bims-proteo Biomed News
on Proteostasis
Issue of 2023–10–22
twenty-one papers selected by
Eric Chevet, INSERM
  1. Removal of hypersignaling endosomes by simaphagy.
  2. Adaptive preservation of orphan ribosomal proteins in chaperone-dispersed condensates.
  3. Targeting selective E3 ligase for healing cardiac fibrosis.
  4. The HACE1 E3 ligase mediates RAC1-dependent control of mTOR signaling complexes.
  5. Ubiquitin ligase CHFR mediated degradation of VE-cadherin through ubiquitylation disrupts endothelial adherens junctions.
  6. SARS-CoV-2 papain-like protease (PLpro) plays multiple roles in regulating cellular proteins in the endoplasmic reticulum.
  7. The regulatory landscape of the yeast phosphoproteome.
  8. Identification of LMAN1- and SURF4-Dependent Secretory Cargoes.
  9. An optimized protocol to detect ubiquitination modification of exogenous or endogenous proteins.
  10. The intrinsically disordered region of eIF5B stimulates IRES usage and nucleates biological granule formation.
  11. Mitochondria-lysosome-related organelles mediate mitochondrial clearance during cellular dedifferentiation.
  12. Expanding PROTACtable genome universe of E3 ligases.
  13. PAICS ubiquitination recruits UBAP2 to trigger phase separation for purinosome assembly.
  14. Ubiquitin signaling and the proteasome drive human DNA-protein crosslink repair.
  15. The role of the N terminus of lipidated human Atg8-family proteins in cis-membrane association.
  16. Pathogenic signal peptide variants in the human genome.
  17. Molecular basis of Wnt biogenesis, secretion, and Wnt7-specific signaling.
  18. A Legionella toxin exhibits tRNA mimicry and glycosyl transferase activity to target the translation machinery and trigger a ribotoxic stress response.
  19. Elucidating the cellular determinants of targeted membrane protein degradation by lysosome-targeting chimeras.
  20. Accurate prediction of protein folding mechanisms by simple structure-based statistical mechanical models.
  21. Methods for computer-assisted PROTAC design.
  1. Autophagy. 2023 Oct 16.
    Removal of hypersignaling endosomes by simaphagy.
    Simona M Migliano, Sebastian W Schultz, Eva M Wenzel, Szabolcs Takáts, Dan Liu, Silje Mørk, Kia Wee Tan, Tor Erik Rusten, Camilla Raiborg, Harald Stenmark.
      Activated transmembrane receptors continue to signal following endocytosis and are only silenced upon ESCRT-mediated internalization of the receptors into intralumenal vesicles (ILVs) of the endosomes. Accordingly, endosomes with dysfunctional receptor internalization into ILVs can cause sustained receptor signaling which has been implicated in cancer progression. Here, we describe a surveillance mechanism that allows cells to detect and clear physically intact endosomes with aberrant receptor accumulation and elevated signaling. Proximity biotinylation and proteomics analyses of ESCRT-0 defective endosomes revealed a strong enrichment of the ubiquitin-binding macroautophagy/autophagy receptors SQSTM1 and NBR1, a phenotype that was confirmed in cell culture and fly tissue. Live cell microscopy demonstrated that loss of the ESCRT-0 subunit HGS/HRS or the ESCRT-I subunit VPS37 led to high levels of ubiquitinated and phosphorylated receptors on endosomes. This was accompanied by dynamic recruitment of NBR1 and SQSTM1 as well as proteins involved in autophagy initiation and autophagosome biogenesis. Light microscopy and electron tomography revealed that endosomes with intact limiting membrane, but aberrant receptor downregulation were engulfed by phagophores. Inhibition of autophagy caused increased intra- and intercellular signaling and directed cell migration. We conclude that dysfunctional endosomes are surveyed and cleared by an autophagic process, simaphagy, which serves as a failsafe mechanism in signal termination.
    Keywords:  ESCRT; autophagy; endosome; receptor degradation; signaling
    DOI:  https://doi.org/10.1080/15548627.2023.2267958
  2. Nat Cell Biol. 2023 Oct 16.
    Adaptive preservation of orphan ribosomal proteins in chaperone-dispersed condensates.
    Asif Ali, Rania Garde, Olivia C Schaffer, Jared A M Bard, Kabir Husain, Samantha Keyport Kik, Kathleen A Davis, Sofia Luengo-Woods, Maya G Igarashi, D Allan Drummond, Allison H Squires, David Pincus.
      Ribosome biogenesis is among the most resource-intensive cellular processes, with ribosomal proteins accounting for up to half of all newly synthesized proteins in eukaryotic cells. During stress, cells shut down ribosome biogenesis in part by halting rRNA synthesis, potentially leading to massive accumulation of aggregation-prone 'orphan' ribosomal proteins (oRPs). Here we show that, during heat shock in yeast and human cells, oRPs accumulate as reversible peri-nucleolar condensates recognized by the Hsp70 co-chaperone Sis1/DnaJB6. oRP condensates are liquid-like in cell-free lysate but solidify upon depletion of Sis1 or inhibition of Hsp70. When cells recover from heat shock, oRP condensates disperse in a Sis1- and Hsp70-dependent manner, and the oRP constituents are incorporated into functional ribosomes in the cytosol, enabling cells to efficiently resume growth. Preserving biomolecules in reversible condensates-like mRNAs in cytosolic stress granules and oRPs at the nucleolar periphery-may be a primary function of the Hsp70 chaperone system.
    DOI:  https://doi.org/10.1038/s41556-023-01253-2
  3. Cell Chem Biol. 2023 Oct 19. pii: S2451-9456(23)00289-1. [Epub ahead of print]30(10): 1188-1190
    Targeting selective E3 ligase for healing cardiac fibrosis.
    Guangdong Yang.
      Protein quality control is often disrupted in heart diseases. In this issue of Cell Chemical Biology, Yang et al.1 describe the selective E3 ligase SPOP as a pro-fibrotic factor for inducing cardiac fibrosis through RACK1 degradation-mediated Smad3 activation.
    DOI:  https://doi.org/10.1016/j.chembiol.2023.08.017
  4. EMBO Rep. 2023 Oct 17. e56815
    The HACE1 E3 ligase mediates RAC1-dependent control of mTOR signaling complexes.
    Busra Turgu, Amal El-Naggar, Melanie Kogler, Luigi Tortola, Hai-Feng Zhang, Mariam Hassan, Michael M Lizardo, Sonia Hy Kung, Wan Lam, Josef M Penninger, Poul H Sorensen.
      HACE1 is a HECT family E3 ubiquitin-protein ligase with broad but incompletely understood tumor suppressor activity. Here, we report a previously unrecognized link between HACE1 and signaling complexes containing mammalian target of rapamycin (mTOR). HACE1 blocks mTORC1 and mTORC2 activities by reducing mTOR stability in an E3 ligase-dependent manner. Mechanistically, HACE1 binds to and ubiquitylates Ras-related C3 botulinum toxin substrate 1 (RAC1) when RAC1 is associated with mTOR complexes, including at focal adhesions, leading to proteasomal degradation of RAC1. This in turn decreases the stability of mTOR to reduce mTORC1 and mTORC2 activity. HACE1 deficient cells show enhanced mTORC1/2 activity, which is reversed by chemical or genetic RAC1 inactivation but not in cells expressing the HACE1-insensitive mutant, RAC1K147R . In vivo, Rac1 deletion reverses enhanced mTOR expression in KRasG12D -driven lung tumors of Hace1-/- mice. HACE1 co-localizes with mTOR and RAC1, resulting in RAC1-dependent loss of mTOR protein stability. Together, our data demonstrate that HACE1 destabilizes mTOR by targeting RAC1 within mTOR-associated complexes, revealing a unique ubiquitin-dependent process to control the activity of mTOR signaling complexes.
    Keywords:  E3 ubiquitin ligase; HACE1; RAC1; mTORC1 and mTORC2; tumor suppressor gene
    DOI:  https://doi.org/10.15252/embr.202356815
  5. Nat Commun. 2023 Oct 18. 14(1): 6582
    Ubiquitin ligase CHFR mediated degradation of VE-cadherin through ubiquitylation disrupts endothelial adherens junctions.
    Chinnaswamy Tiruppathi, Dong-Mei Wang, Mohammad Owais Ansari, Shabana Bano, Yoshikazu Tsukasaki, Amitabha Mukhopadhyay, Jagdish C Joshi, Christian Loch, Hans W M Niessen, Asrar B Malik.
      Vascular endothelial cadherin (VE-cadherin) expressed at endothelial adherens junctions (AJs) is vital for vascular integrity and endothelial homeostasis. Here we identify the requirement of the ubiquitin E3-ligase CHFR as a key mechanism of ubiquitylation-dependent degradation of VE-cadherin. CHFR was essential for disrupting the endothelium through control of the VE-cadherin protein expression at AJs. We observe augmented expression of VE-cadherin in endothelial cell (EC)-restricted Chfr knockout (ChfrΔEC) mice. We also observe abrogation of LPS-induced degradation of VE-cadherin in ChfrΔEC mice, suggesting the pathophysiological relevance of CHFR in regulating the endothelial junctional barrier in inflammation. Lung endothelial barrier breakdown, inflammatory neutrophil extravasation, and mortality induced by LPS were all suppressed in ChfrΔEC mice. We find that the transcription factor FoxO1 is a key upstream regulator of CHFR expression. These findings demonstrate the requisite role of the endothelial cell-expressed E3-ligase CHFR in regulating the expression of VE-cadherin, and thereby endothelial junctional barrier integrity.
    DOI:  https://doi.org/10.1038/s41467-023-42225-2
  6. J Biol Chem. 2023 Oct 12. pii: S0021-9258(23)02374-8. [Epub ahead of print] 105346
    SARS-CoV-2 papain-like protease (PLpro) plays multiple roles in regulating cellular proteins in the endoplasmic reticulum.
    Mei Yang, Jennifer Mariano, Rebecca Su, Christopher E Smith, Sudipto Das, Catherine Gill, Thorkell Andresson, Jadranka Loncarek, Yien Che Tsai, Allan M Weissman.
      Nsp3s are the largest non-structural proteins of coronaviruses. These transmembrane proteins include papain-like proteases (PLpro) that play essential roles in cleaving viral polyproteins into their mature units. The PLpro of SARS-CoV viruses also have deubiquitinating and deISGylating activities. As Nsp3 is an endoplasmic reticulum (ER)-localized protein, we asked if the deubiquitinating activity of SARS-CoV-2 PLpro affects proteins that are substrates for ER-associated degradation (ERAD). Using full-length Nsp3 as well as a truncated transmembrane form we interrogated, by co-expression, three potential ERAD substrates, all of which play roles in regulating lipid biosynthesis. Transmembrane PLpro increases the level of INSIG-1 and decreases its ubiquitination. However, different effects were seen with SREBP-1 and SREBP-2. Transmembrane PLpro cleaves SREBP-1 at three sites, including two non-canonical sites in the N-terminal half of the protein, resulting in a decrease in precursors of the active transcription factor. Conversely, cleavage of SREBP-2 occurs at a single canonical site that disrupts a C-terminal degron, resulting in increased SREBP-2 levels. When this site is mutated and the degron can no longer be interrupted, SREBP-2 is still stabilized by transmembrane PLpro, which correlates with a decrease in SREBP-2 ubiquitination. All of these observations are dependent on PLpro catalytic activity. Our findings demonstrate that, when anchored to the ER membrane, SARS-CoV-2 Nsp3 PLpro can function as a deubiquitinating enzyme to stabilize ERAD substrates. Additionally, SARS-CoV-2 Nsp3 PLpro can cleave ER resident proteins, including at sites that could escape analyses based on the established consensus sequence.
    Keywords:  COVID; cell biology; cholesterol; deubiquitinating enzyme; deubiquitination; lipid biosynthesis; mass spectrometry; proteolysis; ubiquitin; virology; virulence factor
    DOI:  https://doi.org/10.1016/j.jbc.2023.105346
  7. Nat Struct Mol Biol. 2023 Oct 16.
    The regulatory landscape of the yeast phosphoproteome.
    Mario Leutert, Anthony S Barente, Noelle K Fukuda, Ricard A Rodriguez-Mias, Judit Villén.
      The cellular ability to react to environmental fluctuations depends on signaling networks that are controlled by the dynamic activities of kinases and phosphatases. Here, to gain insight into these stress-responsive phosphorylation networks, we generated a quantitative mass spectrometry-based atlas of early phosphoproteomic responses in Saccharomyces cerevisiae exposed to 101 environmental and chemical perturbations. We report phosphosites on 59% of the yeast proteome, with 18% of the proteome harboring a phosphosite that is regulated within 5 min of stress exposure. We identify shared and perturbation-specific stress response programs, uncover loss of phosphorylation as an integral early event, and dissect the interconnected regulatory landscape of kinase-substrate networks, as we exemplify with target of rapamycin signaling. We further reveal functional organization principles of the stress-responsive phosphoproteome based on phosphorylation site motifs, kinase activities, subcellular localizations, shared functions and pathway intersections. This information-rich map of 25,000 regulated phosphosites advances our understanding of signaling networks.
    DOI:  https://doi.org/10.1038/s41594-023-01115-3
  8. J Proteome Res. 2023 Oct 16.
    Identification of LMAN1- and SURF4-Dependent Secretory Cargoes.
    Vi T Tang, Prabhodh S Abbineni, Felipe da Veiga Leprevost, Venkatesha Basrur, Rami Khoriaty, Brian T Emmer, Alexey I Nesvizhskii, David Ginsburg.
      Most proteins secreted into the extracellular space are first recruited from the endoplasmic reticulum into coat protein complex II (COPII)-coated vesicles or tubules that facilitate their transport to the Golgi apparatus. Although several secreted proteins have been shown to be actively recruited into COPII vesicles and tubules by the cargo receptors LMAN1 and SURF4, the full cargo repertoire of these receptors is unknown. We now report mass spectrometry analysis of conditioned media and cell lysates from HuH7 cells CRISPR targeted to inactivate the LMAN1 or SURF4 gene. We found that LMAN1 has limited clients in HuH7 cells, whereas SURF4 traffics a broad range of cargoes. Analysis of putative SURF4 cargoes suggests that cargo recognition is governed by complex mechanisms rather than interaction with a universal binding motif..
    Keywords:  COPII trafficking; cargo receptor; secretome
    DOI:  https://doi.org/10.1021/acs.jproteome.3c00259
  9. STAR Protoc. 2023 Oct 18. pii: S2666-1667(23)00617-2. [Epub ahead of print]4(4): 102650
    An optimized protocol to detect ubiquitination modification of exogenous or endogenous proteins.
    Xinyu Li, Wei Jiang, Min Fang.
      Ubiquitination modification is an important post-translational modification that regulates the stability and function of proteins. Here, we present a protocol to detect the K27-linked polyubiquitination of exogenous and endogenous mitochondrial antiviral signaling protein. We describe steps for detecting ubiquitination of exogenous protein, transfecting the encoding plasmid of the protein, and immunoprecipitating the target protein with an antibody. We then detail procedures for detecting ubiquitin of the target protein by western blot. This protocol applies to other proteins of interest. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2023).1.
    Keywords:  Cell Separation/Fractionation; Cell-Based Assays; Immunology; Molecular Biology
    DOI:  https://doi.org/10.1016/j.xpro.2023.102650
  10. Cell Rep. 2023 Oct 18. pii: S2211-1247(23)01295-0. [Epub ahead of print]42(10): 113283
    The intrinsically disordered region of eIF5B stimulates IRES usage and nucleates biological granule formation.
    Meghan T Harris, Michael T Marr.
      Cells activate stress response pathways to survive adverse conditions. Such responses involve the inhibition of global cap-dependent translation. This inhibition is a block that essential transcripts must escape via alternative methods of translation initiation, e.g., an internal ribosome entry site (IRES). IRESs have distinct structures and generally require a limited repertoire of translation factors. Cellular IRESs have been identified in many critical cellular stress response transcripts. We previously identified cellular IRESs in the murine insulin receptor (Insr) and insulin-like growth factor 1 receptor (Igf1r) transcripts and demonstrated their resistance to eukaryotic initiation factor 4F (eIF4F) inhibition. Here, we find that eIF5B preferentially promotes Insr, Igf1r, and hepatitis C virus IRES activity through a non-canonical mechanism that requires its highly charged and disordered N terminus. We find that the N-terminal region of eIF5B can drive cytoplasmic granule formation. This eIF5B granule is triggered by cellular stress and is sufficient to specifically promote IRES activity.
    Keywords:  CP: Cell biology; CP: Molecular biology; Insulin Receptor (Insr); Insulin-like growth factor receptor (Igf1r); Internal Ribosome Entry Site (IRES); cellular stress; eukaryotic initiation factor 5B (eIF5B); translation
    DOI:  https://doi.org/10.1016/j.celrep.2023.113283
  11. Cell Rep. 2023 Oct 19. pii: S2211-1247(23)01303-7. [Epub ahead of print]42(10): 113291
    Mitochondria-lysosome-related organelles mediate mitochondrial clearance during cellular dedifferentiation.
    Xiaowen Ma, Sharon Manley, Hui Qian, Yuan Li, Chen Zhang, Kevin Li, Benjamin Ding, Fengli Guo, Allen Chen, Xing Zhang, Meilian Liu, Meihua Hao, Benjamin Kugler, E Matthew Morris, John Thyfault, Ling Yang, Hiromi Sesaki, Hong-Min Ni, Heidi McBride, Wen-Xing Ding.
      Dysfunctional mitochondria are removed via multiple pathways, such as mitophagy, a selective autophagy process. Here, we identify an intracellular hybrid mitochondria-lysosome organelle (termed the mitochondria-lysosome-related organelle [MLRO]), which regulates mitochondrial homeostasis independent of canonical mitophagy during hepatocyte dedifferentiation. The MLRO is an electron-dense organelle that has either a single or double membrane with both mitochondria and lysosome markers. Mechanistically, the MLRO is likely formed from the fusion of mitochondria-derived vesicles (MDVs) with lysosomes through a PARKIN-, ATG5-, and DRP1-independent process, which is negatively regulated by transcription factor EB (TFEB) and associated with mitochondrial protein degradation and hepatocyte dedifferentiation. The MLRO, which is galectin-3 positive, is reminiscent of damaged lysosome and could be cleared by overexpression of TFEB, resulting in attenuation of hepatocyte dedifferentiation. Together, results from this study suggest that the MLRO may act as an alternative mechanism for mitochondrial quality control independent of canonical autophagy/mitophagy involved in cell dedifferentiation.
    Keywords:  ATG5; CP: Cell biology; DRP1; autophagy; hepatocytes; lysosome; mitophagy
    DOI:  https://doi.org/10.1016/j.celrep.2023.113291
  12. Nat Commun. 2023 Oct 16. 14(1): 6509
    Expanding PROTACtable genome universe of E3 ligases.
    Yuan Liu, Jingwen Yang, Tianlu Wang, Mei Luo, Yamei Chen, Chengxuan Chen, Ze'ev Ronai, Yubin Zhou, Eytan Ruppin, Leng Han.
      Proteolysis-targeting chimera (PROTAC) and other targeted protein degradation (TPD) molecules that induce degradation by the ubiquitin-proteasome system (UPS) offer new opportunities to engage targets that remain challenging to be inhibited by conventional small molecules. One fundamental element in the degradation process is the E3 ligase. However, less than 2% amongst hundreds of E3 ligases in the human genome have been engaged in current studies in the TPD field, calling for the recruiting of additional ones to further enhance the therapeutic potential of TPD. To accelerate the development of PROTACs utilizing under-explored E3 ligases, we systematically characterize E3 ligases from seven different aspects, including chemical ligandability, expression patterns, protein-protein interactions (PPI), structure availability, functional essentiality, cellular location, and PPI interface by analyzing 30 large-scale data sets. Our analysis uncovers several E3 ligases as promising extant PROTACs. In total, combining confidence score, ligandability, expression pattern, and PPI, we identified 76 E3 ligases as PROTAC-interacting candidates. We develop a user-friendly and flexible web portal ( https://hanlaboratory.com/E3Atlas/ ) aimed at assisting researchers to rapidly identify E3 ligases with promising TPD activities against specifically desired targets, facilitating the development of these therapies in cancer and beyond.
    DOI:  https://doi.org/10.1038/s41467-023-42233-2
  13. Mol Cell. 2023 Oct 10. pii: S1097-2765(23)00755-4. [Epub ahead of print]
    PAICS ubiquitination recruits UBAP2 to trigger phase separation for purinosome assembly.
    Ming-Chieh Chou, Yi-Hsuan Wang, Fei-Yun Chen, Chun-Ying Kung, Kuen-Phon Wu, Jean-Cheng Kuo, Shu-Jou Chan, Mei-Ling Cheng, Chih-Yu Lin, Yu-Chi Chou, Meng-Chiao Ho, Steven Firestine, Jie-Rong Huang, Ruey-Hwa Chen.
      Purinosomes serve as metabolons to enhance de novo purine synthesis (DNPS) efficiency through compartmentalizing DNPS enzymes during stressed conditions. However, the mechanism underpinning purinosome assembly and its pathophysiological functions remains elusive. Here, we show that K6-polyubiquitination of the DNPS enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) by cullin-5/ankyrin repeat and SOCS box containing 11 (Cul5/ASB11)-based ubiquitin ligase plays a driving role in purinosome assembly. Upon several purinosome-inducing cues, ASB11 is upregulated by relieving the H3K9me3/HP1α-mediated transcriptional silencing, thus stimulating PAICS polyubiquitination. The polyubiquitinated PAICS recruits ubiquitin-associated protein 2 (UBAP2), a ubiquitin-binding protein with multiple stretches of intrinsically disordered regions, thereby inducing phase separation to trigger purinosome assembly for enhancing DNPS pathway flux. In human melanoma, ASB11 is highly expressed to facilitate a constitutive purinosome formation to which melanoma cells are addicted for supporting their proliferation, viability, and tumorigenesis in a xenograft model. Our study identifies a driving mechanism for purinosome assembly in response to cellular stresses and uncovers the impact of purinosome formation on human malignancies.
    Keywords:  Cullin-RING ubiquitin ligase; biomolecular condensates; de novo purine synthesis; liquid-liquid phase separation; metabolon; purinosome; ubiquitination
    DOI:  https://doi.org/10.1016/j.molcel.2023.09.028
  14. Nucleic Acids Res. 2023 Oct 16. pii: gkad860. [Epub ahead of print]
    Ubiquitin signaling and the proteasome drive human DNA-protein crosslink repair.
    Maram Essawy, Lisa Chesner, Duha Alshareef, Shaofei Ji, Natalia Tretyakova, Colin Campbell.
      DNA-protein crosslinks (DPCs) are large cytotoxic DNA lesions that form following exposure to chemotherapeutic drugs and environmental chemicals. Nucleotide excision repair (NER) and homologous recombination (HR) promote survival following exposure to DPC-inducing agents. However, it is not known how cells recognize DPC lesions, or what mechanisms selectively target DPC lesions to these respective repair pathways. To address these questions, we examined DPC recognition and repair by transfecting a synthetic DPC lesion comprised of the human oxoguanine glycosylase (OGG1) protein crosslinked to double-stranded M13MP18 into human cells. In wild-type cells, this lesion is efficiently repaired, whereas cells deficient in NER can only repair this lesion if an un-damaged homologous donor is co-transfected. Transfected DPC is subject to rapid K63 polyubiquitination. In NER proficient cells, the DPC is subject to K48 polyubiquitination, and is removed via a proteasome-dependent mechanism. In NER-deficient cells, the DNA-conjugated protein is not subject to K48 polyubiquitination. Instead, the K63 tag remains attached, and is only lost when a homologous donor molecule is present. Taken together, these results support a model in which selective addition of polyubiquitin chains to DNA-crosslinked protein leads to selective recruitment of the proteasome and the cellular NER and recombinational DNA repair machinery.
    DOI:  https://doi.org/10.1093/nar/gkad860
  15. Autophagy. 2023 Oct 17.
    The role of the N terminus of lipidated human Atg8-family proteins in cis-membrane association.
    Hana Popelka, Daniel J Klionsky.
      A multifunctional role of Atg8-family proteins (Atg8 from yeast and human LC3 and GABARAP subfamilies, all referred to here as ATG8s) in macroautophagy/autophagy is carried out by two protein domains, the N-terminal helical domain (NHD) and ubiquitin-like (UBL) domain. Previous studies showed that the NHD of PE-conjugated ATG8s mediates membrane hemifusion via a direct interaction with lipids in trans-membrane association, which would require the NHD in lipidated ATG8s to adopt a solvent-oriented, "open", conformation that unmasks a UBL domain surface needed for membrane tethering. A purpose of the "closed" conformation of the NHD masking the tethering surface on the UBL domain, a conformation seen in the most structures of non-lipidated ATG8s, remained elusive. A recent study by Zhang et al. discussed in this article, showed that the N terminus of lipidated human ATG8s adopts the "closed" conformation when it interacts with the membrane in cis-membrane association, i.e. with the same membrane ATG8 is anchored to. This finding suggests functions for two distinct conformations of the NHD in lipidated ATG8s and raises questions about determinants controlling cis- or trans-membrane associations of the NHD in ATG8-PE.
    Keywords:  ATG8 proteins, cis- and trans-membrane association of lipidated ATG8s; ubiquitin-like
    DOI:  https://doi.org/10.1080/15548627.2023.2272233
  16. NAR Genom Bioinform. 2023 Dec;5(4): lqad093
    Pathogenic signal peptide variants in the human genome.
    Sneider Alexander Gutierrez Guarnizo, Morgana K Kellogg, Sarah C Miller, Elena B Tikhonova, Zemfira N Karamysheva, Andrey L Karamyshev.
      Secreted and membrane proteins represent a third of all cellular proteins and contain N-terminal signal peptides that are required for protein targeting to endoplasmic reticulum (ER). Mutations in signal peptides affect protein targeting, translocation, processing, and stability, and are associated with human diseases. However, only a few of them have been identified or characterized. In this report, we identified pathogenic signal peptide variants across the human genome using bioinformatic analyses and predicted the molecular mechanisms of their pathology. We recovered more than 65 thousand signal peptide mutations, over 11 thousand we classified as pathogenic, and proposed framework for distinction of their molecular mechanisms. The pathogenic mutations affect over 3.3 thousand genes coding for secreted and membrane proteins. Most pathogenic mutations alter the signal peptide hydrophobic core, a critical recognition region for the signal recognition particle, potentially activating the Regulation of Aberrant Protein Production (RAPP) quality control and specific mRNA degradation. The remaining pathogenic variants (about 25%) alter either the N-terminal region or signal peptidase processing site that can result in translocation deficiencies at the ER membrane or inhibit protein processing. This work provides a conceptual framework for the identification of mutations across the genome and their connection with human disease.
    DOI:  https://doi.org/10.1093/nargab/lqad093
  17. Cell. 2023 Oct 10. pii: S0092-8674(23)01075-9. [Epub ahead of print]
    Molecular basis of Wnt biogenesis, secretion, and Wnt7-specific signaling.
    Xiaofeng Qi, Qinli Hu, Nadia Elghobashi-Meinhardt, Tao Long, Hongwen Chen, Xiaochun Li.
      Wnt proteins are enzymatically lipidated by Porcupine (PORCN) in the ER and bind to Wntless (WLS) for intracellular transport and secretion. Mechanisms governing the transfer of these low-solubility Wnts from the ER to the extracellular space remain unclear. Through structural and functional analyses of Wnt7a, a crucial Wnt involved in central nervous system angiogenesis and blood-brain barrier maintenance, we have elucidated the principles of Wnt biogenesis and Wnt7-specific signaling. The Wnt7a-WLS complex binds to calreticulin (CALR), revealing that CALR functions as a chaperone to facilitate Wnt transfer from PORCN to WLS during Wnt biogenesis. Our structures, functional analyses, and molecular dynamics simulations demonstrate that a phospholipid in the core of Wnt-bound WLS regulates the association and dissociation between Wnt and WLS, suggesting a lipid-mediated Wnt secretion mechanism. Finally, the structure of Wnt7a bound to RECK, a cell-surface Wnt7 co-receptor, reveals how RECKCC4 engages the N-terminal domain of Wnt7a to activate Wnt7-specific signaling.
    Keywords:  N-glycan; Porcupine; RECK; Wnt; Wntless; calreticulin; cryo-EM; phospholipid
    DOI:  https://doi.org/10.1016/j.cell.2023.09.021
  18. Nat Cell Biol. 2023 Oct 19.
    A Legionella toxin exhibits tRNA mimicry and glycosyl transferase activity to target the translation machinery and trigger a ribotoxic stress response.
    Advait Subramanian, Lan Wang, Tom Moss, Mark Voorhies, Smriti Sangwan, Erica Stevenson, Ernst H Pulido, Samentha Kwok, Robert J Chalkley, Kathy H Li, Nevan J Krogan, Danielle L Swaney, Alma L Burlingame, Stephen N Floor, Anita Sil, Peter Walter, Shaeri Mukherjee.
      A widespread strategy employed by pathogens to establish infection is to inhibit host-cell protein synthesis. Legionella pneumophila, an intracellular bacterial pathogen and the causative organism of Legionnaires' disease, secretes a subset of protein effectors into host cells that inhibit translation elongation. Mechanistic insights into how the bacterium targets translation elongation remain poorly defined. We report here that the Legionella effector SidI functions in an unprecedented way as a transfer-RNA mimic that directly binds to and glycosylates the ribosome. The 3.1 Å cryo-electron microscopy structure of SidI reveals an N-terminal domain with an 'inverted L' shape and surface-charge distribution characteristic of tRNA mimicry, and a C-terminal domain that adopts a glycosyl transferase fold that licenses SidI to utilize GDP-mannose as a sugar precursor. This coupling of tRNA mimicry and enzymatic action endows SidI with the ability to block protein synthesis with a potency comparable to ricin, one of the most powerful toxins known. In Legionella-infected cells, the translational pausing activated by SidI elicits a stress response signature mimicking the ribotoxic stress response, which is activated by elongation inhibitors that induce ribosome collisions. SidI-mediated effects on the ribosome activate the stress kinases ZAKα and p38, which in turn drive an accumulation of the protein activating transcription factor 3 (ATF3). Intriguingly, ATF3 escapes the translation block imposed by SidI, translocates to the nucleus and orchestrates the transcription of stress-inducible genes that promote cell death, revealing a major role for ATF3 in the response to collided ribosome stress. Together, our findings elucidate a novel mechanism by which a pathogenic bacterium employs tRNA mimicry to hijack a ribosome-to-nuclear signalling pathway that regulates cell fate.
    DOI:  https://doi.org/10.1038/s41556-023-01248-z
  19. Science. 2023 Oct 20. 382(6668): eadf6249
    Elucidating the cellular determinants of targeted membrane protein degradation by lysosome-targeting chimeras.
    Green Ahn, Nicholas M Riley, Roarke A Kamber, Simon Wisnovsky, Salvador Moncayo von Hase, Michael C Bassik, Steven M Banik, Carolyn R Bertozzi.
      Targeted protein degradation can provide advantages over inhibition approaches in the development of therapeutic strategies. Lysosome-targeting chimeras (LYTACs) harness receptors, such as the cation-independent mannose 6-phosphate receptor (CI-M6PR), to direct extracellular proteins to lysosomes. In this work, we used a genome-wide CRISPR knockout approach to identify modulators of LYTAC-mediated membrane protein degradation in human cells. We found that disrupting retromer genes improved target degradation by reducing LYTAC recycling to the plasma membrane. Neddylated cullin-3 facilitated LYTAC-complex lysosomal maturation and was a predictive marker for LYTAC efficacy. A substantial fraction of cell surface CI-M6PR remains occupied by endogenous M6P-modified glycoproteins. Thus, inhibition of M6P biosynthesis increased the internalization of LYTAC-target complexes. Our findings inform design strategies for next-generation LYTACs and elucidate aspects of cell surface receptor occupancy and trafficking.
    DOI:  https://doi.org/10.1126/science.adf6249
  20. Nat Commun. 2023 Oct 19. 14(1): 6338
    Accurate prediction of protein folding mechanisms by simple structure-based statistical mechanical models.
    Koji Ooka, Munehito Arai.
      Recent breakthroughs in highly accurate protein structure prediction using deep neural networks have made considerable progress in solving the structure prediction component of the 'protein folding problem'. However, predicting detailed mechanisms of how proteins fold into specific native structures remains challenging, especially for multidomain proteins constituting most of the proteomes. Here, we develop a simple structure-based statistical mechanical model that introduces nonlocal interactions driving the folding of multidomain proteins. Our model successfully predicts protein folding processes consistent with experiments, without the limitations of protein size and shape. Furthermore, slight modifications of the model allow prediction of disulfide-oxidative and disulfide-intact protein folding. These predictions depict details of the folding processes beyond reproducing experimental results and provide a rationale for the folding mechanisms. Thus, our physics-based models enable accurate prediction of protein folding mechanisms with low computational complexity, paving the way for solving the folding process component of the 'protein folding problem'.
    DOI:  https://doi.org/10.1038/s41467-023-41664-1
  21. Methods Enzymol. 2023 ;pii: S0076-6879(23)00228-8. [Epub ahead of print]690 311-340
    Methods for computer-assisted PROTAC design.
    Evianne Rovers, Matthieu Schapira.
      Proximity-induced pharmacology is an emerging field in chemical biology and drug discovery where a small molecule induces non-natural interactions between two proteins, leading to specific phenotypic responses. Proteolysis targeting chimeras (PROTACs) are the most mature examples, where ligands for an E3 ligase and a target protein are linked to induce the ubiquitination and subsequent degradation of the target. The discovery of PROTACs typically relies on a trial-and-error approach where chemical handles and linker chemistry, length and attachment points are systematically varied in the hope that one of the combinations will produce an active molecule. Novel computational methods and tools are developed in an attempt to rationalize and accelerate this process and differ significantly from traditional structure-based drug design approaches. In this chapter, we review three different solutions for computer-assisted PROTAC design: MOE, ICM and PRosettaC. Specifically, we describe protocols to predict the structure of ternary complexes (E3 ligase-PROTAC-target protein) and to screen virtually libraries of PROTAC candidates. We also provide troubleshooting tips. Rational PROTAC design is still in its infancy. By opening this space to users and developers, we hope that this methods article will contribute to much needed advancement in the field.
    Keywords:  PROTAC; Proximity pharmacology; Virtual screening
    DOI:  https://doi.org/10.1016/bs.mie.2023.06.020
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