bims-proteo Biomed News
on Proteostasis
Issue of 2023‒09‒10
thirty-one papers selected by
Eric Chevet, INSERM
  1. Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10.
  2. eIF3d controls the persistent integrated stress response.
  3. Activation of the integrated stress response by inhibitors of its kinases.
  4. Ubiquitin-driven protein condensation promotes clathrin-mediated endocytosis.
  5. The nonautophagic functions of autophagy-related proteins.
  6. Autophagy-mediated surveillance of Rim4-mRNA interaction safeguards programmed meiotic translation.
  7. The N-end rule pathway regulates ER stress-induced clusterin release to the cytosol where it directs misfolded proteins for degradation.
  8. Degradation of Cyclin-Dependent Kinase 9/Cyclin T1 by Optimized Microtubule-Associated Protein 1 Light Chain 3 Beta-Recruiting Coumarin Analogs.
  9. Multifaceted membrane interactions of human Atg3 promote LC3-phosphatidylethanolamine conjugation during autophagy.
  10. Ufmylation on UFBP1 alleviates non-alcoholic fatty liver disease by modulating hepatic endoplasmic reticulum stress.
  11. The interaction network of the proteasome assembly chaperone PSMD9 regulates proteostasis.
  12. TMEM127 suppresses tumor development by promoting RET ubiquitination, positioning, and degradation.
  13. 3D based generative PROTAC linker design with reinforcement learning.
  14. The separate axes of TECPR1 and ATG16L1 in CASM.
  15. USP2 promotes tumor immune evasion via deubiquitination and stabilization of PD-L1.
  16. Surf4 collaborates with Derlin-2 and Derlin-1 to mediate Cyclooxygenase-2 translocation to the cytosol for degradation.
  17. Avidity-based biosensors for ubiquitylated PCNA reveal choreography of DNA damage bypass.
  18. Design principles for cyclin K molecular glue degraders.
  19. A Biotin Targeting Chimera (BioTAC) System to Map Small Molecule Interactomes in situ.
  20. Structure shows that the BIR2 domain of E3 ligase XIAP binds across the RIPK2 kinase dimer interface.
  21. The endoplasmic reticulum-localized enzyme zDHHC6 mediates S-acylation of short transmembrane constructs from multiple type I and II membrane proteins.
  22. Deciphering the protein ubiquitylation system in plants.
  23. Mammalian autophagosomes form from finger-like phagophores.
  24. The RasGAP-associated endoribonuclease G3BP mediates stress granule assembly.
  25. Lon degrades stable substrates slowly but with enhanced processivity, redefining the attributes of a successful AAA+ protease.
  26. Mechanisms and pathology of protein misfolding and aggregation.
  27. Modulation of the proteostasis network promotes tumor resistance to oncogenic KRAS inhibitors.
  28. Orchestrating vesicular and nonvesicular membrane dynamics by intrinsically disordered proteins.
  29. Caspase-mediated nuclear pore complex trimming in cell differentiation and endoplasmic reticulum stress.
  30. Deciphering the functional landscape of phosphosites with deep neural network.
  31. Lysosomes mediate the mitochondrial UPR via mTORC1-dependent ATF4 phosphorylation.
  1. Cell Rep. 2023 Sep 06. pii: S2211-1247(23)01111-7. [Epub ahead of print]42(9): 113100
    Mechanism and evolutionary origins of alanine-tail C-degron recognition by E3 ligases Pirh2 and CRL2-KLHDC10.
    Pratik Rajendra Patil, A Maxwell Burroughs, Mohit Misra, Federico Cerullo, Carlos Costas Insua, Hao-Chih Hung, Ivan Dikic, L Aravind, Claudio A P Joazeiro.
      In ribosome-associated quality control (RQC), nascent polypeptides produced by interrupted translation are modified with C-terminal polyalanine tails ("Ala-tails") that function outside ribosomes to induce ubiquitylation by E3 ligases Pirh2 (p53-induced RING-H2 domain-containing) or CRL2 (Cullin-2 RING ligase2)-KLHDC10. Here, we investigate the molecular basis of Ala-tail function using biochemical and in silico approaches. We show that Pirh2 and KLHDC10 directly bind to Ala-tails and that structural predictions identify candidate Ala-tail-binding sites, which we experimentally validate. The degron-binding pockets and specific pocket residues implicated in Ala-tail recognition are conserved among Pirh2 and KLHDC10 homologs, suggesting that an important function of these ligases across eukaryotes is in targeting Ala-tailed substrates. Moreover, we establish that the two Ala-tail-binding pockets have convergently evolved, either from an ancient module of bacterial provenance (Pirh2) or via tinkering of a widespread C-degron-recognition element (KLHDC10). These results shed light on the recognition of a simple degron sequence and the evolution of Ala-tail proteolytic signaling.
    Keywords:  Ala-tail; C-end degron; CP: Molecular biology; E3 ubiquitin ligase; KLHDC10; Pirh2; polyalanine; protein degradation; protein quality control; protein-protein interaction; structure prediction
    DOI:  https://doi.org/10.1016/j.celrep.2023.113100
  2. Mol Cell. 2023 Aug 30. pii: S1097-2765(23)00643-3. [Epub ahead of print]
    eIF3d controls the persistent integrated stress response.
    Shaoni Mukhopadhyay, Maria E Amodeo, Amy S Y Lee.
      Cells respond to intrinsic and extrinsic stresses by reducing global protein synthesis and activating gene programs necessary for survival. Here, we show that the integrated stress response (ISR) is driven by the non-canonical cap-binding protein eIF3d that acts as a critical effector to control core stress response orchestrators, the translation factor eIF2α and the transcription factor ATF4. We find that during persistent stress, eIF3d activates the translation of the kinase GCN2, inducing eIF2α phosphorylation and inhibiting general protein synthesis. In parallel, eIF3d upregulates the m6A demethylase ALKBH5 to drive 5' UTR-specific demethylation of stress response genes, including ATF4. Ultimately, this cascade converges on ATF4 expression by increasing mRNA engagement of translation machinery and enhancing ribosome bypass of upstream open reading frames (uORFs). Our results reveal that eIF3d acts in a life-or-death decision point during chronic stress and uncover a synergistic signaling mechanism in which translational cascades complement transcriptional amplification to control essential cellular processes.
    Keywords:  ATF4; GCN2; RNA methylation; eIF3d; integrated stress response; m(6)A; translation regulation
    DOI:  https://doi.org/10.1016/j.molcel.2023.08.008
  3. Nat Commun. 2023 Sep 08. 14(1): 5535
    Activation of the integrated stress response by inhibitors of its kinases.
    Maria Szaruga, Dino A Janssen, Claudia de Miguel, George Hodgson, Agnieszka Fatalska, Aleksandra P Pitera, Antonina Andreeva, Anne Bertolotti.
      Phosphorylation of the translation initiation factor eIF2α to initiate the integrated stress response (ISR) is a vital signalling event. Protein kinases activating the ISR, including PERK and GCN2, have attracted considerable attention for drug development. Here we find that the widely used ATP-competitive inhibitors of PERK, GSK2656157, GSK2606414 and AMG44, inhibit PERK in the nanomolar range, but surprisingly activate the ISR via GCN2 at micromolar concentrations. Similarly, a PKR inhibitor, C16, also activates GCN2. Conversely, GCN2 inhibitor A92 silences its target but induces the ISR via PERK. These findings are pivotal for understanding ISR biology and its therapeutic manipulations because most preclinical studies used these inhibitors at micromolar concentrations. Reconstitution of ISR activation with recombinant proteins demonstrates that PERK and PKR inhibitors directly activate dimeric GCN2, following a Gaussian activation-inhibition curve, with activation driven by allosterically increasing GCN2 affinity for ATP. The tyrosine kinase inhibitors Neratinib and Dovitinib also activate GCN2 by increasing affinity of GCN2 for ATP. Thus, the mechanism uncovered here might be broadly relevant to ATP-competitive inhibitors and perhaps to other kinases.
    DOI:  https://doi.org/10.1038/s41467-023-40823-8
  4. bioRxiv. 2023 Aug 22. pii: 2023.08.21.554139. [Epub ahead of print]
    Ubiquitin-driven protein condensation promotes clathrin-mediated endocytosis.
    Feng Yuan, Sadhana Gollapudi, Kasey J Day, Grant Ashby, Arjun Sangani, Brandon T Malady, Liping Wang, Eileen M Lafer, Jon M Huibregtse, Jeanne C Stachowiak.
      Clathrin-mediated endocytosis is an essential cellular pathway that enables signaling and recycling of transmembrane proteins and lipids. During endocytosis, dozens of cytosolic proteins come together at the plasma membrane, assembling into a highly interconnected network that drives endocytic vesicle biogenesis. Recently, multiple labs have reported that early endocytic proteins form liquid-like condensates, which provide a flexible platform for the efficient assembly of endocytic vesicles. Given the importance of this network in the dynamics of endocytosis, how might cells regulate its stability? Many receptors and endocytic proteins are ubiquitylated, while early endocytic proteins such as Eps15 contain ubiquitin-interacting motifs. Therefore, we examined the influence of ubiquitin on the stability of the early endocytic protein network. In vitro, we found that recruitment of small amounts of polyubiquitin dramatically increased the stability of Eps15 condensates, suggesting that ubiquitylation could nucleate endocytic sites. In live cell imaging experiments, a version of Eps15 that lacked the ubiquitin-interacting motif failed to rescue defects in endocytic initiation created by Eps15 knockout. Furthermore, fusion of Eps15 to a deubiquitinase enzyme destabilized nascent endocytic sites within minutes. These results suggest that ubiquitylation drives assembly of the flexible protein network responsible for catalyzing endocytic events. More broadly, this work illustrates a biophysical mechanism by which ubiquitylated transmembrane proteins at the plasma membrane could regulate the efficiency of endocytic recycling.
    DOI:  https://doi.org/10.1101/2023.08.21.554139
  5. Autophagy. 2023 Sep 08. 1-15
    The nonautophagic functions of autophagy-related proteins.
    Jia-Ni Shang, Chen-Ge Yu, Rui Li, Yan Xi, Yue Jenny Jian, Nan Xu, Su Chen.
      ABBREVIATIONS: ATG: autophagy related; BECN1: beclin 1; cAMP: cyclic adenosine monophosphate; dsDNA: double-stranded DNA; EMT: epithelial-mesenchymal transition; IFN: interferon; ISCs: intestinal stem cells; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAPK/JNK: mitogen-activated protein kinase/c-Jun N-terminal kinases; MTOR: mechanistic target of rapamycin kinase; STING1: stimulator of interferon response cGAMP interactor 1; UVRAG: UV radiation resistance associated; VPS: vacuolar protein sorting.
    Keywords:  autophagy-related proteins; cargo transport; cell growth and senescence; metabolic homeostasis; nonautophagic functions; signal transduction
    DOI:  https://doi.org/10.1080/15548627.2023.2254664
  6. Cell Rep. 2023 Sep 01. pii: S2211-1247(23)01062-8. [Epub ahead of print]42(9): 113051
    Autophagy-mediated surveillance of Rim4-mRNA interaction safeguards programmed meiotic translation.
    Rudian Zhang, Wenzhi Feng, Suhong Qian, Fei Wang.
      In yeast meiosis, autophagy is active and essential. Here, we investigate the fate of Rim4, a meiosis-specific RNA-binding protein (RBP), and its associated transcripts during meiotic autophagy. We demonstrate that Rim4 employs a nuclear localization signal (NLS) to enter the nucleus, where it loads its mRNA substrates before nuclear export. Upon reaching the cytoplasm, active autophagy selectively spares the Rim4-mRNA complex. During meiotic divisions, autophagy preferentially degrades Rim4 in an Atg11-dependent manner, coinciding with the release of Rim4-bound mRNAs for translation. Intriguingly, these released mRNAs also become vulnerable to autophagy. In vitro, purified Rim4 and its RRM-motif-containing variants activate Atg1 kinase in meiotic cell lysates and in immunoprecipitated (IP) Atg1 complexes. This suggests that the conserved RNA recognition motifs (RRMs) of Rim4 are involved in stimulating Atg1 and thereby facilitating selective autophagy. Taken together, our findings indicate that autophagy surveils Rim4-mRNA interaction to ensure stage-specific translation during meiosis.
    Keywords:  Atg1; Atg11; CP: Cell biology; Pab1; RBP; Rim4; mRNAs; meiosis; nucleus; selective autophagy
    DOI:  https://doi.org/10.1016/j.celrep.2023.113051
  7. Cell Rep. 2023 Sep 01. pii: S2211-1247(23)01070-7. [Epub ahead of print]42(9): 113059
    The N-end rule pathway regulates ER stress-induced clusterin release to the cytosol where it directs misfolded proteins for degradation.
    Sandeep Satapathy, Holly Walker, James Brown, Yann Gambin, Mark R Wilson.
      Previous work suggests that cell stress induces release of the normally secreted chaperone clusterin (CLU) into the cytosol. We analyzed the localization of CLU in healthy and stressed cells, the mechanism of its cytosolic release, and its interactions with cytosolic misfolded proteins. Key results of this study are the following: (1) full-length CLU is released to the cytosol during stress, (2) the CLU N-terminal D1 residue is recognized by the N-end rule pathway and together with the enzyme ATE1 is essential for cytosolic release, (3) CLU can form stable complexes with cytosolic misfolded proteins and direct them to the proteasome and autophagosomes, and (4) cytosolic CLU protects cells from hypoxic stress and the cytosolic overexpression of an aggregation-prone protein. Collectively, the results suggest that enhanced cytosolic release of CLU is a stress response that can inhibit the toxicity of misfolded proteins and facilitate their targeted degradation via both autophagy and the proteasome.
    Keywords:  CP: Cell biology; N-End Rule Pathway; autophagy; clusterin; cytoprotection; cytosolic release; intracellular proteostasis; proteasome
    DOI:  https://doi.org/10.1016/j.celrep.2023.113059
  8. J Med Chem. 2023 Sep 06.
    Degradation of Cyclin-Dependent Kinase 9/Cyclin T1 by Optimized Microtubule-Associated Protein 1 Light Chain 3 Beta-Recruiting Coumarin Analogs.
    Yanping Zeng, Jian Xiao, Yuanxin Xu, Fan Wei, Lina Tian, Yinglei Gao, Yi Chen, Youhong Hu.
      Autophagy is an efficient and attractive protein degradation pathway in addition to the ubiquitin-proteasome system. Herein, systematic optimization of coumarin analogs linked with the CDK9 inhibitor SNS-032 is reported that may bind to cyclin-dependent kinase 9 (CDK9) and microtubule-associated protein 1 light chain 3 beta (LC3B) simultaneously, which leads to the selective autophagic degradation of targeted CDK9/cyclin T1 and is different from the PROTAC degrader THAL-SNS-032. Further mechanism studies revealed an autophagy-lysosome pathway, where the degraders possibly formed a ternary complex with CDK9 and LC3B. In addition, degrader 10 showed antitumor efficacy in vivo. Our work optimized a potent LC3B recruiter and demonstrated the feasibility of autophagy-tethering compounds (ATTECs), which could be applied for the degradation of diverse intracellular pathogenic proteins to treat related diseases.
    DOI:  https://doi.org/10.1021/acs.jmedchem.3c00828
  9. Nat Commun. 2023 Sep 07. 14(1): 5503
    Multifaceted membrane interactions of human Atg3 promote LC3-phosphatidylethanolamine conjugation during autophagy.
    Yansheng Ye, Erin R Tyndall, Van Bui, Maria C Bewley, Guifang Wang, Xupeng Hong, Yang Shen, John M Flanagan, Hong-Gang Wang, Fang Tian.
      Autophagosome formation, a crucial step in macroautophagy (autophagy), requires the covalent conjugation of LC3 proteins to the amino headgroup of phosphatidylethanolamine (PE) lipids. Atg3, an E2-like enzyme, catalyzes the transfer of LC3 from LC3-Atg3 to PEs in targeted membranes. Here we show that the catalytically important C-terminal regions of human Atg3 (hAtg3) are conformationally dynamic and directly interact with the membrane, in collaboration with its N-terminal membrane curvature-sensitive helix. The functional relevance of these interactions was confirmed by in vitro conjugation and in vivo cellular assays. Therefore, highly curved phagophoric rims not only serve as a geometric cue for hAtg3 recruitment, but also their interaction with hAtg3 promotes LC3-PE conjugation by targeting its catalytic center to the membrane surface and bringing substrates into proximity. Our studies advance the notion that autophagosome biogenesis is directly guided by the spatial interactions of Atg3 with highly curved phagophoric rims.
    DOI:  https://doi.org/10.1038/s41467-023-41243-4
  10. Cell Death Dis. 2023 09 02. 14(9): 584
    Ufmylation on UFBP1 alleviates non-alcoholic fatty liver disease by modulating hepatic endoplasmic reticulum stress.
    Ziming Mao, Xiaowen Ma, Yu Jing, Minyan Shen, Xirui Ma, Jing Zhu, Huifang Liu, Guangya Zhang, Fengling Chen.
      Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease characterized by lipid accumulation and endoplasmic reticulum (ER) stress, while effective therapies targeting the specific characteristics of NAFLD are limited. Ufmylation is a newly found post-translational modification process that involves the attachment of the Ubiquitin-fold modifier 1 (UFM1) protein to its substrates via ufmylation modification system. Ufmylation regulates ER stress via modifying UFM1 binding protein 1 (UFBP1), suggesting a potential role for ufmylation in NAFLD pathogenesis. However, the precise role of ufmylation in NAFLD remains unclear. Herein, we aim to elucidate the impact of ufmylation on UFBP1 in NAFLD and explore the underlying mechanisms involved. We observed increased expression of UFM1-conjugated proteins and ufmylation modification system components in livers with steatosis derived from NAFLD patients and NAFLD models. Upregulation of ufmylation on hepatic proteins appeared to be an adaptive response to hepatic ER stress in NAFLD. In vitro, knocking down UFBP1 resulted in increased lipid accumulation and lipogenesis in hepatocytes treated with free fatty acids (FFA), which could be rescued by wild-type UFBP1 (WT UFBP1) but not by a mutant form of UFBP1 lacking the main ufmylation site lys267 (UFBP1 K267R). In vivo, ufmylation on UFBP1 ameliorated obesity, hepatic steatosis, hepatic lipogenesis, dyslipidemia, insulin resistance and liver damage in mice with NAFLD induced by a high fat diet (HFD). We also demonstrated that the downregulation of UFBP1 induced ER stress, whereas the reintroduction or overexpression of UFBP1 alleviated ER stress in a manner dependent on ufmylation in NAFLD. This mechanism could be responsible for the amelioration of aberrant hepatic lipogenesis and insulin resistance in NAFLD. Our data reveal a protective role of ufmylation on UFBP1 against NAFLD and offer a specific target for NAFLD treatment.
    DOI:  https://doi.org/10.1038/s41419-023-06095-2
  11. FEBS J. 2023 Sep 04.
    The interaction network of the proteasome assembly chaperone PSMD9 regulates proteostasis.
    Joel Christie, C Merlyn Anthony, Mahalakshmi Harish, Deepti Mudartha, Sheikh Burhan Ud Din Farooqee, Prasanna Venkatraman.
      Functional networks in cells are created by physical, genetic and regulatory interactions. Mapping them and annotating their functions by available methods remains a challenge. We use affinity purification mass spectrometry (AP-MS) coupled with SLiMFinder to discern such a network involving 26S proteasome non-ATPase regulatory subunit 9 (PSMD9), a chaperone of proteasome assembly. Approximately 20% of proteins within the PSMD9 interactome carry a short linear motif (SLiM) of the type 'EXKK'. The binding of purified PSMD9 with the peptide sequence ERKK, proteins heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNPA2B1; containing ERKK) and peroxiredoxin-6 (PRDX6; containing EAKK) provided proof of principle for this motif-driven network. The EXKK motif in the peptide primarily interacts with the coiled-coil N domain of PSMD9, a unique interaction not reported for any coiled-coil domain. PSMD9 knockout (KO) HEK293 cells experience endoplasmic reticulum (ER) stress and respond by increasing the unfolded protein response (UPR) and reducing the formation of aggresomes and lipid droplets. Trans-expression of PSMD9 in the KO cells rescues lipid droplet formation. Overexpression of PSMD9 in HEK293 cells results in reduced UPR, and increased lipid droplet and aggresome formation. The outcome argues for the prominent role of PSMD9 in maintaining proteostasis. Probable mechanisms involve the binding of PSMD9 to binding immunoglobulin protein (BIP/GRP78; containing EDKK), an endoplasmic reticulum chaperone and key regulator of the UPR, and fatty acid synthase (FASN; containing ELKK), involved in fatty acid synthesis/lipid biogenesis. We propose that PSMD9 acts as a buffer in the cellular milieu by moderating the UPR and enhancing aggresome formation to reduce stress-induced proteotoxicity. Akin to waves created in ponds that perpetuate to a distance, perturbing the levels of PSMD9 would cause ripples down the networks, affecting final reactions in the pathway, one of which is altered proteostasis.
    Keywords:  PSMD9; proteasome; protein-protein interaction; proteostasis; short linear motif
    DOI:  https://doi.org/10.1111/febs.16948
  12. Cell Rep. 2023 Sep 01. pii: S2211-1247(23)01081-1. [Epub ahead of print]42(9): 113070
    TMEM127 suppresses tumor development by promoting RET ubiquitination, positioning, and degradation.
    Qianjin Guo, Zi-Ming Cheng, Hector Gonzalez-Cantú, Matthew Rotondi, Gabriela Huelgas-Morales, Purushoth Ethiraj, Zhijun Qiu, Jonathan Lefkowitz, Wan Song, Bethany N Landry, Hector Lopez, Cynthia M Estrada-Zuniga, Shivi Goyal, Mohammad Aasif Khan, Timothy J Walker, Exing Wang, Faqian Li, Yanli Ding, Lois M Mulligan, Ricardo C T Aguiar, Patricia L M Dahia.
      The TMEM127 gene encodes a transmembrane protein of poorly known function that is mutated in pheochromocytomas, neural crest-derived tumors of adrenomedullary cells. Here, we report that, at single-nucleus resolution, TMEM127-mutant tumors share precursor cells and transcription regulatory elements with pheochromocytomas carrying mutations of the tyrosine kinase receptor RET. Additionally, TMEM127-mutant pheochromocytomas, human cells, and mouse knockout models of TMEM127 accumulate RET and increase its signaling. TMEM127 contributes to RET cellular positioning, trafficking, and lysosome-mediated degradation. Mechanistically, TMEM127 binds to RET and recruits the NEDD4 E3 ubiquitin ligase for RET ubiquitination and degradation via TMEM127 C-terminal PxxY motifs. Lastly, increased cell proliferation and tumor burden after TMEM127 loss can be reversed by selective RET inhibitors in vitro and in vivo. Our results define TMEM127 as a component of the ubiquitin system and identify aberrant RET stabilization as a likely mechanism through which TMEM127 loss-of-function mutations cause pheochromocytoma.
    Keywords:  CP: Cancer; NEDD4; RET; TMEM127; degradation; oncogene; paraganglioma; pheochromocytoma; positioning; single-nucleus sequencing; tumor suppressor gene; ubiquitin
    DOI:  https://doi.org/10.1016/j.celrep.2023.113070
  13. Brief Bioinform. 2023 Sep 05. pii: bbad323. [Epub ahead of print]
    3D based generative PROTAC linker design with reinforcement learning.
    Baiqing Li, Ting Ran, Hongming Chen.
      Proteolysis targeting chimera (PROTAC), has emerged as an effective modality to selectively degrade disease-related proteins by harnessing the ubiquitin-proteasome system. Due to PROTACs' hetero-bifunctional characteristics, in which a linker joins a warhead binding to a protein of interest (POI), conferring specificity and a E3-ligand binding to an E3 ubiquitin ligase, this could trigger the ubiquitination and transportation of POI to the proteasome, followed by degradation. The rational PROTAC linker design is challenging due to its relatively large molecular weight and the complexity of maintaining the binding mode of warhead and E3-ligand in the binding pockets of counterpart. Conventional linker generation method can only generate linkers in either 1D SMILES or 2D graph, without taking into account the information of ternary structures. Here we propose a novel 3D linker generative model PROTAC-INVENT which can not only generate SMILES of PROTAC but also its 3D putative binding conformation coupled with the target protein and the E3 ligase. The model is trained jointly with the RL approach to bias the generation of PROTAC structures toward pre-defined 2D and 3D based properties. Examples were provided to demonstrate the utility of the model for generating reasonable 3D conformation of PROTACs. On the other hand, our results show that the associated workflow for 3D PROTAC conformation generation can also be used as an efficient docking protocol for PROTACs.
    Keywords:  PROTAC linker design; generative model; reinforcement learning
    DOI:  https://doi.org/10.1093/bib/bbad323
  14. Autophagy. 2023 Sep 07. 1-2
    The separate axes of TECPR1 and ATG16L1 in CASM.
    Namrita Kaur, Sven R Carlsson, Alf Håkon Lystad.
      Conjugation of ATG8 to single membranes (CASM) is a fundamental cellular process that entails the conjugation of mammalian Atg8 homologs, here referred to as ATG8, to phosphatidylethanolamine (PE) and phosphatidylserine (PS) on endolysosomal compartments. Our current research, together with recent reports from the Randow, Wu, and Wileman labs, has uncovered yet another layer to this process. We discovered that, in addition to ATG16L1-containing complexes, TECPR1 (tectonin beta-propeller repeat containing 1)-containing ATG12-ATG5 E3 complexes can facilitate CASM, thereby providing a broader understanding of this pathway.
    Keywords:  CASM; DysF; SopF; membrane damage; non-canonical autophagy; sphingomyelin
    DOI:  https://doi.org/10.1080/15548627.2023.2255462
  15. Cell Death Differ. 2023 Sep 05.
    USP2 promotes tumor immune evasion via deubiquitination and stabilization of PD-L1.
    Zean Kuang, Xiaojia Liu, Na Zhang, Jingwen Dong, Cuicui Sun, Mingxiao Yin, Yuting Wang, Lu Liu, Dian Xiao, Xinbo Zhou, Yanchun Feng, Danqing Song, Hongbin Deng.
      The abnormal upregulation of programmed death ligand-1 (PD-L1) on tumor cells impedes T-cell mediated cytotoxicity through PD-1 engagement, and further exploring the mechanisms regulation of PD-L1 in cancers may enhance the clinical efficacy of PD-L1 blockade. Here, using single-guide RNAs (sgRNAs) screening system, we identify ubiquitin-specific processing protease 2 (USP2) as a novel regulator of PD-L1 stabilization for tumor immune evasion. USP2 directly interacts with and increases PD-L1 abundance in colorectal and prostate cancer cells. Our results show that Thr288, Arg292 and Asp293 at USP2 control its binding to PD-L1 through deconjugating the K48-linked polyubiquitination at lysine 270 of PD-L1. Depletion of USP2 causes endoplasmic reticulum (ER)-associated degradation of PD-L1, thus attenuates PD-L1/PD-1 interaction and sensitizes cancer cells to T cell-mediated killing. Meanwhile, USP2 ablation-induced PD-L1 clearance enhances antitumor immunity in mice via increasing CD8+ T cells infiltration and reducing immunosuppressive infiltration of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), whereas PD-L1 overexpression reverses the tumor growth suppression by USP2 silencing. USP2-depletion combination with anti-PD-1 also exhibits a synergistic anti-tumor effect. Furthermore, analysis of clinical tissue samples indicates that USP2 is positively associated with PD-L1 expression in cancer. Collectively, our data reveal a crucial role of USP2 for controlling PD-L1 stabilization in tumor cells, and highlight USP2 as a potential therapeutic target for cancer immunotherapy.
    DOI:  https://doi.org/10.1038/s41418-023-01219-9
  16. J Cell Sci. 2023 Sep 07. pii: jcs.260995. [Epub ahead of print]
    Surf4 collaborates with Derlin-2 and Derlin-1 to mediate Cyclooxygenase-2 translocation to the cytosol for degradation.
    Shu-Fen Chen, Chun-Hu Wu, Yen-Ming Lee, Kabik Tam, Jun-Yang Liou, Song-Kun Shyue.
      Derlin family members participate in the retrotranslocation of endoplasmic reticulum (ER) lumen protein to the cytosol for ER-associated degradation (ERAD). However, the protein(s) facilitating their retrotranslocation remains to be explored. Using CRISPR library screening, we found that Derlin-2 and Surf4 were candidates to facilitate cyclooxygenase-2 (COX-2) degradation. Our results showed that Derlin-2 is the upstream of Derlin-1 and Surf4 is the downstream of Derlin-2 and Derlin-1 to facilitate COX-2 degradation. Knockdown of Derlin-2 or Surf4 impedes COX-2 ubiquitination and the interaction of COX-2 with caveolin-1 and p97 in cytosol. Additionally, COX-2 degradation is N-glycosylation-dependent. Although Derlin-2 facilitates N-glycosylated COX-2 degradation, the interaction of Derlin-2 with COX-2 is independent of COX-2 N-glycosylation. Derlin-1, Surf4 and p97 preferentially interact with non-glycosylated COX-2, while caveolin-1 preferentially interacts with N-glycosylated COX-2, regardless of the N-glycosylation patterns. Collectively, our results reveal that Surf4 collaborates with Derlin-2 and Derlin-1 to mediate COX-2 translocation from the ER lumen to the cytosol. The Derlin-2/Derlin-1/Surf4/Cav-1 machinery may represent a unique pathway to accelerate COX-2 degradation in ERAD.
    Keywords:  Cyclooxygenase-2; Derlin-1; Derlin-2; ERAD; N-glycosylation; Surfeit 4
    DOI:  https://doi.org/10.1242/jcs.260995
  17. Sci Adv. 2023 Sep 08. 9(36): eadf3041
    Avidity-based biosensors for ubiquitylated PCNA reveal choreography of DNA damage bypass.
    George Yakoub, Yun-Seok Choi, Ronald P Wong, Tina Strauch, Kezia J Ann, Robert E Cohen, Helle D Ulrich.
      In eukaryotes, the posttranslational modifier ubiquitin is used to regulate the amounts, interactions, or activities of proteins in diverse pathways and signaling networks. Its effects are mediated by monoubiquitin or polyubiquitin chains of varying geometries. We describe the design, validation, and application of a series of avidity-based probes against the ubiquitylated forms of the DNA replication clamp, proliferating cell nuclear antigen (PCNA), in budding yeast. Directed against total ubiquitylated PCNA or specifically K63-polyubiquitylated PCNA, the probes are tunable in their activities and can be used either as biosensors or as inhibitors of the PCNA-dependent DNA damage bypass pathway. Used in live cells, the probes revealed the timing of PCNA ubiquitylation during damage bypass and a particular susceptibility of the ribosomal DNA locus to the activation of the pathway. Our approach is applicable to a wide range of ubiquitin-conjugated proteins, thus representing a generalizable strategy for the design of biosensors for specific (poly)ubiquitylated forms of individual substrates.
    DOI:  https://doi.org/10.1126/sciadv.adf3041
  18. Nat Chem Biol. 2023 Sep 07.
    Design principles for cyclin K molecular glue degraders.
    Zuzanna Kozicka, Dakota J Suchyta, Vivian Focht, Georg Kempf, Georg Petzold, Marius Jentzsch, Charles Zou, Cristina Di Genua, Katherine A Donovan, Seemon Coomar, Marko Cigler, Cristina Mayor-Ruiz, Jonathan L Schmid-Burgk, Daniel Häussinger, Georg E Winter, Eric S Fischer, Mikołaj Słabicki, Dennis Gillingham, Benjamin L Ebert, Nicolas H Thomä.
      Molecular glue degraders are an effective therapeutic modality, but their design principles are not well understood. Recently, several unexpectedly diverse compounds were reported to deplete cyclin K by linking CDK12-cyclin K to the DDB1-CUL4-RBX1 E3 ligase. Here, to investigate how chemically dissimilar small molecules trigger cyclin K degradation, we evaluated 91 candidate degraders in structural, biophysical and cellular studies and reveal all compounds acquire glue activity via simultaneous CDK12 binding and engagement of DDB1 interfacial residues, in particular Arg928. While we identify multiple published kinase inhibitors as cryptic degraders, we also show that these glues do not require pronounced inhibitory properties for activity and that the relative degree of CDK12 inhibition versus cyclin K degradation is tuneable. We further demonstrate cyclin K degraders have transcriptional signatures distinct from CDK12 inhibitors, thereby offering unique therapeutic opportunities. The systematic structure-activity relationship analysis presented herein provides a conceptual framework for rational molecular glue design.
    DOI:  https://doi.org/10.1038/s41589-023-01409-z
  19. bioRxiv. 2023 Aug 22. pii: 2023.08.21.554211. [Epub ahead of print]
    A Biotin Targeting Chimera (BioTAC) System to Map Small Molecule Interactomes in situ.
    Andrew J Tao, Jiewei Jiang, Gillian E Gadbois, Pavitra Goyal, Bridget T Boyle, Elizabeth J Mumby, Samuel A Myers, Justin G English, Fleur M Ferguson.
      Unbiased chemical biology strategies for direct readout of protein interactome remodelling by small molecules provide advantages over target-focused approaches, including the ability to detect previously unknown targets, and the inclusion of chemical off-compete controls leading to high-confidence identifications. We describe the BioTAC system, a small-molecule guided proximity labelling platform, to rapidly identify both direct and complexed small molecule binding proteins. The BioTAC system overcomes a limitation of current approaches, and supports identification of both inhibitor bound and molecular glue bound complexes.
    DOI:  https://doi.org/10.1101/2023.08.21.554211
  20. Life Sci Alliance. 2023 Nov;pii: e202201784. [Epub ahead of print]6(11):
    Structure shows that the BIR2 domain of E3 ligase XIAP binds across the RIPK2 kinase dimer interface.
    Mathilde Lethier, Karine Huard, Michael Hons, Adrien Favier, Bernhard Brutscher, Elisabetta Boeri Erba, Derek W Abbott, Stephen Cusack, Erika Pellegrini.
      RIPK2 is an essential adaptor for NOD signalling and its kinase domain is a drug target for NOD-related diseases, such as inflammatory bowel disease. However, recent work indicates that the phosphorylation activity of RIPK2 is dispensable for signalling and that inhibitors of both RIPK2 activity and RIPK2 ubiquitination prevent the essential interaction between RIPK2 and the BIR2 domain of XIAP, the key RIPK2 ubiquitin E3 ligase. Moreover, XIAP BIR2 antagonists also block this interaction. To reveal the molecular mechanisms involved, we combined native mass spectrometry, NMR, and cryo-electron microscopy to determine the structure of the RIPK2 kinase BIR2 domain complex and validated the interface with in cellulo assays. The structure shows that BIR2 binds across the RIPK2 kinase antiparallel dimer and provides an explanation for both inhibitory mechanisms. It also highlights why phosphorylation of the kinase activation loop is dispensable for signalling while revealing the structural role of RIPK2-K209 residue in the RIPK2-XIAP BIR2 interaction. Our results clarify the features of the RIPK2 conformation essential for its role as a scaffold protein for ubiquitination.
    DOI:  https://doi.org/10.26508/lsa.202201784
  21. J Biol Chem. 2023 Sep 01. pii: S0021-9258(23)02229-9. [Epub ahead of print] 105201
    The endoplasmic reticulum-localized enzyme zDHHC6 mediates S-acylation of short transmembrane constructs from multiple type I and II membrane proteins.
    Christine Salaun, Nicholas C O Tomkinson, Luke H Chamberlain.
      In this study, we investigated the S-acylation of two host cell proteins important for viral infection: TMPRSS2 (transmembrane serine protease 2), which cleaves SARS-CoV-2 spike to facilitate viral entry, and BST2 (Bone marrow stromal antigen 2), a general viral restriction factor. We found that both proteins were S-acylated by zDHHC6, an S-acyltransferase enzyme localized at the endoplasmic reticulum (ER), in co-expression experiments. Mutagenic analysis revealed that zDHHC6 modifies a single cysteine in each protein, which are in proximity to the transmembrane domains (TMDs). For TMPRSS2, the modified cysteine is positioned two residues into the TMD, whereas the modified cysteine in BST2 has a cytosolic location two amino acids upstream of the TMD. Cysteine swapping revealed that repositioning the target cysteine of TMPRSS2 further into the TMD substantially reduced S-acylation by zDHHC6. Interestingly, zDHHC6 efficiently S-acylated truncated forms of these proteins that contained only the TMDs and short juxtamembrane regions. The ability of zDHHC6 to modify short TMD sequences was also seen for the transferrin receptor (another type II membrane protein) and for five different type I membrane protein constructs, including CD4. Collectively, the results of this study show that zDHHC6 can modify diverse membrane proteins (Type I and II) and requires only the presence of the TMD and target cysteine for efficient S-acylation. Thus, zDHHC6 may be a broad specificity S-acyltransferase specialized for the modification of a diverse set of transmembrane proteins at the endoplasmic reticulum.
    Keywords:  S-acylation; endoplasmic reticulum; mPEG-Click; palmitoylation; transmembrane; zDHHC enzymes; zDHHC6
    DOI:  https://doi.org/10.1016/j.jbc.2023.105201
  22. J Exp Bot. 2023 Sep 09. pii: erad354. [Epub ahead of print]
    Deciphering the protein ubiquitylation system in plants.
    Zhihua Hua.
      Protein ubiquitylation is a post-translational modification (PTM) process that covalently modifies a protein substrate with either mono-ubiquitin moieties or poly-ubiquitin chains often at the lysine residues. In Arabidopsis, bioinformatic predictions have suggested that over 5% of its proteome constitutes the protein ubiquitylation system. Despite advancements in functional genomic studies in plants, only a small fraction of this bioinformatically predicted system has been functionally characterized. To expand our understanding about the regulatory function of protein ubiquitylation to that rivaling several other major systems, such as transcription regulation and epigenetics, I described the status, issues, and new approaches of protein ubiquitylation studies in plant biology. I summarized the methods utilized in defining the ubiquitylation machinery by bioinformatics, identifying ubiquitylation substrates by proteomics, and characterizing the ubiquitin E3 ligase-substrate pathways by functional genomics. Based on the functional and evolutionary analyses of the F-box gene superfamily, I proposed a deleterious duplication model for the large expansion of this family in plant genomes. Given this model, I presented new perspectives of future functional genomic studies on the plant ubiquitylation system to focus on core and active groups of Ub E3 ligase genes.
    Keywords:  Bioinformatics; Deleterious duplications; Evolution; Functional genomics; Methods; Protein ubiquitylation; Proteomics
    DOI:  https://doi.org/10.1093/jxb/erad354
  23. Dev Cell. 2023 Aug 30. pii: S1534-5807(23)00410-0. [Epub ahead of print]
    Mammalian autophagosomes form from finger-like phagophores.
    Claudia Puri, Matthew J Gratian, David C Rubinsztein.
      The sequence of morphological intermediates that leads to mammalian autophagosome formation and closure is a crucial yet poorly understood issue. Previous studies have shown that yeast autophagosomes evolve from cup-shaped phagophores with only one closure point, and mammalian studies have inferred that mammalian phagophores also have single openings. Our superresolution microscopy studies in different human cell lines in conditions of basal and nutrient-deprivation-induced autophagy identified autophagosome precursors with multifocal origins that evolved into unexpected finger-like phagophores with multiple openings before becoming more spherical structures. Compatible phagophore structures were observed with whole-mount and conventional electron microscopy. This sequence of events was visualized using advanced SIM2 superresolution live microscopy. The finger-shaped phagophore apertures remained open when ESCRT function was compromised. The efficient closure of autophagic structures is important for their release from the recycling endosome. This has important implications for understanding how autophagosomes form and capture various cargoes.
    Keywords:  CHMP2A; ESCRT complex; RAB11A; VPS4A; autophagosome; autophagy; mitophagy; phagophore; recycling endosome; superresolution microscopy
    DOI:  https://doi.org/10.1016/j.devcel.2023.08.016
  24. J Cell Biol. 2023 Nov 06. pii: e200212128072023new. [Epub ahead of print]222(11):
    The RasGAP-associated endoribonuclease G3BP mediates stress granule assembly.
    Hélène Tourrière, Karim Chebli, Latifa Zekri, Brice Courselaud, Jean Marie Blanchard, Edouard Bertrand, Jamal Tazi.
      Stress granules (SGs) are formed in the cytoplasm in response to various toxic agents and are believed to play a critical role in the regulation of mRNA metabolism during stress. In SGs, mRNAs are stored in an abortive translation initiation complex that can be routed to either translation initiation or degradation. Here, we show that G3BP, a phosphorylation-dependent endoribonuclease that interacts with RasGAP, is recruited to SGs in cells exposed to arsenite. G3BP may thus determine the fate of mRNAs during cellular stress. Remarkably, SG assembly can be either dominantly induced by G3BP overexpression, or on the contrary, inhibited by expressing a central domain of G3BP. This region binds RasGAP and contains serine 149 whose dephosphorylation is induced by arsenite treatment. Critically, a non-phosphorylatable G3BP mutant (S149A) oligomerizes and assembles SG. These results suggest that G3BP is an effector of SG assembly and that Ras signaling contributes to this process by regulating G3BP dephosphorylation.
    DOI:  https://doi.org/10.1083/jcb.200212128072023new
  25. Cell Rep. 2023 Sep 01. pii: S2211-1247(23)01072-0. [Epub ahead of print]42(9): 113061
    Lon degrades stable substrates slowly but with enhanced processivity, redefining the attributes of a successful AAA+ protease.
    Meghann R Kasal, Hema Chandra Kotamarthi, Madeline M Johnson, Hannah M Stephens, Matthew J Lang, Robert T Sauer, Tania A Baker.
      Lon is a widely distributed AAA+ (ATPases associated with diverse cellular activities) protease known for degrading poorly folded and damaged proteins and is often classified as a weak protein unfoldase. Here, using a Lon-degron pair from Mesoplasma florum (MfLon and MfssrA, respectively), we perform ensemble and single-molecule experiments to elucidate the molecular mechanisms underpinning MfLon function. Notably, we find that MfLon unfolds and degrades stably folded substrates and that translocation of these unfolded polypeptides occurs with a ∼6-amino-acid step size. Moreover, the time required to hydrolyze one ATP corresponds to the dwell time between steps, indicating that one step occurs per ATP-hydrolysis-fueled "power stroke." Comparison of MfLon to related AAA+ enzymes now provides strong evidence that HCLR-clade enzymes function using a shared power-stroke mechanism and, surprisingly, that MfLon is more processive than ClpXP and ClpAP. We propose that ample unfoldase strength and substantial processivity are features that contribute to the Lon family's evolutionary success.
    Keywords:  AAA+ enzyme processivity; AAA+ motors; AAA+ unfoldase translocation step size; CP: Molecular biology; protein degradation; single-molecule optical trapping
    DOI:  https://doi.org/10.1016/j.celrep.2023.113061
  26. Nat Rev Mol Cell Biol. 2023 Sep 08.
    Mechanisms and pathology of protein misfolding and aggregation.
    Nikolaos Louros, Joost Schymkowitz, Frederic Rousseau.
      Despite advances in machine learning-based protein structure prediction, we are still far from fully understanding how proteins fold into their native conformation. The conventional notion that polypeptides fold spontaneously to their biologically active states has gradually been replaced by our understanding that cellular protein folding often requires context-dependent guidance from molecular chaperones in order to avoid misfolding. Misfolded proteins can aggregate into larger structures, such as amyloid fibrils, which perpetuate the misfolding process, creating a self-reinforcing cascade. A surge in amyloid fibril structures has deepened our comprehension of how a single polypeptide sequence can exhibit multiple amyloid conformations, known as polymorphism. The assembly of these polymorphs is not a random process but is influenced by the specific conditions and tissues in which they originate. This observation suggests that, similar to the folding of native proteins, the kinetics of pathological amyloid assembly are modulated by interactions specific to cells and tissues. Here, we review the current understanding of how intrinsic protein conformational propensities are modulated by physiological and pathological interactions in the cell to shape protein misfolding and aggregation pathology.
    DOI:  https://doi.org/10.1038/s41580-023-00647-2
  27. Science. 2023 Sep 08. 381(6662): eabn4180
    Modulation of the proteostasis network promotes tumor resistance to oncogenic KRAS inhibitors.
    Xiangdong Lv, Xuan Lu, Jin Cao, Qin Luo, Yao Ding, Fanglue Peng, Apar Pataer, Dong Lu, Dong Han, Eric Malmberg, Doug W Chan, Xiaoran Wang, Sara R Savage, Sufeng Mao, Jingjing Yu, Fei Peng, Liang Yan, Huan Meng, Laure Maneix, Yumin Han, Yiwen Chen, Wantong Yao, Eric C Chang, Andre Catic, Xia Lin, George Miles, Pengxiang Huang, Zheng Sun, Bryan Burt, Huamin Wang, Jin Wang, Qizhi Cathy Yao, Bing Zhang, Jack A Roth, Bert W O'Malley, Matthew J Ellis, Mothaffar F Rimawi, Haoqiang Ying, Xi Chen.
      Despite substantial advances in targeting mutant KRAS, tumor resistance to KRAS inhibitors (KRASi) remains a major barrier to progress. Here, we report proteostasis reprogramming as a key convergence point of multiple KRASi-resistance mechanisms. Inactivation of oncogenic KRAS down-regulated both the heat shock response and the inositol-requiring enzyme 1α (IRE1α) branch of the unfolded protein response, causing severe proteostasis disturbances. However, IRE1α was selectively reactivated in an ER stress-independent manner in acquired KRASi-resistant tumors, restoring proteostasis. Oncogenic KRAS promoted IRE1α protein stability through extracellular signal-regulated kinase (ERK)-dependent phosphorylation of IRE1α, leading to IRE1α disassociation from 3-hydroxy-3-methylglutaryl reductase degradation (HRD1) E3-ligase. In KRASi-resistant tumors, both reactivated ERK and hyperactivated AKT restored IRE1α phosphorylation and stability. Suppression of IRE1α overcame resistance to KRASi. This study reveals a druggable mechanism that leads to proteostasis reprogramming and facilitates KRASi resistance.
    DOI:  https://doi.org/10.1126/science.abn4180
  28. EMBO Rep. 2023 Sep 08. e57758
    Orchestrating vesicular and nonvesicular membrane dynamics by intrinsically disordered proteins.
    Stephan J Sigrist, Volker Haucke.
      Compartmentalization by membranes is a common feature of eukaryotic cells and serves to spatiotemporally confine biochemical reactions to control physiology. Membrane-bound organelles such as the endoplasmic reticulum (ER), the Golgi complex, endosomes and lysosomes, and the plasma membrane, continuously exchange material via vesicular carriers. In addition to vesicular trafficking entailing budding, fission, and fusion processes, organelles can form membrane contact sites (MCSs) that enable the nonvesicular exchange of lipids, ions, and metabolites, or the secretion of neurotransmitters via subsequent membrane fusion. Recent data suggest that biomolecule and information transfer via vesicular carriers and via MCSs share common organizational principles and are often mediated by proteins with intrinsically disordered regions (IDRs). Intrinsically disordered proteins (IDPs) can assemble via low-affinity, multivalent interactions to facilitate membrane tethering, deformation, fission, or fusion. Here, we review our current understanding of how IDPs drive the formation of multivalent protein assemblies and protein condensates to orchestrate vesicular and nonvesicular transport with a special focus on presynaptic neurotransmission. We further discuss how dysfunction of IDPs causes disease and outline perspectives for future research.
    Keywords:  intrinsically disordered proteins; membrane contact sites; neurotransmission; synapse; vesicular and nonvesicular transport
    DOI:  https://doi.org/10.15252/embr.202357758
  29. Elife. 2023 09 04. pii: RP89066. [Epub ahead of print]12
    Caspase-mediated nuclear pore complex trimming in cell differentiation and endoplasmic reticulum stress.
    Ukrae H Cho, Martin W Hetzer.
      During apoptosis, caspases degrade 8 out of ~30 nucleoporins to irreversibly demolish the nuclear pore complex. However, for poorly understood reasons, caspases are also activated during cell differentiation. Here, we show that sublethal activation of caspases during myogenesis results in the transient proteolysis of four peripheral Nups and one transmembrane Nup. 'Trimmed' NPCs become nuclear export-defective, and we identified in an unbiased manner several classes of cytoplasmic, plasma membrane, and mitochondrial proteins that rapidly accumulate in the nucleus. NPC trimming by non-apoptotic caspases was also observed in neurogenesis and endoplasmic reticulum stress. Our results suggest that caspases can reversibly modulate nuclear transport activity, which allows them to function as agents of cell differentiation and adaptation at sublethal levels.
    Keywords:  caspase; cell biology; cell differentiation; developmental biology; mouse; nuclear pore complex
    DOI:  https://doi.org/10.7554/eLife.89066
  30. Cell Rep. 2023 Sep 01. pii: S2211-1247(23)01059-8. [Epub ahead of print]42(9): 113048
    Deciphering the functional landscape of phosphosites with deep neural network.
    Zhongjie Liang, Tonghai Liu, Qi Li, Guangyu Zhang, Bei Zhang, Xikun Du, Jingqiu Liu, Zhifeng Chen, Hong Ding, Guang Hu, Hao Lin, Fei Zhu, Cheng Luo.
      Current biochemical approaches have only identified the most well-characterized kinases for a tiny fraction of the phosphoproteome, and the functional assignments of phosphosites are almost negligible. Herein, we analyze the substrate preference catalyzed by a specific kinase and present a novel integrated deep neural network model named FuncPhos-SEQ for functional assignment of human proteome-level phosphosites. FuncPhos-SEQ incorporates phosphosite motif information from a protein sequence using multiple convolutional neural network (CNN) channels and network features from protein-protein interactions (PPIs) using network embedding and deep neural network (DNN) channels. These concatenated features are jointly fed into a heterogeneous feature network to prioritize functional phosphosites. Combined with a series of in vitro and cellular biochemical assays, we confirm that NADK-S48/50 phosphorylation could activate its enzymatic activity. In addition, ERK1/2 are discovered as the primary kinases responsible for NADK-S48/50 phosphorylation. Moreover, FuncPhos-SEQ is developed as an online server.
    Keywords:  CP: Genomics; CP: Molecular biology; NADK; deep neural network; enzymatic activity; evolutionary features; functional phosphosite; network embedding; protein-protein interaction (PPI)
    DOI:  https://doi.org/10.1016/j.celrep.2023.113048
  31. Cell Discov. 2023 Sep 07. 9(1): 92
    Lysosomes mediate the mitochondrial UPR via mTORC1-dependent ATF4 phosphorylation.
    Terytty Yang Li, Qi Wang, Arwen W Gao, Xiaoxu Li, Yu Sun, Adrienne Mottis, Minho Shong, Johan Auwerx.
      Lysosomes are central platforms for not only the degradation of macromolecules but also the integration of multiple signaling pathways. However, whether and how lysosomes mediate the mitochondrial stress response (MSR) remain largely unknown. Here, we demonstrate that lysosomal acidification via the vacuolar H+-ATPase (v-ATPase) is essential for the transcriptional activation of the mitochondrial unfolded protein response (UPRmt). Mitochondrial stress stimulates v-ATPase-mediated lysosomal activation of the mechanistic target of rapamycin complex 1 (mTORC1), which then directly phosphorylates the MSR transcription factor, activating transcription factor 4 (ATF4). Disruption of mTORC1-dependent ATF4 phosphorylation blocks the UPRmt, but not other similar stress responses, such as the UPRER. Finally, ATF4 phosphorylation downstream of the v-ATPase/mTORC1 signaling is indispensable for sustaining mitochondrial redox homeostasis and protecting cells from ROS-associated cell death upon mitochondrial stress. Thus, v-ATPase/mTORC1-mediated ATF4 phosphorylation via lysosomes links mitochondrial stress to UPRmt activation and mitochondrial function resilience.
    DOI:  https://doi.org/10.1038/s41421-023-00589-1
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