Dynamics of CLIMP-63 S-acylation control ER morphology.
  4. The C-terminus of the cargo receptor Erv14p affects COPII vesicle formation and cargo delivery.
  5. Palmitoylation and PDE6δ regulate membrane-compartment-specific substrate ubiquitylation and degradation.
  6. KLHL12 can form large COPII structures in the absence of CUL3 neddylation.
  7. A bio-orthogonal linear ubiquitin probe identifies STAT3 as a direct substrate of OTULIN in glioblastoma.
  8. ULK1-mediated phosphorylation regulates the conserved role of YKT6 in autophagy.
  9. Impact of Hypermannosylation on the Structure and Functionality of the ER and the Golgi Complex.
  10. Biosynthesis of long polyubiquitin chains in high yield and purity.
  11. Inactive Proteasomes Routed to Autophagic Turnover Are Confined within the Soluble Fraction of the Cell.
  12. HPV is a cargo for the COPI sorting complex during virus entry.
  13. Perturbation of placental protein glycosylation by endoplasmic reticulum stress promotes maladaptation of maternal hepatic glucose metabolism.
  14. HECT Domain Interaction with Ubiquitin Binding Sites on Tsg101-UEV Controls HIV-1 Egress, Maturation and Infectivity.
  15. Capture, release, and identification of newly synthesized proteins for improved profiling of functional translatomes.
  16. Targeted Degradation of PD-L1 and Activation of the STING Pathway by Carbon-Dot-Based PROTACs for Cancer Immunotherapy.
  17. Inhibitors of the ATPase p97/VCP: From basic research to clinical applications.
  18. A molecular network of conserved factors keeps ribosomes dormant in the egg.
  19. Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation.
  20. Structural remodeling of AAA+ ATPase p97 by adaptor protein ASPL facilitates posttranslational methylation by METTL21D.
  21. Inhibition of autophagy in microglia and macrophages exacerbates innate immune responses and worsens brain injury outcomes.
  22. A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast.
  23. DNA repair protein FANCD2 has both ubiquitination-dependent and -independent functions during germ cell development.
  24. De-centralizing the Central Dogma: mRNA translation in space and time.
  25. Hepatic ribosomal protein S6 (Rps6) insufficiency results in failed bile duct development and loss of hepatocyte viability; a ribosomopathy-like phenotype that is partially p53-dependent.
  1. Biochem Soc Trans. 2023 Jan 16. pii: BST20220880. [Epub ahead of print]
    From seeds to trees: how E2 enzymes grow ubiquitin chains.
    Adam J Middleton, Catherine L Day.
      Modification of proteins by ubiquitin is a highly regulated process that plays a critical role in eukaryotes, from the construction of signalling platforms to the control of cell division. Aberrations in ubiquitin transfer are associated with many diseases, including cancer and neurodegenerative disorders. The ubiquitin machinery generates a rich code on substrate proteins, spanning from single ubiquitin modifications to polyubiquitin chains with diverse linkage types. Central to this process are the E2 enzymes, which often determine the exact nature of the ubiquitin code. The focus of this mini-review is on the molecular details of how E2 enzymes can initiate and grow ubiquitin chains. In particular, recent developments and biochemical breakthroughs that help explain how the degradative E2 enzymes, Ube2s, Ube2k, and Ube2r, generate complex ubiquitin chains with exquisite specificity will be discussed.
    Keywords:  E2 enzymes; protein degradation; ubiquitin; ubiquitin chains; ubiquitin signalling
    DOI:  https://doi.org/10.1042/BST20220880
  2. J Cell Sci. 2023 Jan 15. pii: jcs260656. [Epub ahead of print]136(2):
    Distinct role of ERp57 and ERdj5 as a disulfide isomerase and reductase during ER protein folding.
    Philip John Robinson, Marie Anne Pringle, Bethany Fleming, Neil John Bulleid.
      Proteins entering the secretory pathway need to attain native disulfide pairings to fold correctly. For proteins with complex disulfides, this process requires the reduction and isomerisation of non-native disulfides. Two key members of the protein disulfide isomerase (PDI) family, ERp57 and ERdj5 (also known as PDIA3 and DNAJC10, respectively), are thought to be required for correct disulfide formation but it is unknown whether they act as a reductase, an isomerase or both. In addition, it is unclear how reducing equivalents are channelled through PDI family members to substrate proteins. Here, we show that neither enzyme is required for disulfide formation, but ERp57 is required for isomerisation of non-native disulfides within glycoproteins. In addition, alternative PDIs compensate for the absence of ERp57 to isomerise glycoprotein disulfides, but only in the presence of a robust reductive pathway. ERdj5 is required for this alternative pathway to function efficiently indicating its role as a reductase. Our results define the essential cellular functions of two PDIs, highlighting a distinction between formation, reduction and isomerisation of disulfide bonds.
    Keywords:  Disulfide formation; Endoplasmic reticulum; Protein disulfide isomerase; Protein folding; Protein secretion
    DOI:  https://doi.org/10.1242/jcs.260656
  3. Nat Commun. 2023 Jan 17. 14(1): 264
    Dynamics of CLIMP-63 S-acylation control ER morphology.
    Patrick A Sandoz, Robin A Denhardt-Eriksson, Laurence Abrami, Luciano A Abriata, Gard Spreemann, Catherine Maclachlan, Sylvia Ho, Béatrice Kunz, Kathryn Hess, Graham Knott, Francisco S Mesquita, Vassily Hatzimanikatis, F Gisou van der Goot.
      The complex architecture of the endoplasmic reticulum (ER) comprises distinct dynamic features, many at the nanoscale, that enable the coexistence of the nuclear envelope, regions of dense sheets and a branched tubular network that spans the cytoplasm. A key player in the formation of ER sheets is cytoskeleton-linking membrane protein 63 (CLIMP-63). The mechanisms by which CLIMP-63 coordinates ER structure remain elusive. Here, we address the impact of S-acylation, a reversible post-translational lipid modification, on CLIMP-63 cellular distribution and function. Combining native mass-spectrometry, with kinetic analysis of acylation and deacylation, and data-driven mathematical modelling, we obtain in-depth understanding of the CLIMP-63 life cycle. In the ER, it assembles into trimeric units. These occasionally exit the ER to reach the plasma membrane. However, the majority undergoes S-acylation by ZDHHC6 in the ER where they further assemble into highly stable super-complexes. Using super-resolution microscopy and focused ion beam electron microscopy, we show that CLIMP-63 acylation-deacylation controls the abundance and fenestration of ER sheets. Overall, this study uncovers a dynamic lipid post-translational regulation of ER architecture.
    DOI:  https://doi.org/10.1038/s41467-023-35921-6
  4. J Cell Sci. 2023 Jan 18. pii: jcs.260527. [Epub ahead of print]
    The C-terminus of the cargo receptor Erv14p affects COPII vesicle formation and cargo delivery.
    Daniel Lagunas-Gomez, Carolina Yañez-Dominguez, Guadalupe Zavala-Padilla, Charles Barlowe, Omar Pantoja.
      The endoplasmic reticulum (ER) is the start site of the secretory pathway, where newly synthesized secreted and membrane proteins are packaged into COPII vesicles through direct interaction with the COPII coat or aided by specific cargo receptors. Little is known about how post-translational modification events regulate packaging of cargo into COPII vesicles. Erv14/Cornichon belongs to a conserved family of cargo receptors required for the selection and ER export of transmembrane proteins. In this work, we show the importance of a phosphorylation consensus site (Serine-134) at the C-terminus of Erv14. Mimicking phosphorylation of S134 (S134D) prevents the incorporation of Erv14 into COPII vesicles, delays cell growth, exacerbates growth of sec mutants, modifies ER structure, and affects localization of several plasma membrane transporters. In contrast, the dephosphorylated mimic (S134A) had less deleterious effects, but still modifies ER structure and slows cell growth. Our results suggest that a possible cycle of phosphorylation and dephosphorylation is important for the correct functioning of Erv14p.
    Keywords:  COPII vesicles; Phosphorylation; ScErv14; Serine-134
    DOI:  https://doi.org/10.1242/jcs.260527
  5. Cell Rep. 2023 Jan 18. pii: S2211-1247(23)00010-4. [Epub ahead of print]42(1): 111999
    Palmitoylation and PDE6δ regulate membrane-compartment-specific substrate ubiquitylation and degradation.
    David Liang, Liping Jiang, Sameer Ahmed Bhat, Sonia Missiroli, Mariasole Perrone, Angela Lauriola, Ritika Adhikari, Anish Gudur, Zahra Vasi, Ian Ahearn, Daniele Guardavaccaro, Carlotta Giorgi, Mark Philips, Shafi Kuchay.
      Substrate degradation by the ubiquitin proteasome system (UPS) in specific membrane compartments remains elusive. Here, we show that the interplay of two lipid modifications and PDE6δ regulates compartmental substrate targeting via the SCFFBXL2. FBXL2 is palmitoylated in a prenylation-dependent manner on cysteines 417 and 419 juxtaposed to the CaaX motif. Palmitoylation/depalmitoylation regulates its subcellular trafficking for substrate engagement and degradation. To control its subcellular distribution, lipid-modified FBXL2 interacts with PDE6δ. Perturbing the equilibrium between FBXL2 and PDE6δ disrupts the delivery of FBXL2 to all membrane compartments, whereas depalmitoylated FBXL2 is enriched on the endoplasmic reticulum (ER). Depalmitoylated FBXL2(C417S/C419S) promotes the degradation of IP3R3 at the ER, inhibits IP3R3-dependent mitochondrial calcium overload, and counteracts calcium-dependent cell death upon oxidative stress. In contrast, disrupting the PDE6δ-FBXL2 equilibrium has the opposite effect. These findings describe a mechanism underlying spatially-restricted substrate degradation and suggest that inhibition of FBXL2 palmitoylation and/or binding to PDE6δ may offer therapeutic benefits.
    Keywords:  CP: Cell biology; CP: Molecular biology; E3-ligases; PDE6δ; calcium homeostasis; cell membranes; oxidative stress; palmitoylation; prenylation; protein degradation; trafficking; ubiquitylation
    DOI:  https://doi.org/10.1016/j.celrep.2023.111999
  6. Mol Biol Cell. 2023 Jan 18. mbcE22080383
    KLHL12 can form large COPII structures in the absence of CUL3 neddylation.
    Tamara Moretti, Kyungho Kim, Astha Tuladhar, Jinoh Kim.
      CUL3-RING ubiquitin ligases (CRL3s) are involved in various cellular processes through different Bric a brac, Tramtrack and Broad-Complex (BTB)-domain proteins. KLHL12, a BTB-domain protein, is suggested to play an essential role in the export of large cargo molecules like procollagen from the endoplasmic reticulum (ER). CRL3KLHL12 mono-ubiquitylates SEC31, leading to an increase in COPII vesicle dimension. Enlarged COPII vesicles can accommodate procollagen molecules. Thus, CRL3KLHL12 is essential for the assembly of large COPII structures and collagen secretion. CRL3s are activated by CUL3 neddylation. Here, we evaluated the importance of CUL3 neddylation in COPII assembly and collagen secretion. Unexpectedly, the assembly of large COPII-KLHL12 structures persisted and cellular collagen levels decreased by the treatment of MLN4924, a potent inhibitor of NEDD8-activating enzyme. When we introduced mutations to KLHL12 at the CUL3 interface, these KLHL12 variants did not interact with neddylated CUL3, but one (Mut A) of them still supported large COPII-KLHL12 structures. Overexpression of wild-type KLHL12, but not Mut A, lowered cellular collagen levels most likely via lysosomal degradation. Our results suggest that CUL3 neddylation is not necessary for the formation of large COPII-KLHL12 structures, but active CRL3KLHL12 contributes to the maintenance of collagen levels in the cell.
    DOI:  https://doi.org/10.1091/mbc.E22-08-0383
  7. Nucleic Acids Res. 2023 Jan 20. pii: gkad002. [Epub ahead of print]
    A bio-orthogonal linear ubiquitin probe identifies STAT3 as a direct substrate of OTULIN in glioblastoma.
    Xianli Du, Jing Pang, Bin Gu, Tian Si, Yan Chang, Tianqi Li, Min Wu, Zicheng Wang, Yuxia Wang, Jiannan Feng, Ning Wu, Jianghong Man, Huiyan Li, Ailing Li, Tong Zhang, Bo Wang, Xiaotao Duan.
      While linear ubiquitin plays critical roles in multiple cell signaling pathways, few substrates have been identified. Global profiling of linear ubiquitin substrates represents a significant challenge because of the low endogenous level of linear ubiquitination and the background interference arising from highly abundant ubiquitin linkages (e.g. K48- and K63-) and from the non-specific attachment of interfering proteins to the linear polyubiquitin chain. We developed a bio-orthogonal linear ubiquitin probe by site-specific encoding of a norbornene amino acid on ubiquitin (NAEK-Ub). This probe facilitates covalent labeling of linear ubiquitin substrates in live cells and enables selective enrichment and identification of linear ubiquitin-modified proteins. Given the fact that the frequent overexpression of the linear linkage-specific deubiquitinase OTULIN correlates with poor prognosis in glioblastoma, we demonstrated the feasibility of the NAEK-Ub strategy by identifying and validating substrates of linear ubiquitination in patient-derived glioblastoma stem-like cells (GSCs). We identified STAT3 as a bona fide substrate of linear ubiquitin, and showed that linear ubiquitination negatively regulates STAT3 activity by recruitment of the phosphatase TC-PTP to STAT3. Furthermore, we demonstrated that preferential expression of OTULIN in GSCs restricts linear ubiquitination on STAT3 and drives persistent STAT3 signaling, and thereby maintains the stemness and self-renewal of GSCs.
    DOI:  https://doi.org/10.1093/nar/gkad002
  8. J Cell Sci. 2023 Jan 16. pii: jcs.260546. [Epub ahead of print]
    ULK1-mediated phosphorylation regulates the conserved role of YKT6 in autophagy.
    Pablo Sánchez-Martín, Franziska Kriegenburg, Ludovico Alves, Julius Adam, Jana Elsaesser, Riccardo Babic, Hector Mancilla, Mariya Licheva, Georg Tascher, Christian Münch, Stefan Eimer, Claudine Kraft.
      Autophagy is a catabolic process during which cytosolic material is enwrapped in a newly formed double membrane structure called the autophagosome, and subsequently targeted for degradation in the lytic compartment of the cell. The fusion of autophagosomes with the lytic compartment is a tightly regulated step and involves membrane-bound SNARE proteins. These play a crucial role as they promote lipid mixing and fusion of the opposing membranes. Among the SNARE proteins implicated in autophagy, the essential SNARE protein YKT6 is the only SNARE protein evolutionary conserved from yeast to humans. Here we show that alterations in YKT6 function, in both mammalian cells and nematodes, produce early and late autophagy defects that result in reduced survival. Moreover, mammalian autophagosomal YKT6 is phospho-regulated by the ULK1 kinase, preventing premature bundling with the lysosomal SNARE proteins and thereby inhibiting autophagosome-lysosome fusion. Together, our findings reveal that timely regulation of the YKT6 phosphorylation status is crucial throughout autophagy progression and cell survival.
    Keywords:  ULK1; autophagosome; autophagy; SNARE; YKT6
    DOI:  https://doi.org/10.1242/jcs.260546
  9. Biomedicines. 2023 Jan 06. pii: 146. [Epub ahead of print]11(1):
    Impact of Hypermannosylation on the Structure and Functionality of the ER and the Golgi Complex.
    Patricia Franzka, Svenja Caren Schüler, Takfarinas Kentache, Robert Storm, Andrea Bock, Istvan Katona, Joachim Weis, Katrin Buder, Christoph Kaether, Christian A Hübner.
      Proteins of the secretory pathway undergo glycosylation in the endoplasmic reticulum (ER) and the Golgi apparatus. Altered protein glycosylation can manifest in serious, sometimes fatal malfunctions. We recently showed that mutations in GDP-mannose pyrophosphorylase A (GMPPA) can cause a syndrome characterized by alacrima, achalasia, mental retardation, and myopathic alterations (AAMR syndrome). GMPPA acts as a feedback inhibitor of GDP-mannose pyrophosphorylase B (GMPPB), which provides GDP-mannose as a substrate for protein glycosylation. Loss of GMPPA thus enhances the incorporation of mannose into glycochains of various proteins, including α-dystroglycan (α-DG), a protein that links the extracellular matrix with the cytoskeleton. Here, we further characterized the consequences of loss of GMPPA for the secretory pathway. This includes a fragmentation of the Golgi apparatus, which comes along with a regulation of the abundance of several ER- and Golgi-resident proteins. We further show that the activity of the Golgi-associated endoprotease furin is reduced. Moreover, the fraction of α-DG, which is retained in the ER, is increased. Notably, WT cells cultured at a high mannose concentration display similar changes with increased retention of α-DG, altered structure of the Golgi apparatus, and a decrease in furin activity. In summary, our data underline the importance of a balanced mannose homeostasis for the secretory pathway.
    Keywords:  Golgi network; endoplasmic reticulum; mannosylation
    DOI:  https://doi.org/10.3390/biomedicines11010146
  10. Anal Biochem. 2023 Jan 12. pii: S0003-2697(23)00009-X. [Epub ahead of print]664 115044
    Biosynthesis of long polyubiquitin chains in high yield and purity.
    Chaoqiang Li, Bin Song, Wenjia Shi, Xin Liu, Ning Song, Jie Zheng.
      As one of the most prevalent protein post-translational modifications, ubiquitin modification plays a momentous role in regulating varied cellular functions. Different polyubiquitin linkage types have diverse effects on cell signaling. However, compared with short ubiquitin chains, the preparation of long ubiquitin chains remains difficult and expensive to purchase commercially. In this study, we constructed an enzyme library of ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin-ligase E3, which are specific for synthesizing K63, K48, and M1 linked polyubiquitin chains. We demonstrate that these distinctly linked polyubiquitin chains could be synthesized and purified with high yield and purity. More importantly, this method can synthesize longer ubiquitin chains, the longest can reach more than fifteen ubiquitin molecules, which provides great convenience for ubiquitin-related structural and functional studies.
    Keywords:  In vitro biosynthesis; K48 linked polyubiquitin; K63 linked polyubiquitin; Long polyubiquitin chain; M1 linked polyubiquitin
    DOI:  https://doi.org/10.1016/j.ab.2023.115044
  11. Biomolecules. 2022 Dec 30. pii: 77. [Epub ahead of print]13(1):
    Inactive Proteasomes Routed to Autophagic Turnover Are Confined within the Soluble Fraction of the Cell.
    Keren Friedman, Ofri Karmon, Uri Fridman, Yair Goldberg, Ophry Pines, Shay Ben-Aroya.
      Previous studies demonstrated that dysfunctional yeast proteasomes accumulate in the insoluble protein deposit (IPOD), described as the final deposition site for amyloidogenic insoluble proteins and that this compartment also mediates proteasome ubiquitination, a prerequisite for their targeted autophagy (proteaphagy). Here, we examined the solubility state of proteasomes subjected to autophagy as a result of their inactivation, or under nutrient starvation. In both cases, only soluble proteasomes could serve as a substrate to autophagy, suggesting a modified model whereby substrates for proteaphagy are dysfunctional proteasomes in their near-native soluble state, and not as previously believed, those sequestered at the IPOD. Furthermore, the insoluble fraction accumulating in the IPOD represents an alternative pathway, enabling the removal of inactive proteasomes that escaped proteaphagy when the system became saturated. Altogether, we suggest that the relocalization of proteasomes to soluble aggregates represents a general stage of proteasome recycling through autophagy.
    Keywords:  autophagy; proteasome; protein quality control
    DOI:  https://doi.org/10.3390/biom13010077
  12. Sci Adv. 2023 Jan 20. 9(3): eadc9830
    HPV is a cargo for the COPI sorting complex during virus entry.
    Mara C Harwood, Tai-Ting Woo, Yuka Takeo, Daniel DiMaio, Billy Tsai.
      During entry, human papillomavirus (HPV) traffics from the cell surface to the endosome and then to the trans-Golgi network (TGN) and Golgi apparatus. HPV must transit across the TGN/Golgi and exit these compartments to reach the nucleus to cause infection, although how these steps are accomplished is unclear. Combining cellular fractionation, unbiased proteomics, and gene knockdown strategies, we identified the coat protein complex I (COPI), a highly conserved protein complex that facilitates retrograde trafficking of cellular cargos, as a host factor required for HPV infection. Upon TGN/Golgi arrival, the cytoplasmic segment of HPV L2 binds directly to COPI. COPI depletion causes the accumulation of HPV in the TGN/Golgi, resembling the fate of a COPI binding-defective L2 mutant. We propose that the L2-COPI interaction drives HPV trafficking through the TGN and Golgi stacks during virus entry. This shows that an incoming virus is a cargo of the COPI complex.
    DOI:  https://doi.org/10.1126/sciadv.adc9830
  13. iScience. 2023 Jan 20. 26(1): 105911
    Perturbation of placental protein glycosylation by endoplasmic reticulum stress promotes maladaptation of maternal hepatic glucose metabolism.
    Hong Wa Yung, Xiaohui Zhao, Luke Glover, Charlotte Burrin, Poh-Choo Pang, Carolyn J P Jones, Carolyn Gill, Kate Duhig, Matts Olovsson, Lucy C Chappell, Stuart M Haslam, Anne Dell, Graham J Burton, D Stephen Charnock-Jones.
      Placental hormones orchestrate maternal metabolic adaptations to support pregnancy. We hypothesized that placental ER stress, which characterizes early-onset pre-eclampsia (ePE), compromises glycosylation, reducing hormone bioactivity and these maladaptations predispose the mother to metabolic disease in later life. We demonstrate ER stress reduces the complexity and sialylation of trophoblast protein N-glycosylation, while aberrant glycosylation of vascular endothelial growth factor reduced its bioactivity. ER stress alters the expression of 66 of the 146 genes annotated with "protein glycosylation" and reduces the expression of sialyltransferases. Using mouse placental explants, we show ER stress promotes the secretion of mis-glycosylated glycoproteins. Pregnant mice carrying placentas with junctional zone-specific ER stress have reduced blood glucose, anomalous hepatic glucose metabolism, increased cellular stress and elevated DNA methyltransferase 3A. Using pregnancy-specific glycoproteins as a readout, we also demonstrate aberrant glycosylation of placental proteins in women with ePE, thus providing a mechanistic link between ePE and subsequent maternal metabolic disorders.
    Keywords:  Biological sciences; Human metabolism; Pregnancy
    DOI:  https://doi.org/10.1016/j.isci.2022.105911
  14. J Biol Chem. 2023 Jan 12. pii: S0021-9258(23)00033-9. [Epub ahead of print] 102901
    HECT Domain Interaction with Ubiquitin Binding Sites on Tsg101-UEV Controls HIV-1 Egress, Maturation and Infectivity.
    David A Nyenhuis, Rohith Rajasekaran, Susan Watanabe, Marie-Paule Strub, Mahfuz Khan, Michael Powell, Carol A Carter, Nico Tjandra.
      The HECT domain of HECT E3 ligases consists of flexibly linked N- and C-terminal lobes, with a ubiquitin (Ub) donor site on the C-lobe that is directly involved in substrate modification. HECT ligases also possess a secondary Ub binding site in the N-lobe, which is thought to play a role in processivity, specificity, or regulation. Here, we report the use of paramagnetic solution NMR to characterize a complex formed between the isolated HECT domain of neural precursor cell-expressed developmentally downregulated 4-1 (Nedd4-1) and the ubiquitin E2 variant (UEV) domain of tumor susceptibility gene 101 (Tsg101). Both proteins are involved in endosomal trafficking, a process driven by Ub signaling, and are hijacked by viral pathogens for particle assembly, however, a direct interaction between them has not been described and the mechanism by which the HECT E3 ligase contributes to pathogen formation has not been elucidated. We provide evidence for their association, consisting of multiple sites on the Nedd4-1 HECT domain and elements of the Tsg101 UEV domain involved in non-covalent ubiquitin binding. Furthermore, we show using an established reporter assay that HECT residues perturbed by UEV proximity define determinants of viral maturation and infectivity. These results suggest the UEV interaction is a determinant of HECT activity in Ub signaling. As the endosomal trafficking pathway is hijacked by several human pathogens for egress, the HECT-UEV interaction could represent a potential novel target for therapeutic intervention.
    Keywords:  HECT; NMR; Nedd4; Tsg101; UEV; Ubiquitin; endocytic sorting; viral budding
    DOI:  https://doi.org/10.1016/j.jbc.2023.102901
  15. Mol Cell Proteomics. 2023 Jan 12. pii: S1535-9476(23)00006-3. [Epub ahead of print] 100497
    Capture, release, and identification of newly synthesized proteins for improved profiling of functional translatomes.
    Nancy J Phillips, Bala M Vinaithirthan, Juan A Oses-Prieto, Robert J Chalkley, Alma L Burlingame.
      New protein synthesis is regulated both at the level of mRNA transcription and translation. RNA-Seq is effective at measuring levels of mRNA expression, but techniques to monitor mRNA translation are much more limited. Previously, we reported results from O-propargyl-puromycin (OPP) labeling of proteins undergoing active translation in a 2-hour time frame, followed by biotinylation using click chemistry, affinity purification, and on-bead digestion to identify nascent proteins by mass spectrometry (OPP-ID). As with any on-bead digestion protocol, the problem of nonspecific binders complicated the rigorous categorization of nascent proteins by OPP-ID. Here, we incorporate a chemically cleavable linker, Dde biotin-azide, into the protocol (OPP-IDCL) to provide specific release of modified proteins from the streptavidin beads. Following capture, the Dde moiety is readily cleaved with 2% hydrazine, releasing nascent polypeptides bearing OPP plus a residual C3H8N4 tag. When results are compared side-by-side with the original OPP-ID method, change to a cleavable linker led to a dramatic reduction in the number of background proteins detected in controls and a concomitant increase in the number of proteins that could be characterized as newly synthesized. We evaluated the method's ability to detect nascent proteins at various sub-mg protein input levels and showed that when starting with only 100 μg of protein, ∼1500 nascent proteins could be identified with low background. Upon treatment of K562 cells with MLN128, a potent inhibitor of the mammalian target of rapamycin (mTOR), prior to OPP treatment, we identified 1915 nascent proteins, the majority of which were downregulated upon inhibitor treatment. Repressed proteins with log2 FC <-1 revealed a complex network of functionally interacting proteins, with the largest cluster associated with translational initiation. Overall, incorporation of the Dde biotin-azide cleavable linker into our protocol has increased the depth and accuracy of profiling of nascent protein networks.
    DOI:  https://doi.org/10.1016/j.mcpro.2023.100497
  16. Angew Chem Int Ed Engl. 2023 Jan 17.
    Targeted Degradation of PD-L1 and Activation of the STING Pathway by Carbon-Dot-Based PROTACs for Cancer Immunotherapy.
    Wen Su, Mixiao Tan, Zhihang Wang, Jie Zhang, Wenping Huang, Haohao Song, Xingye Wang, Haitao Ran, Yanfeng Gao, Guangjun Nie, Hai Wang.
      Proteolysis targeting chimeras (PROTACs) technology is an emerging approach to degrade disease-associated proteins. Here, we report a carbon-dot (CD)-based PROTAC (CDTAC) that degrade membrane proteins via ubiquitin-proteasome system. CDTACs can bind to programmed cell death ligand 1 (PD-L1), recruit cereblon (CRBN) to induce PD-L1 ubiquitination, and degrade them with proteasomes. Fasting-mimicking diet (FMD) is also used to enhance the cellular uptake and proteasome activity. More than 99% or 90% of PD-L1 in CT26 or B16-F10 tumor cells can be degraded by CDTACs, respectively. Furthermore, CDTACs can activate the stimulator of interferon genes (STING) pathway to trigger immune responses. Thus, CDTACs with FMD treatment effectively inhibit the growth of CT26 and B16-F10 tumors. Compared with small molecule-based PROTACs, CDTACs offer several advantages, such as efficient membrane protein degradation, targeted tumor accumulation, immune system activation, and in vivo detection.
    Keywords:  Cancer therapy; Carbon dots; Nanomedicine; PROTACs
    DOI:  https://doi.org/10.1002/anie.202218128
  17. Cell Chem Biol. 2023 Jan 19. pii: S2451-9456(22)00457-3. [Epub ahead of print]30(1): 3-21
    Inhibitors of the ATPase p97/VCP: From basic research to clinical applications.
    Susan Kilgas, Kristijan Ramadan.
      Protein homeostasis deficiencies underlie various cancers and neurodegenerative diseases. The ubiquitin-proteasome system (UPS) and autophagy are responsible for most of the protein degradation in mammalian cells and, therefore, represent attractive targets for cancer therapy and that of neurodegenerative diseases. The ATPase p97, also known as VCP, is a central component of the UPS that extracts and disassembles its substrates from various cellular locations and also regulates different steps in autophagy. Several UPS- and autophagy-targeting drugs are in clinical trials. In this review, we focus on the development of various p97 inhibitors, including the ATPase inhibitors CB-5083 and CB-5339, which reached clinical trials by demonstrating effective anti-tumor activity across various tumor models, providing an effective alternative to targeting protein degradation for cancer therapy. Here, we provide an overview of how different p97 inhibitors have evolved over time both as basic research tools and effective UPS-targeting cancer therapies in the clinic.
    Keywords:  CB-5083; CB-5339; anti-inflammatory; antimicrobial; cancer therapy; p97 inhibitors; p97/VCP
    DOI:  https://doi.org/10.1016/j.chembiol.2022.12.007
  18. Nature. 2023 Jan 18.
    A molecular network of conserved factors keeps ribosomes dormant in the egg.
    Friederike Leesch, Laura Lorenzo-Orts, Carina Pribitzer, Irina Grishkovskaya, Josef Roehsner, Anastasia Chugunova, Manuel Matzinger, Elisabeth Roitinger, Katarina Belačić, Susanne Kandolf, Tzi-Yang Lin, Karl Mechtler, Anton Meinhart, David Haselbach, Andrea Pauli.
      Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed1-4. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4-eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1 in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.
    DOI:  https://doi.org/10.1038/s41586-022-05623-y
  19. Cell Rep. 2023 Jan 10. pii: S2211-1247(22)01869-1. [Epub ahead of print] 111965
    Protein folding stress potentiates NLRP1 and CARD8 inflammasome activation.
    Elizabeth L Orth-He, Hsin-Che Huang, Sahana D Rao, Qinghui Wang, Qifeng Chen, Claire M O'Mara, Ashley J Chui, Michelle Saoi, Andrew R Griswold, Abir Bhattacharjee, Daniel P Ball, Justin R Cross, Daniel A Bachovchin.
      NLRP1 and CARD8 are related pattern-recognition receptors (PRRs) that detect intracellular danger signals and form inflammasomes. Both undergo autoproteolysis, generating N-terminal (NT) and C-terminal (CT) fragments. The proteasome-mediated degradation of the NT releases the CT from autoinhibition, but the stimuli that trigger NT degradation have not been fully elucidated. Here, we show that several distinct agents that interfere with protein folding, including aminopeptidase inhibitors, chaperone inhibitors, and inducers of the unfolded protein response, accelerate NT degradation. However, these agents alone do not trigger inflammasome formation because the released CT fragments are physically sequestered by the serine dipeptidase DPP9. We show that DPP9-binding ligands must also be present to disrupt these complexes and allow the CT fragments to oligomerize into inflammasomes. Overall, these results indicate that NLRP1 and CARD8 detect a specific perturbation that induces both protein folding stress and DPP9 ligand accumulation.
    Keywords:  CARD8; CP: Immunology; DPP8/9; NLRP1; aminopeptidases; inflammasomes; peptides; protein folding
    DOI:  https://doi.org/10.1016/j.celrep.2022.111965
  20. Proc Natl Acad Sci U S A. 2023 Jan 24. 120(4): e2208941120
    Structural remodeling of AAA+ ATPase p97 by adaptor protein ASPL facilitates posttranslational methylation by METTL21D.
    Saša Petrović, Yvette Roske, Biria Rami, Mai Hoang Quynh Phan, Daniela Panáková, Udo Heinemann.
      p97 is an essential AAA+ ATPase that extracts and unfolds substrate proteins from membranes and protein complexes. Through its mode of action, p97 contributes to various cellular processes, such as membrane fusion, ER-associated protein degradation, DNA repair, and many others. Diverse p97 functions and protein interactions are regulated by a large number of adaptor proteins. Alveolar soft part sarcoma locus (ASPL) is a unique adaptor protein that regulates p97 by disassembling functional p97 hexamers to smaller entities. An alternative mechanism to regulate the activity and interactions of p97 is by posttranslational modifications (PTMs). Although more than 140 PTMs have been identified in p97, only a handful of those have been described in detail. Here we present structural and biochemical data to explain how the p97-remodeling adaptor protein ASPL enables the metastasis promoting methyltransferase METTL21D to bind and trimethylate p97 at a single lysine side chain, which is deeply buried inside functional p97 hexamers. The crystal structure of a heterotrimeric p97:ASPL:METTL21D complex in the presence of cofactors ATP and S-adenosyl homocysteine reveals how structural remodeling by ASPL exposes the crucial lysine residue of p97 to facilitate its trimethylation by METTL21D. The structure also uncovers a role of the second region of homology (SRH) present in the first ATPase domain of p97 in binding of a modifying enzyme to the AAA+ ATPase. Investigation of this interaction in the human, fish, and plant reveals fine details on the mechanism and significance of p97 trimethylation by METTL21D across different organisms.
    Keywords:  AAA+ ATPase; p97; protein methylation; protein remodeling
    DOI:  https://doi.org/10.1073/pnas.2208941120
  21. Autophagy. 2023 Jan 18. 1-19
    Inhibition of autophagy in microglia and macrophages exacerbates innate immune responses and worsens brain injury outcomes.
    Nivedita Hegdekar, Chinmoy Sarkar, Sabrina Bustos, Rodney M Ritzel, Marie Hanscom, Prarthana Ravishankar, Deepika Philkana, Junfang Wu, David J Loane, Marta M Lipinski.
      Excessive and prolonged neuroinflammation following traumatic brain injury (TBI) contributes to long-term tissue damage and poor functional outcomes. However, the mechanisms contributing to exacerbated inflammatory responses after brain injury remain poorly understood. Our previous work showed that macroautophagy/autophagy flux is inhibited in neurons following TBI in mice and contributes to neuronal cell death. In the present study, we demonstrate that autophagy is also inhibited in activated microglia and infiltrating macrophages, and that this potentiates injury-induced neuroinflammatory responses. Macrophage/microglia-specific knockout of the essential autophagy gene Becn1 led to overall increase in neuroinflammation after TBI. In particular, we observed excessive activation of the innate immune responses, including both the type-I interferon and inflammasome pathways. Defects in microglial and macrophage autophagy following injury were associated with decreased phagocytic clearance of danger/damage-associated molecular patterns (DAMP) responsible for activation of the cellular innate immune responses. Our data also demonstrated a role for precision autophagy in targeting and degradation of innate immune pathways components, such as the NLRP3 inflammasome. Finally, inhibition of microglial/macrophage autophagy led to increased neurodegeneration and worse long-term cognitive outcomes after TBI. Conversely, increasing autophagy by treatment with rapamycin decreased inflammation and improved outcomes in wild-type mice after TBI. Overall, our work demonstrates that inhibition of autophagy in microglia and infiltrating macrophages contributes to excessive neuroinflammation following brain injury and in the long term may prevent resolution of inflammation and tissue regeneration.Abbreviations: Becn1/BECN1, beclin 1, autophagy related; CCI, controlled cortical impact; Cybb/CYBB/NOX2: cytochrome b-245, beta polypeptide; DAMP, danger/damage-associated molecular patterns; Il1b/IL1B/Il-1β, interleukin 1 beta; LAP, LC3-associated phagocytosis; Map1lc3b/MAP1LC3/LC3, microtubule-associated protein 1 light chain 3 beta; Mefv/MEFV/TRIM20: Mediterranean fever; Nos2/NOS2/iNOS: nitric oxide synthase 2, inducible; Nlrp3/NLRP3, NLR family, pyrin domain containing 3; Sqstm1/SQSTM1/p62, sequestosome 1; TBI, traumatic brain injury; Tnf/TNF/TNF-α, tumor necrosis factor; Ulk1/ULK1, unc-51 like kinase 1.
    Keywords:  Autophagy; innate immunity; macrophage; microglia; neuroinflammation; traumatic brain injury
    DOI:  https://doi.org/10.1080/15548627.2023.2167689
  22. Mol Syst Biol. 2023 Jan 18. e11084
    A toolbox for systematic discovery of stable and transient protein interactors in baker's yeast.
    Emma J Fenech, Nir Cohen, Meital Kupervaser, Zohar Gazi, Maya Schuldiner.
      Identification of both stable and transient interactions is essential for understanding protein function and regulation. While assessing stable interactions is more straightforward, capturing transient ones is challenging. In recent years, sophisticated tools have emerged to improve transient interactor discovery, with many harnessing the power of evolved biotin ligases for proximity labelling. However, biotinylation-based methods have lagged behind in the model eukaryote, Saccharomyces cerevisiae, possibly due to the presence of several abundant, endogenously biotinylated proteins. In this study, we optimised robust biotin-ligation methodologies in yeast and increased their sensitivity by creating a bespoke technique for downregulating endogenous biotinylation, which we term ABOLISH (Auxin-induced BiOtin LIgase diminiSHing). We used the endoplasmic reticulum insertase complex (EMC) to demonstrate our approaches and uncover new substrates. To make these tools available for systematic probing of both stable and transient interactions, we generated five full-genome collections of strains in which every yeast protein is tagged with each of the tested biotinylation machineries, some on the background of the ABOLISH system. This comprehensive toolkit enables functional interactomics of the entire yeast proteome.
    Keywords:  TurboID; interaction profiling; substrate discovery; yeast libraries
    DOI:  https://doi.org/10.15252/msb.202211084
  23. J Biol Chem. 2023 Jan 12. pii: S0021-9258(23)00037-6. [Epub ahead of print] 102905
    DNA repair protein FANCD2 has both ubiquitination-dependent and -independent functions during germ cell development.
    Simin Zhao, Chengzi Huang, Yajuan Yang, Weiwei Xu, Yongze Yu, Canxin Wen, Lili Cao, Fei Gao, Yingying Qin, Zi-Jiang Chen, Ting Guo, Shidou Zhao.
      When DNA inter-strand crosslink lesions occur, a core complex of Fanconi anemia proteins promotes the ubiquitination of FANCD2 and FANCI, which recruit downstream factors to repair the lesion. However, FANCD2 maintains genome stability not only through its ubiquitination-dependent but also its ubiquitination -independent functions in various DNA damage response pathways. Increasing evidence suggests that FANCD2 is essential for fertility, but its ubiquitination-dependent and -independent roles during germ cell development are not well characterized. In this study, we analyzed germ cell development in Fancd2 knockout and ubiquitination-deficient mutant (Fancd2K559R/K559R) mice. We showed that in the embryonic stage, both the ubiquitination-dependent and -independent functions of FANCD2 were required for expansion of primordial germ cells and establishment of the reproductive reserve by reducing transcription-replication conflicts and thus maintaining genome stability in primordial germ cells. Furthermore, we found that during meiosis in spermatogenesis, FANCD2 promoted chromosome synapsis and regulated crossover formation independently of its ubiquitination, but that both ubiquitinated and non-ubiquitinated FANCD2 functioned in programmed double-strand break repair. Finally, we revealed that on meiotic XY chromosomes, H3K4me2 accumulation required ubiquitination-independent functionality of FANCD2, while the regulation of H3K9me2 and H3K9me3 depended on FANCD2 ubiquitination. Taken together, our findings suggest that FANCD2 has distinct functions that are both dependent on and independent of its ubiquitination during germ cell development.
    Keywords:  FANCD2; Fanconi anemia; infertility; meiosis; primordial germ cell
    DOI:  https://doi.org/10.1016/j.jbc.2023.102905
  24. Mol Cell. 2023 Jan 13. pii: S1097-2765(22)01213-8. [Epub ahead of print]
    De-centralizing the Central Dogma: mRNA translation in space and time.
    Ashley M Bourke, Andre Schwarz, Erin M Schuman.
      As our understanding of the cell interior has grown, we have come to appreciate that most cellular operations are localized, that is, they occur at discrete and identifiable locations or domains. These cellular domains contain enzymes, machines, and other components necessary to carry out and regulate these localized operations. Here, we review these features of one such operation: the localization and translation of mRNAs within subcellular compartments observed across cell types and organisms. We describe the conceptual advantages and the "ingredients" and mechanisms of local translation. We focus on the nature and features of localized mRNAs, how they travel and get localized, and how this process is regulated. We also evaluate our current understanding of protein synthesis machines (ribosomes) and their cadre of regulatory elements, that is, the translation factors.
    Keywords:  local translation; mRNA localization; protein synthesis; ribosome; translation factor
    DOI:  https://doi.org/10.1016/j.molcel.2022.12.030
  25. PLoS Genet. 2023 Jan 19. 19(1): e1010595
    Hepatic ribosomal protein S6 (Rps6) insufficiency results in failed bile duct development and loss of hepatocyte viability; a ribosomopathy-like phenotype that is partially p53-dependent.
    Sarah A Comerford, Elizabeth A Hinnant, Yidong Chen, Robert E Hammer.
      Defective ribosome biogenesis (RiBi) underlies a group of clinically diverse human diseases collectively known as the ribosomopathies, core manifestations of which include cytopenias and developmental abnormalities that are believed to stem primarily from an inability to synthesize adequate numbers of ribosomes and concomitant activation of p53. The importance of a correctly functioning RiBi machinery for maintaining tissue homeostasis is illustrated by the observation that, despite having a paucity of certain cell types in early life, ribosomopathy patients have an increased risk for developing cancer later in life. This suggests that hypoproliferative states trigger adaptive responses that can, over time, become maladaptive and inadvertently drive unchecked hyperproliferation and predispose to cancer. Here we describe an experimentally induced ribosomopathy in the mouse and show that a normal level of hepatic ribosomal protein S6 (Rps6) is required for proper bile duct development and preservation of hepatocyte viability and that its insufficiency later promotes overgrowth and predisposes to liver cancer which is accelerated in the absence of the tumor-suppressor PTEN. We also show that the overexpression of c-Myc in the liver ameliorates, while expression of a mutant hyperstable form of p53 partially recapitulates specific aspects of the hepatopathies induced by Rps6 deletion. Surprisingly, co-deletion of p53 in the Rps6-deficient background fails to restore biliary development or significantly improve hepatic function. This study not only reveals a previously unappreciated dependence of the developing liver on adequate levels of Rps6 and exquisitely controlled p53 signaling, but suggests that the increased cancer risk in ribosomopathy patients may, in part, stem from an inability to preserve normal tissue homeostasis in the face of chronic injury and regeneration.
    DOI:  https://doi.org/10.1371/journal.pgen.1010595
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