bims-proteo Biomed News
on Proteostasis
Issue of 2024‒03‒03
forty-two papers selected by
Eric Chevet, INSERM
  1. Combinatorial selective ER-phagy remodels the ER during neurogenesis.
  2. Modulation of PDI Functions by Localization: The Example of the Anterior Gradient Family.
  3. Bidirectional substrate shuttling between the 26S proteasome and the Cdc48 ATPase promotes protein degradation.
  4. NEMF-mediated Listerin-independent mitochondrial translational surveillance by E3 ligase Pirh2 and mitochondrial protease ClpXP.
  5. The HSP90 chaperone code regulates the crosstalk between proteostasis and autophagy.
  6. Subunits of an E3 Ligase Complex as Degrons for Efficient Degradation of Cytosolic, Nuclear, and Membrane Proteins.
  7. Collective cell migration relies on PPP1R15-mediated regulation of the endoplasmic reticulum stress response.
  8. A ratiometric ER calcium sensor for quantitative comparisons across cell types and subcellular regions.
  9. Maintenance of proteostasis by Drosophila Rer1 is essential for competitive cell survival and Myc-driven overgrowth.
  10. UPF1 ATPase autoinhibition and activation modulate RNA binding kinetics and NMD efficiency.
  11. Ribosomal quality control factors inhibit repeat-associated non-AUG translation from GC-rich repeats.
  12. Transient disome complex formation in native polysomes during ongoing protein synthesis captured by cryo-EM.
  13. Proteasome condensate formation is driven by multivalent interactions with shuttle factors and ubiquitin chains.
  14. The CRL5-SPSB3 ubiquitin ligase targets nuclear cGAS for degradation.
  15. The α-crystallin Chaperones Undergo a Quasi-ordered Co-aggregation Process in Response to Saturating Client Interaction.
  16. Degron-based bioPROTACs for controlling signaling in CAR T cells.
  17. UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation.
  18. A uniquely efficacious type of CFTR corrector with complementary mode of action.
  19. Intraluminal vesicle trafficking is involved in the secretion of base excision repair protein APE1.
  20. Tom20 gates PINK1 activity and mediates its tethering of the TOM and TIM23 translocases upon mitochondrial stress.
  21. Sequestrase chaperones protect against oxidative stress-induced protein aggregation and [PSI+] prion formation.
  22. Translation is an emerging constraint on protein homeostasis in ageing.
  23. ATF6 protects against protein misfolding during cardiac hypertrophy.
  24. A quantitative ultrastructural timeline of nuclear autophagy reveals a role for dynamin-like protein 1 at the nuclear envelope.
  25. Neuronal waste management: new roles for autophagy genes in the extrusion of protein aggregates and in longevity.
  26. Triaging of α-helical proteins to the mitochondrial outer membrane by distinct chaperone machinery based on substrate topology.
  27. Rapid simulation of glycoprotein structures by grafting and steric exclusion of glycan conformer libraries.
  28. Elucidating the EXOSC3-IRE1α interaction: a convergent study incorporating computational, in vitro and in vivo studies.
  29. ESCRT-I protein UBAP1 controls ventricular expansion and cortical neurogenesis via modulating adherens junctions of radial glial cells.
  30. ER: a critical hub for STING signaling regulation.
  31. Fitness effects of phenotypic mutations at proteome-scale reveal optimality of translation machinery.
  32. Targeting the IRE1α-XBP1s axis confers selective vulnerability in hepatocellular carcinoma with activated Wnt signaling.
  33. Deregulated protein homeostasis constrains fetal hematopoietic stem cell pool expansion in Fanconi anemia.
  34. The proteomic landscape of genotoxic stress-induced micronuclei.
  35. SEPN1-related myopathy depends on the oxidoreductase ERO1A and is druggable with the chemical chaperone TUDCA.
  36. Discovery of metal-binding proteins by thermal proteome profiling.
  37. HERC5-catalyzed ISGylation potentiates cGAS-mediated innate immunity.
  38. Hepatitis B surface antigen expression impairs endoplasmic reticulum stress-related autophagic flux by decreasing LAMP2.
  39. ATF4-dependent increase in mitochondrial-endoplasmic reticulum tethering following OPA1 deletion in skeletal muscle.
  40. DUB3 is a MAGEA3 deubiquitinase and a potential therapeutic target in hepatocellular carcinoma.
  41. Experimental and data analysis advances in thermal proteome profiling.
  42. PRMT1 acts as a suppressor of MHC-I and anti-tumor immunity.
  1. Nat Cell Biol. 2024 Mar 01.
    Combinatorial selective ER-phagy remodels the ER during neurogenesis.
    Melissa J Hoyer, Cristina Capitanio, Ian R Smith, Julia C Paoli, Anna Bieber, Yizhi Jiang, Joao A Paulo, Miguel A Gonzalez-Lozano, Wolfgang Baumeister, Florian Wilfling, Brenda A Schulman, J Wade Harper.
      The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions, ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis, where a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodelling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, our understanding of the mechanisms underlying selective removal of the ER and the role of individual ER-phagy receptors is limited. Here we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodelling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodelling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both the magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodelling and versatile genetic toolkit provide a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.
    DOI:  https://doi.org/10.1038/s41556-024-01356-4
  2. Antioxid Redox Signal. 2024 Feb 27.
    Modulation of PDI Functions by Localization: The Example of the Anterior Gradient Family.
    Arvin Pierre, Noa Gavriel, Marianne Guilbard, Eric Ogier-Denis, Eric Chevet, Frederic Delom, Aeid Igbaria.
      SIGNIFICANCE: Oxidative folding within the endoplasmic reticulum (ER) introduces disulfide bonds into nascent polypeptides, ensuring proteins' stability and proper functioning. Consequently, this process is critical for maintaining proteome integrity and overall health. The productive folding of thousands of secretory proteins requires stringent quality control measures, such as the Unfolded Protein Response (UPR) and ER-Associated Degradation (ERAD), which contribute significantly to maintaining ER homeostasis. ER-localised protein disulfide isomerases (PDI) play an essential role in each of these processes, thereby contributing to various aspects of ER homeostasis, including maintaining redox balance, proper protein folding, and signaling from the ER to the nucleus.RECENT ADVANCES: Over the years, there have been increasing reports of the (re)localization of PDI family members and other ER-localized proteins to various compartments. A prime example is the Anterior gradient (AGR) family of PDI proteins, which have been reported to relocate to the cytosol or the extracellular environment, acquiring gain of functions that intersect with various cellular signaling pathways.
    CRITICAL ISSUES: Here, we summarize the functions of PDIs and their gain or loss of functions in non-ER locations. We will focus on the activity, localization, and function of the AGR proteins: AGR1, AGR2, and AGR3.
    FUTURE DIRECTION: Targeting PDIs in general and AGRs in particular is a promising strategy in different human diseases. Thus, there is a need for innovative strategies and tools aimed at targeting PDIs; those strategies should integrate the specific localization and newly acquired functions of these PDIs rather than solely focusing on their canonical roles.
    DOI:  https://doi.org/10.1089/ars.2024.0561
  3. Mol Cell. 2024 Feb 22. pii: S1097-2765(24)00096-0. [Epub ahead of print]
    Bidirectional substrate shuttling between the 26S proteasome and the Cdc48 ATPase promotes protein degradation.
    Hao Li, Zhejian Ji, Joao A Paulo, Steven P Gygi, Tom A Rapoport.
      Most eukaryotic proteins are degraded by the 26S proteasome after modification with a polyubiquitin chain. Substrates lacking unstructured segments cannot be degraded directly and require prior unfolding by the Cdc48 ATPase (p97 or VCP in mammals) in complex with its ubiquitin-binding partner Ufd1-Npl4 (UN). Here, we use purified yeast components to reconstitute Cdc48-dependent degradation of well-folded model substrates by the proteasome. We show that a minimal system consists of the 26S proteasome, the Cdc48-UN ATPase complex, the proteasome cofactor Rad23, and the Cdc48 cofactors Ubx5 and Shp1. Rad23 and Ubx5 stimulate polyubiquitin binding to the 26S proteasome and the Cdc48-UN complex, respectively, allowing these machines to compete for substrates before and after their unfolding. Shp1 stimulates protein unfolding by the Cdc48-UN complex rather than substrate recruitment. Experiments in yeast cells confirm that many proteins undergo bidirectional substrate shuttling between the 26S proteasome and Cdc48 ATPase before being degraded.
    Keywords:  AAA ATPase; Cdc48; ERAD; p97; proteasome; protein degradation; protein unfolding; shuttling factor; ubiquitin
    DOI:  https://doi.org/10.1016/j.molcel.2024.01.029
  4. Cell Rep. 2024 Feb 26. pii: S2211-1247(24)00188-8. [Epub ahead of print]43(3): 113860
    NEMF-mediated Listerin-independent mitochondrial translational surveillance by E3 ligase Pirh2 and mitochondrial protease ClpXP.
    Liang Lv, Jinyou Mo, Yumin Qing, Shuchao Wang, Leijie Chen, Anna Mei, Ru Xu, Hualin Huang, Jieqiong Tan, Yifu Li, Jia Liu.
      The ribosome-associated protein quality control (RQC) pathway acts as a translational surveillance mechanism to maintain proteostasis. In mammalian cells, the cytoplasmic RQC pathway involves nuclear export mediator factor (NEMF)-dependent recruitment of the E3 ligase Listerin to ubiquitinate ribosome-stalled nascent polypeptides on the lysine residue for degradation. However, the quality control of ribosome-stalled nuclear-encoded mitochondrial nascent polypeptides remains elusive, as these peptides can be partially imported into mitochondria through translocons, restricting accessibility to the lysine by Listerin. Here, we identify a Listerin-independent organelle-specific mitochondrial RQC pathway that acts on NEMF-mediated carboxy-terminal poly-alanine modification. In the pathway, mitochondrial proteins carrying C-end poly-Ala tails are recognized by the cytosolic E3 ligase Pirh2 and the ClpXP protease in the mitochondria, which coordinately clear ribosome-stalled mitochondrial nascent polypeptides. Defects in this elimination pathway result in NEMF-mediated aggregates and mitochondrial integrity failure, thus providing a potential molecular mechanism of the RQC pathway in mitochondrial-associated human diseases.
    Keywords:  CP: Microbiology; CP: Molecular biology; ClpXP; Listerin; NEMF; Pirh2; mitochondrion
    DOI:  https://doi.org/10.1016/j.celrep.2024.113860
  5. Autophagy. 2024 Feb 27.
    The HSP90 chaperone code regulates the crosstalk between proteostasis and autophagy.
    Sarah J Backe, Jennifer A Heritz, Mehdi Mollapour.
      Proteostasis, the maintenance of proper protein folding, stability, and degradation within cells, is fundamental for cellular function. Two key players in this intricate cellular process are macroautophagy/autophagy and chaperoning of nascent proteins. Here, we explore the crosstalk between autophagy and the HSP90 chaperone in maintaining proteostasis, highlighting their interplay and significance in cellular homeostasis.
    Keywords:  Atg1; ULK1; chaperone; chaperone code; co-chaperone; phosphorylation
    DOI:  https://doi.org/10.1080/15548627.2024.2323259
  6. ACS Synth Biol. 2024 Feb 26.
    Subunits of an E3 Ligase Complex as Degrons for Efficient Degradation of Cytosolic, Nuclear, and Membrane Proteins.
    Anže Verbič, Tina Lebar, Arne Praznik, Roman Jerala.
      Protein degradation is a highly regulated cellular process crucial to enable the high dynamic range of the response to external and internal stimuli and to balance protein biosynthesis to maintain cell homeostasis. Within mammalian cells, hundreds of E3 ubiquitin ligases target specific protein substrates and could be repurposed for synthetic biology. Here, we present a systematic analysis of the four protein subunits of the multiprotein E3 ligase complex as scaffolds for the designed degrons. While all of them were functional, the fusion of a fragment of Skp1 with the target protein enabled the most effective degradation. Combination with heterodimerizing peptides, protease substrate sites, and chemically inducible dimerizers enabled the regulation of protein degradation. While the investigated subunits of E3 ligases showed variable degradation efficiency of the membrane and cytosolic and nuclear proteins, the bipartite SSD (SOCSbox-Skp1(ΔC111)) degron enabled fast degradation of protein targets in all tested cellular compartments, including the nucleus and plasma membrane, in different cell lines and could be chemically regulated. These subunits could be employed for research as well as for diverse applications, as demonstrated in the regulation of Cas9 and chimeric antigen receptor proteins.
    Keywords:  E3 ligase; control of protein expression; degrons; synthetic biology
    DOI:  https://doi.org/10.1021/acssynbio.3c00588
  7. Curr Biol. 2024 Feb 27. pii: S0960-9822(24)00156-8. [Epub ahead of print]
    Collective cell migration relies on PPP1R15-mediated regulation of the endoplasmic reticulum stress response.
    Yujun Chen, Jocelyn A McDonald.
      Collective cell migration is integral to many developmental and disease processes. Previously, we discovered that protein phosphatase 1 (Pp1) promotes border cell collective migration in the Drosophila ovary. We now report that the Pp1 phosphatase regulatory subunit dPPP1R15 is a critical regulator of border cell migration. dPPP1R15 is an ortholog of mammalian PPP1R15 proteins that attenuate the endoplasmic reticulum (ER) stress response. We show that, in collectively migrating border cells, dPPP1R15 phosphatase restrains an active physiological protein kinase R-like ER kinase- (PERK)-eIF2α-activating transcription factor 4 (ATF4) stress pathway. RNAi knockdown of dPPP1R15 blocks border cell delamination from the epithelium and subsequent migration, increases eIF2α phosphorylation, reduces translation, and drives expression of the stress response transcription factor ATF4. We observe similar defects upon overexpression of ATF4 or the eIF2α kinase PERK. Furthermore, we show that normal border cells express markers of the PERK-dependent ER stress response and require PERK and ATF4 for efficient migration. In many other cell types, unresolved ER stress induces initiation of apoptosis. In contrast, border cells with chronic RNAi knockdown of dPPP1R15 survive. Together, our results demonstrate that the PERK-eIF2α-ATF4 pathway, regulated by dPPP1R15 activity, counteracts the physiological ER stress that occurs during collective border cell migration. We propose that in vivo collective cell migration is intrinsically "stressful," requiring tight homeostatic control of the ER stress response for collective cell cohesion, dynamics, and movement.
    Keywords:  cell migration; cell protrusions; development; integrated stress response; oogenesis
    DOI:  https://doi.org/10.1016/j.cub.2024.02.014
  8. bioRxiv. 2024 Feb 16. pii: 2024.02.15.580492. [Epub ahead of print]
    A ratiometric ER calcium sensor for quantitative comparisons across cell types and subcellular regions.
    Ryan J Farrell, Kirsten G Bredvik, Michael B Hoppa, S Thomas Hennigan, Timothy A Brown, Timothy A Ryan.
      The endoplasmic reticulum (ER) is an important regulator of Ca2+ in cells and dysregulation of ER calcium homeostasis can lead to numerous pathologies. Understanding how various pharmacological and genetic perturbations of ER Ca2+ homeostasis impacts cellular physiology would likely be facilitated by more quantitative measurements of ER Ca2+ levels that allow easier comparisons across conditions. Here, we developed a ratiometric version of our original ER-GCaMP probe that allows for more quantitative comparisons of the concentration of Ca2+ in the ER across cell types and sub-cellular compartments. Using this approach we show that the resting concentration of ER Ca2+ in primary dissociated neurons is substantially lower than that in measured in embryonic fibroblasts.
    DOI:  https://doi.org/10.1101/2024.02.15.580492
  9. PLoS Genet. 2024 Feb 26. 20(2): e1011171
    Maintenance of proteostasis by Drosophila Rer1 is essential for competitive cell survival and Myc-driven overgrowth.
    Pranab Kumar Paul, Shruti Umarvaish, Shivani Bajaj, Rishana Farin S, Hrudya Mohan, Wim Annaert, Varun Chaudhary.
      Defects in protein homeostasis can induce proteotoxic stress, affecting cellular fitness and, consequently, overall tissue health. In various growing tissues, cell competition based mechanisms facilitate detection and elimination of these compromised, often referred to as 'loser', cells by the healthier neighbors. The precise connection between proteotoxic stress and competitive cell survival remains largely elusive. Here, we reveal the function of an endoplasmic reticulum (ER) and Golgi localized protein Rer1 in the regulation of protein homeostasis in the developing Drosophila wing epithelium. Our results show that loss of Rer1 leads to proteotoxic stress and PERK-mediated phosphorylation of eukaryotic initiation factor 2α. Clonal analysis showed that rer1 mutant cells are identified as losers and eliminated through cell competition. Interestingly, we find that Rer1 levels are upregulated upon Myc-overexpression that causes overgrowth, albeit under high proteotoxic stress. Our results suggest that increased levels of Rer1 provide cytoprotection to Myc-overexpressing cells by alleviating the proteotoxic stress and thereby supporting Myc-driven overgrowth. In summary, these observations demonstrate that Rer1 acts as a novel regulator of proteostasis in Drosophila and reveal its role in competitive cell survival.
    DOI:  https://doi.org/10.1371/journal.pgen.1011171
  10. Nucleic Acids Res. 2024 Feb 27. pii: gkae136. [Epub ahead of print]
    UPF1 ATPase autoinhibition and activation modulate RNA binding kinetics and NMD efficiency.
    Joseph H Chapman, Alice M Youle, Acadia L Grimme, Keir C Neuman, J Robert Hogg.
      The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited 'closed' state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active 'open' state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants (i.e. poorly processive, slow, and mechanochemically uncoupled) can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower RNA binding kinetics and enhanced ATP-stimulated RNA dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2 and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.
    DOI:  https://doi.org/10.1093/nar/gkae136
  11. Nucleic Acids Res. 2024 Feb 27. pii: gkae137. [Epub ahead of print]
    Ribosomal quality control factors inhibit repeat-associated non-AUG translation from GC-rich repeats.
    Yi-Ju Tseng, Amy Krans, Indranil Malik, Xiexiong Deng, Evrim Yildirim, Sinem Ovunc, Elizabeth M H Tank, Karen Jansen-West, Ross Kaufhold, Nicolas B Gomez, Roger Sher, Leonard Petrucelli, Sami J Barmada, Peter K Todd.
      A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1 and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter assays and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation-suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders.
    DOI:  https://doi.org/10.1093/nar/gkae137
  12. Nat Commun. 2024 Feb 26. 15(1): 1756
    Transient disome complex formation in native polysomes during ongoing protein synthesis captured by cryo-EM.
    Timo Flügel, Magdalena Schacherl, Anett Unbehaun, Birgit Schroeer, Marylena Dabrowski, Jörg Bürger, Thorsten Mielke, Thiemo Sprink, Christoph A Diebolder, Yollete V Guillén Schlippe, Christian M T Spahn.
      Structural studies of translating ribosomes traditionally rely on in vitro assembly and stalling of ribosomes in defined states. To comprehensively visualize bacterial translation, we reactivated ex vivo-derived E. coli polysomes in the PURE in vitro translation system and analyzed the actively elongating polysomes by cryo-EM. We find that 31% of 70S ribosomes assemble into disome complexes that represent eight distinct functional states including decoding and termination intermediates, and a pre-nucleophilic attack state. The functional diversity of disome complexes together with RNase digest experiments suggests that paused disome complexes transiently form during ongoing elongation. Structural analysis revealed five disome interfaces between leading and queueing ribosomes that undergo rearrangements as the leading ribosome traverses through the elongation cycle. Our findings reveal at the molecular level how bL9's CTD obstructs the factor binding site of queueing ribosomes to thwart harmful collisions and illustrate how translation dynamics reshape inter-ribosomal contacts.
    DOI:  https://doi.org/10.1038/s41467-024-46092-3
  13. Proc Natl Acad Sci U S A. 2024 Mar 05. 121(10): e2310756121
    Proteasome condensate formation is driven by multivalent interactions with shuttle factors and ubiquitin chains.
    Kenrick A Waite, Gabrielle Vontz, Stella Y Lee, Jeroen Roelofs.
      Stress conditions can cause the relocalization of proteasomes to condensates in yeast and mammalian cells. The interactions that facilitate the formation of proteasome condensates, however, are unclear. Here, we show that the formation of proteasome condensates in yeast depends on ubiquitin chains together with the proteasome shuttle factors Rad23 and Dsk2. These shuttle factors colocalize to these condensates. Strains deleted for the third shuttle factor gene, DDI1, show proteasome condensates in the absence of cellular stress, consistent with the accumulation of substrates with long K48-linked ubiquitin chains that accumulate in this mutant. We propose a model where the long K48-linked ubiquitin chains function as a scaffold for the ubiquitin-binding domains of the shuttle factors and the proteasome, allowing for the multivalent interactions that further drive condensate formation. Indeed, we determined different intrinsic ubiquitin receptors of the proteasome-Rpn1, Rpn10, and Rpn13-and the Ubl domains of Rad23 and Dsk2 are critical under different condensate-inducing conditions. In all, our data support a model where the cellular accumulation of substrates with long ubiquitin chains, potentially due to reduced cellular energy, allows for proteasome condensate formation. This suggests that proteasome condensates are not simply for proteasome storage, but function to sequester soluble ubiquitinated substrates together with inactive proteasomes.
    Keywords:  liquid–liquid phase separation (LLPS); proteasome; proteasome storage granule (PSG); shuttle factors; yeast
    DOI:  https://doi.org/10.1073/pnas.2310756121
  14. Nature. 2024 Feb 28.
    The CRL5-SPSB3 ubiquitin ligase targets nuclear cGAS for degradation.
    Pengbiao Xu, Ying Liu, Chong Liu, Baptiste Guey, Lingyun Li, Pauline Melenec, Jonathan Ricci, Andrea Ablasser.
      Cyclic GMP-AMP synthase (cGAS) senses aberrant DNA during infection, cancer and inflammatory disease, and initiates potent innate immune responses through the synthesis of 2'3'-cyclic GMP-AMP (cGAMP)1-7. The indiscriminate activity of cGAS towards DNA demands tight regulatory mechanisms that are necessary to maintain cell and tissue homeostasis under normal conditions. Inside the cell nucleus, anchoring to nucleosomes and competition with chromatin architectural proteins jointly prohibit cGAS activation by genomic DNA8-15. However, the fate of nuclear cGAS and its role in cell physiology remains unclear. Here we show that the ubiquitin proteasomal system (UPS) degrades nuclear cGAS in cycling cells. We identify SPSB3 as the cGAS-targeting substrate receptor that associates with the cullin-RING ubiquitin ligase 5 (CRL5) complex to ligate ubiquitin onto nuclear cGAS. A cryo-electron microscopy structure of nucleosome-bound cGAS in a complex with SPSB3 reveals a highly conserved Asn-Asn (NN) minimal degron motif at the C terminus of cGAS that directs SPSB3 recruitment, ubiquitylation and cGAS protein stability. Interference with SPSB3-regulated nuclear cGAS degradation primes cells for type I interferon signalling, conferring heightened protection against infection by DNA viruses. Our research defines protein degradation as a determinant of cGAS regulation in the nucleus and provides structural insights into an element of cGAS that is amenable to therapeutic exploitation.
    DOI:  https://doi.org/10.1038/s41586-024-07112-w
  15. J Mol Biol. 2024 Feb 23. pii: S0022-2836(24)00071-8. [Epub ahead of print]436(8): 168499
    The α-crystallin Chaperones Undergo a Quasi-ordered Co-aggregation Process in Response to Saturating Client Interaction.
    Adam P Miller, Susan E O'Neill, Kirsten J Lampi, Steve L Reichow.
      Small heat shock proteins (sHSPs) are ATP-independent chaperones vital to cellular proteostasis, preventing protein aggregation events linked to various human diseases including cataract. The α-crystallins, αA-crystallin (αAc) and αB-crystallin (αBc), represent archetypal sHSPs that exhibit complex polydispersed oligomeric assemblies and rapid subunit exchange dynamics. Yet, our understanding of how this plasticity contributes to chaperone function remains poorly understood. Using biochemical and biophysical analyses combined with single-particle electron microscopy (EM), we examined structural changes in αAc, αBc and native heteromeric lens α-crystallins (αLc) in their apo-states and at varying degree of chaperone saturation leading to co-aggregation, using lysozyme and insulin as model clients. Quantitative single-particle analysis unveiled a continuous spectrum of oligomeric states formed during the co-aggregation process, marked by significant client-triggered expansion and quasi-ordered elongation of the sHSP oligomeric scaffold, whereby the native cage-like sHSP assembly displays a directional growth to accommodate saturating conditions of client sequestration. These structural modifications culminated in an apparent amorphous collapse of chaperone-client complexes, resulting in the creation of co-aggregates capable of scattering visible light. Intriguingly, these co-aggregates maintain internal morphological features of highly elongated sHSP oligomers with striking resemblance to polymeric α-crystallin species isolated from aged lens tissue. This mechanism appears consistent across αAc, αBc and αLc, albeit with varying degrees of susceptibility to client-induced co-aggregation. Importantly, our findings suggest that client-induced co-aggregation follows a distinctive mechanistic and quasi-ordered trajectory, distinct from a purely amorphous process. These insights reshape our understanding of the physiological and pathophysiological co-aggregation processes of α-crystallins, carrying potential implications for a pathway toward cataract formation.
    Keywords:  HSPB4; HSPB5; chaperone; small heat shock protein (sHSP); α-crystallin
    DOI:  https://doi.org/10.1016/j.jmb.2024.168499
  16. bioRxiv. 2024 Feb 17. pii: 2024.02.16.580396. [Epub ahead of print]
    Degron-based bioPROTACs for controlling signaling in CAR T cells.
    Matthew S Kim, Hersh K Bhargava, Gavin E Shavey, Wendell A Lim, Hana El-Samad, Andrew H Ng.
      Chimeric antigen receptor (CAR) T cells have made a tremendous impact in the clinic, but potent signaling through the CAR can be detrimental to treatment safety and efficacy. The use of protein degradation to control CAR signaling can address these issues in pre-clinical models. Existing strategies for regulating CAR stability rely on small molecules to induce systemic degradation. In contrast to small molecule regulation, genetic circuits offer a more precise method to control CAR signaling in an autonomous, cell-by-cell fashion. Here, we describe a programmable protein degradation tool that adopts the framework of bioPROTACs, heterobifunctional proteins that are composed of a target recognition domain fused to a domain that recruits the endogenous ubiquitin proteasome system. We develop novel bioPROTACs that utilize a compact four residue degron and demonstrate degradation of cytosolic and membrane protein targets using either a nanobody or synthetic leucine zipper as a protein binder. Our bioPROTACs exhibit potent degradation of CARs and can inhibit CAR signaling in primary human T cells. We demonstrate the utility of our bioPROTACs by constructing a genetic circuit to degrade the tyrosine kinase ZAP70 in response to recognition of a specific membrane-bound antigen. This circuit is able to disrupt CAR T cell signaling only in the presence of a specific cell population. These results suggest that bioPROTACs are a powerful tool for expanding the cell engineering toolbox for CAR T cells.
    DOI:  https://doi.org/10.1101/2024.02.16.580396
  17. bioRxiv. 2024 Feb 16. pii: 2024.02.13.580169. [Epub ahead of print]
    UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation.
    Rochelle L Tiedemann, Joel Hrit, Qian Du, Ashley K Wiseman, Hope E Eden, Bradley M Dickson, Xiangqian Kong, Alison A Chomiak, Robert M Vaughan, Jakob M Hebert, Yael David, Wanding Zhou, Stephen B Baylin, Peter A Jones, Susan J Clark, Scott B Rothbart.
      The RING E3 ubiquitin ligase UHRF1 is an established cofactor for DNA methylation inheritance. Nucleosomal engagement through histone and DNA interactions directs UHRF1 ubiquitin ligase activity toward lysines on histone H3 tails, creating binding sites for DNMT1 through ubiquitin interacting motifs (UIM1 and UIM2). Here, we profile contributions of UHRF1 and DNMT1 to genome-wide DNA methylation inheritance and dissect specific roles for ubiquitin signaling in this process. We reveal DNA methylation maintenance at low-density CpGs is vulnerable to disruption of UHRF1 ubiquitin ligase activity and DNMT1 ubiquitin reading activity through UIM1. Hypomethylation of low-density CpGs in this manner induces formation of partially methylated domains (PMD), a methylation signature observed across human cancers. Furthermore, disrupting DNMT1 UIM2 function abolishes DNA methylation maintenance. Collectively, we show DNMT1-dependent DNA methylation inheritance is a ubiquitin-regulated process and suggest a disrupted UHRF1-DNMT1 ubiquitin signaling axis contributes to the development of PMDs in human cancers.
    DOI:  https://doi.org/10.1101/2024.02.13.580169
  18. Sci Adv. 2024 Mar;10(9): eadk1814
    A uniquely efficacious type of CFTR corrector with complementary mode of action.
    Valentina Marchesin, Lucile Monnier, Peter Blattmann, Florent Chevillard, Christine Kuntz, Camille Forny, Judith Kamper, Rolf Studer, Alexandre Bossu, Eric A Ertel, Oliver Nayler, Christine Brotschi, Jodi T Williams, John Gatfield.
      Three distinct pharmacological corrector types (I, II, III) with different binding sites and additive behavior only partially rescue the F508del-cystic fibrosis transmembrane conductance regulator (CFTR) folding and trafficking defect observed in cystic fibrosis. We describe uniquely effective, macrocyclic CFTR correctors that were additive to the known corrector types, exerting a complementary "type IV" corrector mechanism. Macrocycles achieved wild-type-like folding efficiency of F508del-CFTR at the endoplasmic reticulum and normalized CFTR currents in reconstituted patient-derived bronchial epithelium. Using photo-activatable macrocycles, docking studies and site-directed mutagenesis a highly probable binding site and pose for type IV correctors was identified in a cavity between lasso helix-1 (Lh1) and transmembrane helix-1 of membrane spanning domain (MSD)-1, distinct from the known corrector binding sites. Since only F508del-CFTR fragments spanning from Lh1 until MSD2 responded to type IV correctors, these likely promote cotranslational assembly of Lh1, MSD1, and MSD2. Previously corrector-resistant CFTR folding mutants were also robustly rescued, suggesting substantial therapeutic potential for type IV correctors.
    DOI:  https://doi.org/10.1126/sciadv.adk1814
  19. FEBS J. 2024 Feb 24.
    Intraluminal vesicle trafficking is involved in the secretion of base excision repair protein APE1.
    Isabella Parolini, Monica Degrassi, Francesca Spadaro, Federica Caponnetto, Katia Fecchi, Serena Mastantuono, Xue Zhouyiyuan, Bruce Demple, Daniela Cesselli, Gianluca Tell.
      The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an essential enzyme of the base excision repair pathway of non-distorting DNA lesions. In response to genotoxic treatments, APE1 is highly secreted (sAPE1) in association with small-extracellular vesicles (EVs). Interestingly, its presence in the serum of patients with hepatocellular or non-small-cell-lung cancers may represent a prognostic biomarker. The mechanism driving APE1 to associate with EVs is unknown, but is of paramount importance in better understanding the biological roles of sAPE1. Because APE1 lacks an endoplasmic reticulum-targeting signal peptide, it can be secreted through an unconventional protein secretion endoplasmic reticulum-Golgi-independent pathway, which includes an endosome-based secretion of intraluminal vesicles, mediated by multivesicular bodies (MVBs). Using HeLa and A549 cell lines, we investigated the role of endosomal sorting complex required for transport protein pathways (either-dependent or -independent) in the constitutive or trichostatin A-induced secretion of sAPE1, by means of manumycin A and GW 4869 treatments. Through an in-depth biochemical analysis of late-endosomes (LEs) and early-endosomes (EEs), we observed that the distribution of APE1 on density gradient corresponded to that of LE-CD63, LE-Rab7, EE-EEA1 and EE-Rab 5. Interestingly, the secretion of sAPE1, induced by cisplatin genotoxic stress, involved an autophagy-based unconventional secretion requiring MVBs. The present study enlightens the central role played by MVBs in the secretion of sAPE1 under various stimuli, and offers new perspectives in understanding the biological relevance of sAPE1 in cancer cells.
    Keywords:  APE1; BER pathway; autophagy; extracellular vesicles; multivesicular bodies
    DOI:  https://doi.org/10.1111/febs.17088
  20. Proc Natl Acad Sci U S A. 2024 Mar 05. 121(10): e2313540121
    Tom20 gates PINK1 activity and mediates its tethering of the TOM and TIM23 translocases upon mitochondrial stress.
    Mohamed A Eldeeb, Andrew N Bayne, Armaan Fallahi, Thomas Goiran, Emma J MacDougall, Andrea Soumbasis, Cornelia E Zorca, Jace-Jones Tabah, Rhalena A Thomas, Nathan Karpilovsky, Meghna Mathur, Thomas M Durcan, Jean-François Trempe, Edward A Fon.
      Mutations in PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive early-onset Parkinson's disease (PD). PINK1 is a Ser/Thr kinase that regulates mitochondrial quality control by triggering mitophagy mediated by the ubiquitin (Ub) ligase Parkin. Upon mitochondrial damage, PINK1 accumulates on the outer mitochondrial membrane forming a high-molecular-weight complex with the translocase of the outer membrane (TOM). PINK1 then phosphorylates Ub, which enables recruitment and activation of Parkin followed by autophagic clearance of the damaged mitochondrion. Thus, Parkin-dependent mitophagy hinges on the stable accumulation of PINK1 on the TOM complex. Yet, the mechanism linking mitochondrial stressors to PINK1 accumulation and whether the translocases of the inner membrane (TIMs) are also involved remain unclear. Herein, we demonstrate that mitochondrial stress induces the formation of a PINK1-TOM-TIM23 supercomplex in human cultured cell lines, dopamine neurons, and midbrain organoids. Moreover, we show that PINK1 is required to stably tether the TOM to TIM23 complexes in response to stress such that the supercomplex fails to accumulate in cells lacking PINK1. This tethering is dependent on an interaction between the PINK1 N-terminal-C-terminal extension module and the cytosolic domain of the Tom20 subunit of the TOM complex, the disruption of which, by either designer or PD-associated PINK1 mutations, inhibits downstream mitophagy. Together, the findings provide key insight into how PINK1 interfaces with the mitochondrial import machinery, with important implications for the mechanisms of mitochondrial quality control and PD pathogenesis.
    Keywords:  PINK1; mitochondrial import; mitochondrial quality control; mitophagy; proteolysis
    DOI:  https://doi.org/10.1073/pnas.2313540121
  21. PLoS Genet. 2024 Feb 29. 20(2): e1011194
    Sequestrase chaperones protect against oxidative stress-induced protein aggregation and [PSI+] prion formation.
    Zorana Carter, Declan Creamer, Aikaterini Kouvidi, Chris M Grant.
      Misfolded proteins are usually refolded to their functional conformations or degraded by quality control mechanisms. When misfolded proteins evade quality control, they can be sequestered to specific sites within cells to prevent the potential dysfunction and toxicity that arises from protein aggregation. Btn2 and Hsp42 are compartment-specific sequestrases that play key roles in the assembly of these deposition sites. Their exact intracellular functions and substrates are not well defined, particularly since heat stress sensitivity is not observed in deletion mutants. We show here that Btn2 and Hsp42 are required for tolerance to oxidative stress conditions induced by exposure to hydrogen peroxide. Btn2 and Hsp42 act to sequester oxidized proteins into defined PQC sites following ROS exposure and their absence leads to an accumulation of protein aggregates. The toxicity of protein aggregate accumulation causes oxidant sensitivity in btn2 hsp42 sequestrase mutants since overexpression of the Hsp104 disaggregase rescues oxidant tolerance. We have identified the Sup35 translation termination factor as an in vivo sequestrase substrate and show that Btn2 and Hsp42 act to suppress oxidant-induced formation of the yeast [PSI+] prion, which is the amyloid form of Sup35. [PSI+] prion formation in sequestrase mutants does not require IPOD (insoluble protein deposit) localization which is the site where amyloids are thought to undergo fragmentation and seeding to propagate their heritable prion form. Instead, both amorphous and amyloid Sup35 aggregates are increased in btn2 hsp42 mutants consistent with the idea that prion formation occurs at multiple intracellular sites during oxidative stress conditions in the absence of sequestrase activity. Taken together, our data identify protein sequestration as a key antioxidant defence mechanism that functions to mitigate the damaging consequences of protein oxidation-induced aggregation.
    DOI:  https://doi.org/10.1371/journal.pgen.1011194
  22. Trends Cell Biol. 2024 Feb 28. pii: S0962-8924(24)00024-2. [Epub ahead of print]
    Translation is an emerging constraint on protein homeostasis in ageing.
    Jack Llewellyn, Simon J Hubbard, Joe Swift.
      Proteins are molecular machines that provide structure and perform vital transport, signalling and enzymatic roles. Proteins expressed by cells require tight regulation of their concentration, folding, localisation, and modifications; however, this state of protein homeostasis is continuously perturbed by tissue-level stresses. While cells in healthy tissues are able to buffer against these perturbations, for example, by expression of chaperone proteins, protein homeostasis is lost in ageing, and can lead to protein aggregation characteristic of protein folding diseases. Here, we review reports of a progressive disconnect between transcriptomic and proteomic regulation during cellular ageing. We discuss how age-associated changes to cellular responses to specific stressors in the tissue microenvironment are exacerbated by loss of ribosomal proteins, ribosomal pausing, and mistranslation.
    Keywords:  cellular senescence; mistranslation; molecular chaperones; protein turnover; proteostasis network; ribosomal activity
    DOI:  https://doi.org/10.1016/j.tcb.2024.02.001
  23. J Mol Cell Cardiol. 2024 Feb 23. pii: S0022-2828(24)00018-X. [Epub ahead of print]189 12-24
    ATF6 protects against protein misfolding during cardiac hypertrophy.
    Christoph Hofmann, Marjan Aghajani, Cecily D Alcock, Erik A Blackwood, Clara Sandmann, Nicole Herzog, Julia Groß, Lars Plate, R Luke Wiseman, Randal J Kaufman, Hugo A Katus, Tobias Jakobi, Mirko Völkers, Christopher C Glembotski, Shirin Doroudgar.
      Cardiomyocytes activate the unfolded protein response (UPR) transcription factor ATF6 during pressure overload-induced hypertrophic growth. The UPR is thought to increase ER protein folding capacity and maintain proteostasis. ATF6 deficiency during pressure overload leads to heart failure, suggesting that ATF6 protects against myocardial dysfunction by preventing protein misfolding. However, conclusive evidence that ATF6 prevents toxic protein misfolding during cardiac hypertrophy is still pending. Here, we found that activation of the UPR, including ATF6, is a common response to pathological cardiac hypertrophy in mice. ATF6 KO mice failed to induce sufficient levels of UPR target genes in response to chronic isoproterenol infusion or transverse aortic constriction (TAC), resulting in impaired cardiac growth. To investigate the effects of ATF6 on protein folding, the accumulation of poly-ubiquitinated proteins as well as soluble amyloid oligomers were directly quantified in hypertrophied hearts of WT and ATF6 KO mice. Whereas only low levels of protein misfolding was observed in WT hearts after TAC, ATF6 KO mice accumulated increased quantities of misfolded protein, which was associated with impaired myocardial function. Collectively, the data suggest that ATF6 plays a critical adaptive role during cardiac hypertrophy by protecting against protein misfolding.
    Keywords:  ATF6; Cardiac hypertrophy; Protein misfolding; Proteostasis; UPR; Unfolded protein response
    DOI:  https://doi.org/10.1016/j.yjmcc.2024.02.001
  24. bioRxiv. 2024 Feb 15. pii: 2024.02.14.580336. [Epub ahead of print]
    A quantitative ultrastructural timeline of nuclear autophagy reveals a role for dynamin-like protein 1 at the nuclear envelope.
    Philip J Mannino, Andrew Perun, Ivan Surovstev, Nicholas R Ader, Lin Shao, Thomas J Melia, Megan C King, C Patrick Lusk.
      Autophagic mechanisms that maintain nuclear envelope homeostasis are bulwarks to aging and disease. By leveraging 4D lattice light sheet microscopy and correlative light and electron tomography, we define a quantitative and ultrastructural timeline of a nuclear macroautophagy (nucleophagy) pathway in yeast. Nucleophagy initiates with a rapid local accumulation of the nuclear cargo adaptor Atg39 at the nuclear envelope adjacent to the nucleus-vacuole junction and is delivered to the vacuole in ~300 seconds through an autophagosome intermediate. Mechanistically, nucleophagy incorporates two consecutive and genetically defined membrane fission steps: inner nuclear membrane (INM) fission generates a lumenal vesicle in the perinuclear space followed by outer nuclear membrane (ONM) fission to liberate a double membraned vesicle to the cytosol. ONM fission occurs independently of phagophore engagement and instead relies surprisingly on dynamin-like protein1 (Dnm1), which is recruited to sites of Atg39 accumulation at the nuclear envelope. Loss of Dnm1 compromises nucleophagic flux by stalling nucleophagy after INM fission. Our findings reveal how nuclear and INM cargo are removed from an intact nucleus without compromising its integrity, achieved in part by a non-canonical role for Dnm1 in nuclear envelope remodeling.
    DOI:  https://doi.org/10.1101/2024.02.14.580336
  25. Autophagy. 2024 Feb 27.
    Neuronal waste management: new roles for autophagy genes in the extrusion of protein aggregates and in longevity.
    Ling-Hsuan Sun, Caitlin Lange, Malene Hansen, Caroline Kumsta.
      A decline in macroautophagic/autophagic activity with age contributes to the accumulation of damaged molecules and is associated with the impairment of neuronal functions and the onset of age-related diseases, particularly neurodegenerative disorders. To learn about the neuronal-specific roles of autophagy genes in aging, we specifically inhibited autophagy genes pan-neuronally in C. elegans, which leads to unexpected positive impacts on neuronal homeostasis including polyQ aggregate load and organismal lifespan. These improvements are independent of canonical, degradative autophagy in neurons and instead correlated with an increase in the secretion of large, extracellular vesicles, known as exophers. We found that the ATG-16.2 WD40 domain, a conserved domain critical for at least some noncanonical autophagy functions of ATG16L1 in mammalian cells, is required for the increased exopher biogenesis, reduction in polyQ aggregate load, and lifespan extension induced by neuronal inhibition of early-acting autophagy genes. Our study suggests that noncanonical functions of ATG-16.2, and potentially other early-acting autophagy genes, may play a role in neuronal exopher formation and in C. elegans aging, extending beyond their canonical degradative functions in the autophagy process.
    Keywords:  ATG-16.2/ATG16L1; Aging; c. elegans; exophers; non-canonical autophagy
    DOI:  https://doi.org/10.1080/15548627.2024.2322420
  26. Mol Cell. 2024 Feb 27. pii: S1097-2765(24)00095-9. [Epub ahead of print]
    Triaging of α-helical proteins to the mitochondrial outer membrane by distinct chaperone machinery based on substrate topology.
    Gayathri Muthukumar, Taylor A Stevens, Alison J Inglis, Theodore K Esantsi, Reuben A Saunders, Fabian Schulte, Rebecca M Voorhees, Alina Guna, Jonathan S Weissman.
      Mitochondrial outer membrane ⍺-helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing the targeting and insertion of these biophysically diverse proteins remain unknown. Here, we first defined the complement of required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct membrane proteins. Systematic analysis of nine identified factors across 21 diverse ⍺-helical substrates reveals that these components are organized into distinct targeting pathways that act on substrates based on their topology. NAC is required for the efficient targeting of polytopic proteins, whereas signal-anchored proteins require TTC1, a cytosolic chaperone that physically engages substrates. Biochemical and mutational studies reveal that TTC1 employs a conserved TPR domain and a hydrophobic groove in its C-terminal domain to support substrate solubilization and insertion into mitochondria. Thus, the targeting of diverse mitochondrial membrane proteins is achieved through topological triaging in the cytosol using principles with similarities to ER membrane protein biogenesis systems.
    Keywords:  CRISPR; NAC; TTC1; cell biology; cytosolic targeting; genetic screens; membrane protein insertion; mitochondrial outer membrane; topology
    DOI:  https://doi.org/10.1016/j.molcel.2024.01.028
  27. Cell. 2024 Feb 29. pii: S0092-8674(24)00101-6. [Epub ahead of print]187(5): 1296-1311.e26
    Rapid simulation of glycoprotein structures by grafting and steric exclusion of glycan conformer libraries.
    Yu-Xi Tsai, Ning-En Chang, Klaus Reuter, Hao-Ting Chang, Tzu-Jing Yang, Sören von Bülow, Vidhi Sehrawat, Noémie Zerrouki, Matthieu Tuffery, Michael Gecht, Isabell Louise Grothaus, Lucio Colombi Ciacchi, Yong-Sheng Wang, Min-Feng Hsu, Kay-Hooi Khoo, Gerhard Hummer, Shang-Te Danny Hsu, Cyril Hanus, Mateusz Sikora.
      Most membrane proteins are modified by covalent addition of complex sugars through N- and O-glycosylation. Unlike proteins, glycans do not typically adopt specific secondary structures and remain very mobile, shielding potentially large fractions of protein surface. High glycan conformational freedom hinders complete structural elucidation of glycoproteins. Computer simulations may be used to model glycosylated proteins but require hundreds of thousands of computing hours on supercomputers, thus limiting routine use. Here, we describe GlycoSHIELD, a reductionist method that can be implemented on personal computers to graft realistic ensembles of glycan conformers onto static protein structures in minutes. Using molecular dynamics simulation, small-angle X-ray scattering, cryoelectron microscopy, and mass spectrometry, we show that this open-access toolkit provides enhanced models of glycoprotein structures. Focusing on N-cadherin, human coronavirus spike proteins, and gamma-aminobutyric acid receptors, we show that GlycoSHIELD can shed light on the impact of glycans on the conformation and activity of complex glycoproteins.
    Keywords:  GABAA receptor; N-cadherin; SAXS; coronavirus S protein; cryo-EM; glycans; glycoproteins; mass spectrometry; molecular dynamics
    DOI:  https://doi.org/10.1016/j.cell.2024.01.034
  28. J Biomol Struct Dyn. 2024 Feb 26. 1-11
    Elucidating the EXOSC3-IRE1α interaction: a convergent study incorporating computational, in vitro and in vivo studies.
    Ariha Ashraf, Younis Majeed, Irfan Gul, Mariam Banday, Khalida Ramzan, Khalid Majid Fazili.
      The Unfolded protein response (UPR) is an adaptive signalling pathway which is triggered by accumulation of unfolded/misfolded protein in ER lumen. The UPR consist of three transmembrane proteins-IRE1α, PERK and ATF6 that sense ER stress which leads to activation and downstream signaling from ER lumen to cytosol to restore homeostasis. IRE1α is an evolutionary conserved arm of UPR and acts as an interaction platform for many potential proteins that become activated under ER stress conditions. We investigated potential partners of IRE1 α through MS studies and found EXOSC3 as one of the binding partner of IRE1α. Exosomal complex proteins have 3'5' exonuclease properties (EXOSC3) that play an important role in mRNA surveillance. This property of exosomal proteins coincides with IRE1α ribonuclease activities and its mechanism of action is similar to that of IRE1α-RIDD pathway which degrades any unstable mRNA that disrupts cellular homeostasis. At the same time, studies have shown that knockdown of EXOSC3 causes ER stress in human cells, so we speculated that there might be a functional crosstalk between IRE1α and EXOSC3 under ER stress conditions. Therefore, we employed computational tools to predict and explore the stability and dynamics of the IRE1α-EXOSC3 complex. The analysis indicates that IRE1α and EXOSC3 exhibit potential interaction with the involvement of ScanNet, predicting binding pockets between the two proteins. Further, the interaction was validated via co-immunoprecipitation and yeast two-hybrid assays, thus suggesting EXOSC3 as a component of the UPRosome complex. Hence, this functional crosstalk might influence the dynamic functional output of IRE1α.Communicated by Ramaswamy H. Sarma.
    Keywords:  Endoplasmic reticulum (ER) stress; Exosomal complex protein 3; IRE1α; Protein Data Bank; Unfolded protein response
    DOI:  https://doi.org/10.1080/07391102.2024.2320252
  29. Cell Rep. 2024 Feb 24. pii: S2211-1247(24)00146-3. [Epub ahead of print]43(3): 113818
    ESCRT-I protein UBAP1 controls ventricular expansion and cortical neurogenesis via modulating adherens junctions of radial glial cells.
    Danping Lu, Yiqiang Zhi, Huizhen Su, Xiang Lin, Jingjing Lin, Yan Shi, Wenxiang Yi, Chaoyin Hong, Tongtong Zhang, Zhifei Fu, Li-Yu Chen, Zhiqi Zhao, Rong Li, Zhiheng Xu, Wanjin Chen, Ning Wang, Dan Xu.
      Intricate cerebral cortex formation is orchestrated by the precise behavior and division dynamics of radial glial cells (RGCs). Endocytosis functions in the recycling and remodeling of adherens junctions (AJs) in response to changes in RGC activity and function. Here, we show that conditional disruption of ubiquitin-associated protein 1 (UBAP1), a component of endosomal sorting complex required for transport (ESCRT), causes severe brain dysplasia and prenatal ventriculomegaly. UBAP1 depletion disrupts the AJs and polarity of RGCs, leading to failure of apically directed interkinetic nuclear migration. Accordingly, UBAP1 knockout or knockdown results in reduced proliferation and precocious differentiation of neural progenitor cells. Mechanistically, UBAP1 regulates the expression and surface localization of cell adhesion molecules, and β-catenin over-expression significantly rescues the phenotypes of Ubap1 knockdown in vivo. Our study reveals a critical physiological role of the ESCRT machinery in cortical neurogenesis by regulating AJs of RGCs.
    Keywords:  CP: Developmental biology; CP: Neuroscience; ESCRT; UBAP1; adherens junctions; cortical neurogenesis; interkinetic nuclear migration; prenatal ventriculomegaly; radial glial cell; β-catenin
    DOI:  https://doi.org/10.1016/j.celrep.2024.113818
  30. Trends Cell Biol. 2024 Feb 28. pii: S0962-8924(24)00029-1. [Epub ahead of print]
    ER: a critical hub for STING signaling regulation.
    Yuan Luo, Lei Chang, Yewei Ji, Tingbo Liang.
      The Stimulator of Interferon Genes (STING) has a crucial role in mediating the immune response against cytosolic double-stranded DNA (dsDNA) and its activation is critically involved in various diseases. STING is synthesized, modified, and resides in the endoplasmic reticulum (ER), and its ER exit is intimately connected with its signaling. The ER, primarily known for its roles in protein folding, lipid synthesis, and calcium storage, has been identified as a pivotal platform for the regulation of a wide range of STING functions. In this review, we discuss the emerging factors that regulate STING in the ER and examine the interplay between STING signaling and ER pathways, highlighting the impacts of such regulations on immune responses and their potential implications in STING-related disorders.
    Keywords:  STING; cell biology; endoplasmic reticulum; innate immunity
    DOI:  https://doi.org/10.1016/j.tcb.2024.02.006
  31. Mol Biol Evol. 2024 Feb 29. pii: msae048. [Epub ahead of print]
    Fitness effects of phenotypic mutations at proteome-scale reveal optimality of translation machinery.
    Cedric Landerer, Jonas Pöhls, Agnes Toth-Petroczy.
      Errors in protein translation can lead to non-genetic, phenotypic mutations, including amino acid misincorporations. While phenotypic mutations can increase protein diversity, the systematic characterization of their proteome-wide frequencies and their evolutionary impact has been lacking. Here, we developed a mechanistic model of translation errors to investigate how selection acts on protein populations produced by amino acid misincorporations. We fitted the model to empirical observations of misincorporations obtained from over a hundred mass spectrometry datasets of E. coli and S. cerevisiae. We found that on average 20-23% of proteins synthesized in the cell are expected to harbour at least one amino acid misincorporation, and that deleterious misincorporations are less likely to occur. Combining misincorporation probabilities and the estimated fitness effects of amino acid substitutions in a population genetics framework, we found 74% of mistranslation events in E. coli and 94% in S. cerevisiae to be neutral. We further show that the set of available synonymous tRNAs is subject to evolutionary pressure, as the presence of missing tRNAs would increase codon-anticodon cross-reactivity and misincorporation error rates. Overall, we find that the translation machinery is likely optimal in E. coli and S. cerevisiae and that both local solutions at the level of codons and a global solution such as the tRNA pool can mitigate the impact of translation errors. We provide a framework to study the evolutionary impact of codon specific translation errors and a method for their proteome-wide detection across organisms and conditions.
    Keywords:  amino acid misincorporation; mass spectrometry; population genetics; translation error landscape; translation fidelity
    DOI:  https://doi.org/10.1093/molbev/msae048
  32. Oncogene. 2024 Feb 28.
    Targeting the IRE1α-XBP1s axis confers selective vulnerability in hepatocellular carcinoma with activated Wnt signaling.
    Tingting Zhang, Faming Zhao, Yi Zhang, Ji-Hua Shi, Fengzhen Cui, Weixiang Ma, Kai Wang, Chuanrui Xu, Qingping Zeng, Rong Zhong, Ningning Li, Yong Liu, Yang Jin, Xia Sheng.
      Liver-specific Ern1 knockout impairs tumor progression in mouse models of hepatocellular carcinoma (HCC). However, the mechanistic role of IRE1α in human HCC remains unclear. In this study, we show that XBP1s, the major downstream effector of IRE1α, is required for HCC cell survival both in vitro and in vivo. Mechanistically, XBP1s transactivates LEF1, a key co-factor of β-catenin, by binding to its promoter. Moreover, XBP1s physically interacts with LEF1, forming a transcriptional complex that enhances classical Wnt signaling. Consistently, the activities of XBP1s and LEF1 are strongly correlated in human HCC and with disease prognosis. Notably, selective inhibition of XBP1 splicing using an IRE1α inhibitor significantly repressed the viability of tumor explants as well as the growth of tumor xenografts derived from patients with distinct Wnt/LEF1 activities. Finally, machine learning algorithms developed a powerful prognostic signature based on the activities of XBP1s/LEF1. In summary, our study uncovers a key mechanistic role for the IRE1α-XBP1s pathway in human HCC. Targeting this axis could provide a promising therapeutic strategy for HCC with hyperactivated Wnt/LEF1 signaling.
    DOI:  https://doi.org/10.1038/s41388-024-02988-4
  33. Nat Commun. 2024 Feb 29. 15(1): 1852
    Deregulated protein homeostasis constrains fetal hematopoietic stem cell pool expansion in Fanconi anemia.
    Narasaiah Kovuru, Makiko Mochizuki-Kashio, Theresa Menna, Greer Jeffrey, Yuning Hong, Young Me Yoon, Zhe Zhang, Peter Kurre.
      Demand-adjusted and cell type specific rates of protein synthesis represent an important safeguard for fate and function of long-term hematopoietic stem cells. Here, we identify increased protein synthesis rates in the fetal hematopoietic stem cell pool at the onset of hematopoietic failure in Fanconi Anemia, a prototypical DNA repair disorder that manifests with bone marrow failure. Mechanistically, the accumulation of misfolded proteins in Fancd2-/- fetal liver hematopoietic stem cells converges on endoplasmic reticulum stress, which in turn constrains midgestational expansion. Restoration of protein folding by the chemical chaperone tauroursodeoxycholic acid, a hydrophilic bile salt, prevents accumulation of unfolded proteins and rescues Fancd2-/- fetal liver long-term hematopoietic stem cell numbers. We find that proteostasis deregulation itself is driven by excess sterile inflammatory activity in hematopoietic and stromal cells within the fetal liver, and dampened Type I interferon signaling similarly restores fetal Fancd2-/- long-term hematopoietic stem cells to wild type-equivalent numbers. Our study reveals the origin and pathophysiological trigger that gives rise to Fanconi anemia hematopoietic stem cell pool deficits. More broadly, we show that fetal protein homeostasis serves as a physiological rheostat for hematopoietic stem cell fate and function.
    DOI:  https://doi.org/10.1038/s41467-024-46159-1
  34. Mol Cell. 2024 Feb 22. pii: S1097-2765(24)00097-2. [Epub ahead of print]
    The proteomic landscape of genotoxic stress-induced micronuclei.
    Kate M MacDonald, Shahbaz Khan, Brian Lin, Rose Hurren, Aaron D Schimmer, Thomas Kislinger, Shane M Harding.
      Micronuclei (MN) are induced by various genotoxic stressors and amass nuclear- and cytoplasmic-resident proteins, priming the cell for MN-driven signaling cascades. Here, we measured the proteome of micronuclear, cytoplasmic, and nuclear fractions from human cells exposed to a panel of six genotoxins, comprehensively profiling their MN protein landscape. We find that MN assemble a proteome distinct from both surrounding cytoplasm and parental nuclei, depleted of spliceosome and DNA damage repair components while enriched for a subset of the replisome. We show that the depletion of splicing machinery within transcriptionally active MN contributes to intra-MN DNA damage, a known precursor to chromothripsis. The presence of transcription machinery in MN is stress-dependent, causing a contextual induction of MN DNA damage through spliceosome deficiency. This dataset represents a unique resource detailing the global proteome of MN, guiding mechanistic studies of MN generation and MN-associated outcomes of genotoxic stress.
    Keywords:  DNA damage; chromothripsis; genotoxin; mass spectrometry; micronuclei; mitotic errors; proteomics; radiation; spliceosome
    DOI:  https://doi.org/10.1016/j.molcel.2024.02.001
  35. Cell Rep Med. 2024 Feb 19. pii: S2666-3791(24)00062-4. [Epub ahead of print] 101439
    SEPN1-related myopathy depends on the oxidoreductase ERO1A and is druggable with the chemical chaperone TUDCA.
    Serena Germani, Andrew Tri Van Ho, Alessandro Cherubini, Ersilia Varone, Alexander Chernorudskiy, Giorgia Maria Renna, Stefano Fumagalli, Marco Gobbi, Jacopo Lucchetti, Marco Bolis, Luca Guarrera, Ilaria Craparotta, Giorgia Rastelli, Giorgia Piccoli, Cosimo de Napoli, Leonardo Nogara, Elena Poggio, Marisa Brini, Angela Cattaneo, Angela Bachi, Thomas Simmen, Tito Calì, Susana Quijano-Roy, Simona Boncompagni, Bert Blaauw, Ana Ferreiro, Ester Zito.
      Selenoprotein N (SEPN1) is a protein of the endoplasmic reticulum (ER) whose inherited defects originate SEPN1-related myopathy (SEPN1-RM). Here, we identify an interaction between SEPN1 and the ER-stress-induced oxidoreductase ERO1A. SEPN1 and ERO1A, both enriched in mitochondria-associated membranes (MAMs), are involved in the redox regulation of proteins. ERO1A depletion in SEPN1 knockout cells restores ER redox, re-equilibrates short-range MAMs, and rescues mitochondrial bioenergetics. ERO1A knockout in a mouse background of SEPN1 loss blunts ER stress and improves multiple MAM functions, including Ca2+ levels and bioenergetics, thus reversing diaphragmatic weakness. The treatment of SEPN1 knockout mice with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) mirrors the results of ERO1A loss. Importantly, muscle biopsies from patients with SEPN1-RM exhibit ERO1A overexpression, and TUDCA-treated SEPN1-RM patient-derived primary myoblasts show improvement in bioenergetics. These findings point to ERO1A as a biomarker and a viable target for intervention and to TUDCA as a pharmacological treatment for SEPN1-RM.
    Keywords:  ER stress; ERO1; SEPN1; TUDCA; core myopathy; multi mini-core disease
    DOI:  https://doi.org/10.1016/j.xcrm.2024.101439
  36. Nat Chem Biol. 2024 Feb 26.
    Discovery of metal-binding proteins by thermal proteome profiling.
    Xin Zeng, Tiantian Wei, Xianghe Wang, Yuan Liu, Zhenshu Tan, Yihai Zhang, Tianyu Feng, Yao Cheng, Fengzhang Wang, Bin Ma, Wei Qin, Chuanping Gao, Junyu Xiao, Chu Wang.
      Metal-binding proteins (MBPs) have various and important biological roles in all living species and many human diseases are intricately linked to dysfunctional MBPs. Here, we report a chemoproteomic method named 'metal extraction-triggered agitation logged by thermal proteome profiling' (METAL-TPP) to globally profile MBPs in proteomes. The method involves the extraction of metals from MBPs using chelators and monitoring the resulting protein stability changes through thermal proteome profiling. Applying METAL-TPP to the human proteome with a broad-spectrum chelator, EDTA, revealed a group of proteins with reduced thermal stability that contained both previously known MBPs and currently unannotated MBP candidates. Biochemical characterization of one potential target, glutamine-fructose-6-phosphate transaminase 2 (GFPT2), showed that zinc bound the protein, inhibited its enzymatic activity and modulated the hexosamine biosynthesis pathway. METAL-TPP profiling with another chelator, TPEN, uncovered additional MBPs in proteomes. Collectively, this study developed a robust tool for proteomic discovery of MBPs and provides a rich resource for functional studies of metals in cell biology.
    DOI:  https://doi.org/10.1038/s41589-024-01563-y
  37. Cell Rep. 2024 Feb 28. pii: S2211-1247(24)00198-0. [Epub ahead of print]43(3): 113870
    HERC5-catalyzed ISGylation potentiates cGAS-mediated innate immunity.
    Lei Chu, Li Qian, Yu Chen, Shengnan Duan, Ming Ding, Wu Sun, Wei Meng, Juanjuan Zhu, Quanyi Wang, Haiping Hao, Chen Wang, Shufang Cui.
      The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) is essential to elicit type I interferon cascade response; thus, the activity of cGAS must be strictly regulated to boost the antiviral innate immunity. Here, we report that cGAS is responsible for the DNA-induced ISG15 conjugation system. The E3 HERC5 catalyzes the ISGylation of cytoplasmic cGAS at lysine 21, 187, 219, and 458, whereas Ubl carboxy-terminal hydrolase 18 removes the ISGylation of cGAS. The interaction of cGAS and HERC5 depends on the cGAS C-terminal domain and the RRC1-4 and RRC1-5 domains of HERC5. Mechanically, HERC5-catalyzed ISGylation promotes DNA-induced cGAS oligomerization and enhances cGAS enzymatic activity. Deficiency of ISGylation attenuates the downstream inflammatory gene expression induced by the cGAS-STING axis and the antiviral ability in mouse and human cells. Mice deficient in Isg15 or Herc6 are more vulnerable to herpes simplex virus 1 infection. Collectively, our study shows a positive feedback regulation of the cGAS-mediated innate immune pathway by ISGylation.
    Keywords:  CP: Immunology; DNA virus; HERC5; ISGylation; cGAS; innate immunity
    DOI:  https://doi.org/10.1016/j.celrep.2024.113870
  38. JHEP Rep. 2024 Apr;6(4): 101012
    Hepatitis B surface antigen expression impairs endoplasmic reticulum stress-related autophagic flux by decreasing LAMP2.
    Yaojie Liang, Xufeng Luo, Stefan Schefczyk, Lorraine T Muungani, Hui Deng, Baoju Wang, Hideo A Baba, Mengji Lu, Heiner Wedemeyer, Hartmut H Schmidt, Ruth Broering.
      Background & Aims: Hepatitis B surface antigen (HBsAg) drives hepatocarcinogenesis. Factors and mechanisms involved in this progression remain poorly defined, hindering the development of effective therapeutic strategies. Therefore, the mechanisms involved in the HBsAg-induced transformation of normal liver into hepatocellular carcinoma (HCC) were investigated.Methods: Hemizygous Tg(Alb1HBV)44Bri/J mice were examined for HBsAg-induced carcinogenic events. Gene set-enrichment analysis identified significant signatures in HBsAg-transgenic mice that correlated with endoplasmic reticulum (ER) stress, unfolded protein response, autophagy and proliferation. These events were investigated by western blotting, immunohistochemical and immunocytochemical staining in 2-, 8- and 12-month-old HBsAg-transgenic mice. The results were verified in HBsAg-overexpressing Hepa1-6 cells and validated in human HBV-related HCC samples.
    Results: Increased BiP expression in HBsAg-transgenic mice indicated induction of the unfolded protein response. In addition, early-phase autophagy was enhanced (increased BECN1 and LC3B) and late-phase autophagy blocked (increased p62) in HBsAg-transgenic mice. Finally, HBsAg altered lysosomal acidification via ATF4- and ATF6-mediated downregulation of lysosome-associated membrane protein 2 (LAMP2) expression. In patients, HBV-related HCC and adjacent tissues showed increased BiP, p62 and downregulated LAMP2 compared to uninfected controls. In vitro, the use of ER stress inhibitors reversed the HBsAg-related suppression of LAMP2. Furthermore, HBsAg promoted hepatocellular proliferation as indicated by Ki67, cleaved caspase-3 and AFP staining in paraffin-embedded liver sections from HBsAg-transgenic mice. These results were further verified by colony formation assays in HBsAg-expressing Hepa1-6 cells. Interestingly, inhibition of ER stress in HBsAg-overexpressing Hepa1-6 cells suppressed HBsAg-mediated cell proliferation.
    Conclusions: These data showed that HBsAg directly induces ER stress, impairs autophagy and promotes proliferation, thereby driving hepatocarcinogenesis. In addition, this study expanded the understanding of HBsAg-mediated intracellular events in carcinogenesis.
    Impact and implications: Factors and mechanisms involved in hepatocarcinogenesis driven by hepatitis B surface antigen (HBsAg) are poorly defined, hindering the development of effective therapeutic strategies. This study showed that HBsAg-induced endoplasmic reticulum stress suppressed LAMP2, thereby mediating autophagic injury. The present data suggest that restoring LAMP2 function in chronic HBV infection may have both antiviral and anti-cancer effects. This study has provided insights into the role of HBsAg-mediated intracellular events in carcinogenesis and thereby has relevance for future drug development.
    Keywords:  HBV surface protein; cellular homeostasis; incomplete autophagy; liver cancer; lysosome; tumorigenesis
    DOI:  https://doi.org/10.1016/j.jhepr.2024.101012
  39. J Cell Physiol. 2024 Feb 28.
    ATF4-dependent increase in mitochondrial-endoplasmic reticulum tethering following OPA1 deletion in skeletal muscle.
    Antentor Hinton, Prasanna Katti, Margaret Mungai, Duane D Hall, Olha Koval, Jianqiang Shao, Zer Vue, Edgar Garza Lopez, Rahmati Rostami, Kit Neikirk, Jessica Ponce, Jennifer Streeter, Brandon Schickling, Serif Bacevac, Chad Grueter, Andrea Marshall, Heather K Beasley, Young Do Koo, Sue C Bodine, Nayeli G Reyes Nava, Anita M Quintana, Long-Sheng Song, Isabella M Grumbach, Renata O Pereira, Brian Glancy, E Dale Abel.
      Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are protein- and lipid-enriched hubs that mediate interorganellar communication by contributing to the dynamic transfer of Ca2+ , lipid, and other metabolites between these organelles. Defective MERCs are associated with cellular oxidative stress, neurodegenerative disease, and cardiac and skeletal muscle pathology via mechanisms that are poorly understood. We previously demonstrated that skeletal muscle-specific knockdown (KD) of the mitochondrial fusion mediator optic atrophy 1 (OPA1) induced ER stress and correlated with an induction of Mitofusin-2, a known MERC protein. In the present study, we tested the hypothesis that Opa1 downregulation in skeletal muscle cells alters MERC formation by evaluating multiple myocyte systems, including from mice and Drosophila, and in primary myotubes. Our results revealed that OPA1 deficiency induced tighter and more frequent MERCs in concert with a greater abundance of MERC proteins involved in calcium exchange. Additionally, loss of OPA1 increased the expression of activating transcription factor 4 (ATF4), an integrated stress response (ISR) pathway effector. Reducing Atf4 expression prevented the OPA1-loss-induced tightening of MERC structures. OPA1 reduction was associated with decreased mitochondrial and sarcoplasmic reticulum, a specialized form of ER, calcium, which was reversed following ATF4 repression. These data suggest that mitochondrial stress, induced by OPA1 deficiency, regulates skeletal muscle MERC formation in an ATF4-dependent manner.
    Keywords:  activating transcription factor 4; endoplasmic reticulum; integrated stress response; interorganelle communication; mitochondria
    DOI:  https://doi.org/10.1002/jcp.31204
  40. iScience. 2024 Mar 15. 27(3): 109181
    DUB3 is a MAGEA3 deubiquitinase and a potential therapeutic target in hepatocellular carcinoma.
    Yuanhong Chen, Feng Gao, Yan He, Meijun Liu, Yuan Quan, Peijing Zhang.
      Although melanoma-associated antigen A3 and A6 (MAGEA3/6)-specific tumor vaccines have shown antitumor effects in melanoma and non-small cell lung cancer (NSCLC), many cancers do not respond because MAGEA3 can promote cancer without triggering an immune response. Here, we identified DUB3 as the MAGEA3 deubiquitinase. DUB3 interacts with, deubiquitinates and stabilizes MAGEA3. Depletion of DUB3 in hepatocellular carcinoma (HCC) cells results in MAGEA3 degradation and P53-dependent growth inhibition. Moreover, DUB3 knockout attenuates HCC tumorigenesis in vivo, which can be rescued by restoration of MAGEA3. Intriguingly, pharmacological inhibition of DUB3 by palbociclib promotes degradation of MAGEA3 and inhibits tumor growth in preclinical models implanted with parental HCC cells but not with DUB3 knockout HCC cells. In patients with HCC, DUB3 is highly expressed, and its levels positively correlate with MAGEA3 levels. Taken together, DUB3 is a MAGEA3 deubiquitinase, and abrogating DUB3 enzymatic activity by palbociclib is a promising therapeutic strategy for HCC.
    Keywords:  Cancer; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2024.109181
  41. Cell Rep Methods. 2024 Feb 26. pii: S2667-2375(24)00032-8. [Epub ahead of print]4(2): 100717
    Experimental and data analysis advances in thermal proteome profiling.
    Amanda M Figueroa-Navedo, Alexander R Ivanov.
      Method development for mass spectrometry (MS)-based thermal shift proteomic assays have advanced to probe small molecules with known and unknown protein-ligand interaction mechanisms and specificity, which is predominantly used in characterization of drug-protein interactions. In the discovery of target and off-target protein-ligand interactions, a thorough investigation of method development and their impact on the sensitivity and accuracy of protein-small molecule and protein-protein interactions is warranted. In this review, we discuss areas of improvement at each stage of thermal proteome profiling data analysis that includes processing of MS-based data, method development, and their effect on the overall quality of thermal proteome profiles. We also overview the optimization of experimental strategies and prioritization of an increased number of independent biological replicates over the number of evaluated temperatures.
    Keywords:  CP: biotechnology; cellular thermal shift assays; drug discovery; protein-ligand interactions; target engagement; thermal proteomic profiling
    DOI:  https://doi.org/10.1016/j.crmeth.2024.100717
  42. Cell Rep. 2024 Feb 23. pii: S2211-1247(24)00159-1. [Epub ahead of print]43(3): 113831
    PRMT1 acts as a suppressor of MHC-I and anti-tumor immunity.
    Tirta M Djajawi, Lizzy Pijpers, Akash Srivaths, David Chisanga, Kok Fei Chan, Simon J Hogg, Liam Neil, Sarahi Mendoza Rivera, Nenad Bartonicek, Sarah L Ellis, Terry C C Lim Kam Sian, Pouya Faridi, Yang Liao, Bhupinder Pal, Andreas Behren, Wei Shi, Stephin J Vervoort, Ricky W Johnstone, Conor J Kearney.
      Cancer immunotherapies have demonstrated remarkable success; however, the majority of patients do not respond or develop resistance. Here, we conduct epigenetic gene-targeted CRISPR-Cas9 screens to identify epigenomic factors that limit CD8+ T cell-mediated anti-tumor immunity. We identify that PRMT1 suppresses interferon gamma (Ifnγ)-induced MHC-I expression, thus dampening CD8+ T cell-mediated killing. Indeed, PRMT1 knockout or pharmacological targeting of type I PRMT with the clinical inhibitor GSK3368715 enhances Ifnγ-induced MHC-I expression through elevated STAT1 expression and activation, while re-introduction of PRMT1 in PRMT1-deficient cells reverses this effect. Importantly, loss of PRMT1 enhances the efficacy of anti-PD-1 immunotherapy, and The Cancer Genome Atlas analysis reveals that PRMT1 expression in human melanoma is inversely correlated with expression of human leukocyte antigen molecules, infiltration of CD8+ T cells, and overall survival. Taken together, we identify PRMT1 as a negative regulator of anti-tumor immunity, unveiling clinical type I PRMT inhibitors as immunotherapeutic agents or as adjuncts to existing immunotherapies.
    Keywords:  CP: Cancer; CP: Immunology; PRMT1; STAT1; argenine methylation; cancer; immunology
    DOI:  https://doi.org/10.1016/j.celrep.2024.113831
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