bims-proteo Biomed News
on Proteostasis
Issue of 2022‒07‒31
28 papers selected by
Eric Chevet

  1. Essays Biochem. 2022 Jul 26. pii: EBC20210094. [Epub ahead of print]
      Ubiquitination is an essential post-translational signal that allows cells to adapt and respond to environmental stimuli. Substrate modifications range from a single ubiquitin molecule to complex polyubiquitin chains, where diverse chain topologies constitute a code that is utilized to modify the functions of proteins in numerous cellular signalling pathways. Diverse ubiquitin chain topologies are generated by linking the C-terminus of ubiquitin to one of seven lysine residues or the N-terminal methionine 1 residue of the preceding ubiquitin. Cooperative action between a large array of E2 conjugating and E3 ligase enzymes supports the formation of not only homotypic ubiquitin chains but also heterotypic mixed or branched chains. This complex array of chain topologies is recognized by proteins containing linkage-specific ubiquitin-binding domains and regulates numerous cellular pathways. Although many functions of the ubiquitin code in plants remain unknown, recent work suggests that specific chain topologies are associated with particular molecular processes. Deciphering the ubiquitin code and how plants utilize it to cope with the changing environment is essential to understand the regulatory mechanisms that underpin myriad stress responses and establishment of environmental tolerance.
    Keywords:  E2 enzyme; E3 ligase; chain topology; plant stress responses; proteasome; ubiquitin
  2. J Cell Sci. 2022 Jul 28. pii: jcs.260299. [Epub ahead of print]
      During Hedgehog signaling, the ciliary levels of Ptch1 and Smo are regulated by the pathway. At the basal state, Ptch1 localizes to cilia and prevents the ciliary accumulation and activation of Smo. Upon binding Hedgehog ligand Ptch1 exits cilia, relieving inhibition of Smo. Smo then concentrates in cilia, becomes activated and activates downstream signaling. Loss of the ubiquitin E3 ligase Arih2 elevates basal Hedgehog signaling, elevates the cellular level of Smo, and increases basal levels of ciliary Smo. Mice express two isoforms of Arih2 with Arih2α found primarily in the nucleus and Arih2β found on the cytoplasmic face of the endoplasmic reticulum (ER). Re-expression of ER-localized Arih2β but not nuclear-localized Arih2α rescues the Hedgehog phenotypes. When Arih2 is defective, protein aggregates accumulate in the ER and the unfolded protein response is activated. Arih2β appears to regulate the ER-associated degradation of Smo preventing excess and potentially misfolded Smo from reaching the cilium and interfering with pathway regulation.
    Keywords:  Arih2; Cilia; Hedgehog signaling; Ubiquitin
  3. Trends Biochem Sci. 2022 Jul 20. pii: S0968-0004(22)00168-2. [Epub ahead of print]
      In eukaryotic cells, oxidative protein folding occurs in the lumen of the endoplasmic reticulum (ER), catalyzed by ER sulfhydryl oxidase 1 (Ero1) and protein disulfide isomerase (PDI). The efficiency and fidelity of oxidative protein folding are vital for the function of secretory cells. Here, we summarize oxidative protein folding in yeast, plants, and mammals, and discuss how the conformation and activity of human Ero1-PDI machinery is regulated through various post-translational modifications (PTMs). We propose that oxidative protein folding fidelity and ER redox homeostasis are maintained by both the precise control of Ero1 oxidase activity and the division of labor between PDI family members. We also discuss how deregulated Ero1-PDI functions contribute to human diseases and can be leveraged for therapeutic interventions.
    Keywords:  ER sulfhydryl oxidase 1 (Ero1); disulfide bond; endoplasmic reticulum (ER); oxidative protein folding; protein disulfide isomerase (PDI); redox homeostasis
  4. J Mol Biol. 2022 Jul 26. pii: S0022-2836(22)00364-3. [Epub ahead of print] 167762
      Hsp90 and Hsp70 are highly conserved molecular chaperones that promote the proper folding and activation of substrate proteins that are often referred to as clients. The two chaperones functionally collaborate to fold specific clients in an ATP-dependent manner. In eukaryotic cytosol, initial client folding is done by Hsp70 and its co-chaperones, followed by a direct transfer of client refolding intermediates to Hsp90 for final client processing. However, the mechanistic details of collaboration of organelle specific Hsp70 and Hsp90 are lacking. This work investigates the collaboration of the endoplasmic reticulum (ER) Hsp70 and Hsp90, BiP and Grp94 respectively, in protein remodeling using in vitro refolding assays. We show that under milder denaturation conditions, BiP collaborates with its co-chaperones to refold misfolded proteins in an ATP-dependent manner. Grp94 does not play a major role in this refolding reaction. However, under stronger denaturation conditions that favor aggregation, Grp94 works in an ATP-independent manner to bind and hold misfolded clients in a folding competent state for subsequent remodeling by the BiP system. We also show that the collaboration of Grp94 and BiP is not simply a reversal of the eukaryotic refolding mechanism since a direct interaction of Grp94 and BiP is not required for client transfer. Instead, ATP binding but not hydrolysis by Grp94 facilitates the release of the bound client, which is then picked up by the BiP system for subsequent refolding in a Grp94-independent manner.
    Keywords:  DnaJB11; Grp170; Hsp70; Hsp90; molecular chaperones
  5. Proc Natl Acad Sci U S A. 2022 Aug 02. 119(31): e2202080119
      Protein secretion is an essential process that drives cell growth, movement, and communication. Protein traffic within the secretory pathway occurs via transport intermediates that bud from one compartment and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein-protein interactions that drive capture into transport vesicles. Human proprotein convertase subtilisin/kexin type 9 (PCSK9) is a determinant of cholesterol metabolism whose secretion is mediated by a specific cargo adaptor protein, SEC24A. We map a series of protein-protein interactions between PCSK9, its endoplasmic reticulum (ER) export receptor SURF4, and SEC24A that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-phenylbutyrate (4-PBA), a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small-molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins.
    Keywords:  COPII vesicles; ER export; membrane traffic
  6. J Biochem. 2022 Jul 26. pii: mvac060. [Epub ahead of print]
      Endoplasmic reticulum (ER) tubules are interconnected by three-way junctions, resulting in the formation of a tubular ER network. Lunapark (Lnp) localizes to and stabilizes the three-way junctions. The N-terminal cytoplasmic domain in Lnp has a ubiquitin ligase activity. However, the molecular mechanism of how the ubiquitin ligase activity of Lnp is involved in the formation of the tubular ER network remains unknown. In this study, we examined whether the ER membrane proteins responsible for the formation of the tubular ER network are ubiquitinated by Lnp. We found that atlastin-2 (ATL2), an isoform of the ATL family mediating the generation of the three-way junctions by connecting the ER tubules, is a novel substrate for ubiquitination by Lnp. The localization of Lnp at the three-way junctions is important for ubiquitination of ATL2. Lysine 56, 57, 282, and 302 are the potential ubiquitination sites by Lnp. Silencing ATL2 decreased the number of the three-way junctions, and the expression of the ATL2 mutant in which the lysine residues are substituted with arginine failed to rescue the decrease of the three-way junctions in the ATL2 knocked-down cells. These results suggest that Lnp ubiquitinates ATL2 at the three-way junctions for the proper tubular ER network formation.
    Keywords:  amphipathic helix; endoplasmic reticulum; three-way junction; ubiquitin ligase
  7. Elife. 2022 Jul 29. pii: e80911. [Epub ahead of print]11
      Deciphering mechanisms controlling SNARE localization within the Golgi complex is crucial to understanding protein trafficking patterns within the secretory pathway. SNAREs are also thought to prime COPI assembly to ensure incorporation of these essential cargoes into vesicles, but the regulation of these events is poorly understood. Here we report roles for ubiquitin recognition by COPI in SNARE trafficking and in stabilizing interactions between Arf, COPI, and Golgi SNAREs in Saccharomyces cerevisiae. The ability of COPI to bind ubiquitin, but not the dilysine motif, through its N-terminal WD repeat domain of β'COP or through an unrelated ubiquitin-binding domain (UBD) is essential for the proper localization of Golgi SNAREs Bet1 and Gos1. We find that COPI, the ArfGAP Glo3 and multiple Golgi SNAREs are ubiquitinated. Notably, the binding of Arf and COPI to Gos1 is markedly enhanced by ubiquitination of these components. Glo3 is proposed to prime COPI-SNARE interactions; however, Glo3 is not enriched in the ubiquitin-stabilized SNARE-Arf-COPI complex but is instead enriched with COPI complexes that lack SNAREs. These results support a new model for how posttranslational modifications drive COPI priming events crucial for Golgi SNARE localization.
    Keywords:  S. cerevisiae; cell biology
  8. Cell Death Dis. 2022 Jul 28. 13(7): 655
      X-linked inhibitor of apoptosis-associated factor-1 (XAF1) is a stress-inducible tumor suppressor that is commonly inactivated in many human cancers. Despite accumulating evidence for the pro-apoptotic role for XAF1 under various stressful conditions, its involvement in endoplasmic reticulum (ER) stress response remains undefined. Here, we report that XAF1 increases cell sensitivity to ER stress and acts as a molecular switch in unfolded protein response (UPR)-mediated cell-fate decisions favoring apoptosis over adaptive autophagy. Mechanistically, XAF1 interacts with and destabilizes ER stress sensor GRP78 through the assembly of zinc finger protein 313 (ZNF313)-mediated destruction complex. Moreover, XAF1 expression is activated through PERK-Nrf2 signaling and destabilizes C-terminus of Hsc70-interacting protein (CHIP) ubiquitin E3 ligase, thereby blocking CHIP-mediated K63-linked ubiquitination and subsequent phosphorylation of inositol-required enzyme-1α (IRE1α) that is involved in in the adaptive ER stress response. In tumor xenograft assays, XAF1-/- tumors display substantially lower regression compared to XAF1+/+ tumors in response to cytotoxic dose of ER stress inducer. XAF1 and GRP78 expression show an inverse correlation in human cancer cell lines and primary breast carcinomas. Collectively this study uncovers an important role for XAF1 as a linchpin to govern the sensitivity to ER stress and the outcomes of UPR signaling, illuminating the mechanistic consequence of XAF1 inactivation in tumorigenesis.
  9. Cancers (Basel). 2022 Jul 19. pii: 3501. [Epub ahead of print]14(14):
      Ubiquitin-fold modifier 1 (UFM1), a newly identified ubiquitin-like molecule (UBLs), is evolutionarily expressed in multiple species except yeast. Similarly to ubiquitin, UFM1 is covalently attached to its substrates through a well-orchestrated three-step enzymatic reaction involving E1, the UFM1-activating enzyme (ubiquitin-like modifier-activating enzyme 5, UBA5); E2, the UFM1-conjugating enzyme 1 (UFC1); and E3, the UFM1-specific ligase 1 (UFL1). To date, numerous studies have shown that UFM1 modification is implicated in various cellular processes, including endoplasmic reticulum (ER) stress, DNA damage response and erythroid development. An abnormal UFM1 cascade is closely related to a variety of diseases, especially tumors. Herein, we summarize the process and functions of UFM1 modification, illustrating the relationship and mechanisms between aberrant UFMylation and diversified tumors, aiming to provide novel diagnostic biomarkers or therapeutic targets for cancer treatments.
    Keywords:  UFM1; UFMylation; post-translational modifications; tumorigenesis; ubiquitin-like molecules
  10. Nat Chem Biol. 2022 Aug;18(8): 802-811
      The ubiquitin system has become synonymous with the modification of lysine residues. However, the substrate scope and diversity of the conjugation machinery have been underappreciated, bringing us to an epoch in ubiquitin system research. The striking discoveries of metazoan enzymes dedicated toward serine and threonine ubiquitination have revealed the important role of nonlysine ubiquitination in endoplasmic reticulum-associated degradation, immune signaling and neuronal processes, while reports of nonproteinaceous substrates have extended ubiquitination beyond the proteome. Bacterial effectors that bypass the canonical ubiquitination machinery and form unprecedented linkage chemistry further redefine long-standing dogma. While chemical biology approaches have advanced our understanding of the canonical ubiquitin system, further study of noncanonical ubiquitination has been hampered by a lack of suitable tools. This Perspective aims to consolidate and contextualize recent discoveries and to propose potential applications of chemical biology, which will be instrumental in unraveling this new frontier of ubiquitin research.
  11. Elife. 2022 Jul 25. pii: e77706. [Epub ahead of print]11
      Signal-anchored (SA) proteins are anchored into the mitochondrial outer membrane (OM) via a single transmembrane segment at their N-terminus while the bulk of the proteins is facing the cytosol. These proteins are encoded by nuclear DNA, translated on cytosolic ribosomes, and are then targeted to the organelle and inserted into its OM by import factors. Recently, research on the insertion mechanisms of these proteins into the mitochondrial OM have gained a lot of attention. In contrast, the early cytosolic steps of their biogenesis are unresolved. Using various proteins from this category and a broad set of in vivo, in organello, and in vitro assays, we reconstituted the early steps of their biogenesis. We identified a subset of molecular (co)chaperones that interact with newly synthesized SA proteins, namely, Hsp70 and Hsp90 chaperones and co-chaperones from the Hsp40 family like Ydj1 and Sis1. These interactions were mediated by the hydrophobic transmembrane segments of the SA proteins. We further demonstrate that interfering with these interactions inhibits the biogenesis of SA proteins to a various extent. Finally, we could demonstrate direct interaction of peptides corresponding to the transmembrane segments of SA proteins with the (co)chaperones and reconstitute in vitro the transfer of such peptides from the Hsp70 chaperone to the mitochondrial Tom70 receptor. Collectively, this study unravels an array of cytosolic chaperones and mitochondrial import factors that facilitates the targeting and membrane integration of mitochondrial SA proteins.
    Keywords:  S. cerevisiae; biochemistry; chemical biology
  12. Autophagy. 2022 Jul 28.
      The NLRP3 inflammasome is involved in a diverse range of inflammatory diseases. The activation of inflammasomes must be tightly regulated to prevent excessive inflammation, and the protein ubiquitination system is reported to be one of the ways in which inflammasome activation is regulated. However, the deubiquitination regulatory mechanisms of inflammasome activation remain elusive. Here, we demonstrated that USP22 (ubiquitin specific peptidase 22) promotes NLRP3 degradation and inhibits NLRP3 inflammasome activation. USP22 deficiency or in vivo silencing significantly increases alum-induced peritonitis and lipopolysaccharide-induced systemic inflammation. Mechanistically, USP22 inhibits NLRP3 inflammasome activation via the promotion of ATG5-mediated macroautophagy/autophagy. USP22 stabilizes ATG5 via decreasing K27- and K48-linked ubiquitination of ATG5 at the Lys118 site. Taken together, these findings reveal the role USP22 plays in the regulation of NLRP3 inflammasome activation and suggest a potential therapeutic target to treat NLRP3 inflammasome-related diseases.
    Keywords:  Autophagy; NLRP3; autophagy related 5; inflammasome; ubiquitin specific peptidase 22
  13. EMBO J. 2022 Jul 25. e109205
      Patient-derived organoids and cellular spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a reconstituted basement membrane in three dimensions (3D) supports the polarized, stress resilient tissue phenotype of mammary epithelial spheroids. Cells interacting with reconstituted basement membrane in 3D had reduced levels of total and actin-associated filamin and decreased cortical actin tension that increased plasma membrane protrusions to promote negative plasma membrane curvature and plasma membrane protein associations linked to protein secretion. By contrast, cells engaging a reconstituted basement membrane in 2D had high cortical actin tension that forced filamin unfolding and endoplasmic reticulum (ER) associations. Enhanced filamin-ER interactions increased levels of PKR-like ER kinase effectors and ER-plasma membrane contact sites that compromised calcium homeostasis and diminished cell viability. Consequently, cells with decreased cortical actin tension had reduced ER stress and survived better. Consistently, cortical actin tension in cellular spheroids regulated polarized basement membrane membrane deposition and sensitivity to exogenous stress. The findings implicate cortical actin tension-mediated filamin unfolding in ER function and underscore the importance of tissue mechanics in organoid homeostasis.
    Keywords:  actin tension; endoplasmic reticulum; extracellular matrix; membrane contact sites; spheroids
  14. Autophagy. 2022 Jul 27. 1-2
      Macroautophagy/autophagy is a conserved lysosome-dependent metabolic recycling pathway. ULK1 plays an essential role in autophagy initiation through a complex formed with ATG13, RB1CC1/FIP200, and ATG101 in mammalian cells. However, while autophagy is triggered by nutrient starvation where it is essential for cell survival, such conditions lead to the rapid degradation of ULK1, indicating that autophagy must be tightly controlled. Nevertheless, the precise mechanisms regulating the ULK1 complex are still largely unknown. Here we reveal the critical roles played by two novel ULK1 complex binding proteins in autophagy regulation, TRIM27 and STK38L. We show that basal autophagy is maintained through TRIM27-mediated ubiquitination and proteasomal degradation of ULK1, whereas under starvation conditions, excessive autophagy is restrained by the combined actions of TRIM27 and STK38L. TRIM27 ubiquitinates and activates STK38L which in turn phosphorylates ULK1, delivering ULK1 in a permissive state for hyper-ubiquitination by TRIM27. Thus, TRIM27 and STK38L kinase act in concert as a rheostat to control ULK1 levels. We further demonstrate increased basal autophagy in trim27 knockout mice and establish physiological relevance in the context of breast cancer. Our study highlights the STK38L-TRIM27-ULK1 axis as a potential treatment avenue to explore for activating autophagy in various disease states.
    Keywords:  Autophagy; STK38L; TRIM27; ULK1; breast cancer; metastasis; tumorigenesis; ubiquitination
  15. Nat Commun. 2022 Jul 29. 13(1): 4394
      Dobzhansky-Muller incompatibilities represent a major driver of reproductive isolation between species. They are caused when interacting components encoded by alleles from different species cannot function properly when mixed. At incipient stages of speciation, complex incompatibilities involving multiple genetic loci with weak effects are frequently observed, but the underlying mechanisms remain elusive. Here we show perturbed proteostasis leading to compromised mitosis and meiosis in Saccharomyces cerevisiae hybrid lines carrying one or two chromosomes from Saccharomyces bayanus var. uvarum. Levels of proteotoxicity are correlated with the number of protein complexes on replaced chromosomes. Proteomic approaches reveal that multi-protein complexes with subunits encoded by replaced chromosomes tend to be unstable. Furthermore, hybrid defects can be alleviated or aggravated, respectively, by up- or down-regulating the ubiquitin-proteasomal degradation machinery, suggesting that destabilized complex subunits overburden the proteostasis machinery and compromise hybrid fitness. Our findings reveal the general role of impaired protein complex assembly in complex incompatibilities.
  16. Plant Direct. 2022 Jul;6(7): e431
      The endoplasmic reticulum (ER) houses sensors that respond to environmental stress and underly plants' adaptative responses. These sensors transduce signals that lead to changes in nuclear gene expression. The ER to nuclear signaling pathways are primarily attributed to the unfolded protein response (UPR) and are also integrated with a wide range of development, hormone, immune, and stress signaling pathways. Understanding the role of the UPR in signaling network mechanisms that associate with particular phenotypes is crucially important. While UPR-associated genes are the subject of ongoing investigations in a few model plant systems, most remain poorly annotated, hindering the identification of candidates across plant species. This open-source curated database provides a centralized resource of peer reviewed knowledge of ER to nuclear signaling pathways for the plant community. We provide a UPRome interactive viewer for users to navigate through the pathways and to access annotated information. The plant ER UPRome website is located at We welcome contributions from the researchers studying the ER UPR to incorporate additional genes into the database through the "contact us" page.
    Keywords:  curated database; plant protein interaction maps; plant signal transduction; unfolded protein response
  17. Nat Commun. 2022 Jul 29. 13(1): 4355
      Transcriptional regulation by RNA polymerase II is associated with changes in chromatin structure. Activated and promoter-bound heat shock transcription factor 1 (HSF1) recruits transcriptional co-activators, including histone-modifying enzymes; however, the mechanisms underlying chromatin opening remain unclear. Here, we demonstrate that HSF1 recruits the TRRAP-TIP60 acetyltransferase complex in HSP72 promoter during heat shock in a manner dependent on phosphorylation of HSF1-S419. TRIM33, a bromodomain-containing ubiquitin ligase, is then recruited to the promoter by interactions with HSF1 and a TIP60-mediated acetylation mark, and cooperates with the related factor TRIM24 for mono-ubiquitination of histone H2B on K120. These changes in histone modifications are triggered by phosphorylation of HSF1-S419 via PLK1, and stabilize the HSF1-transcription complex in HSP72 promoter. Furthermore, HSF1-S419 phosphorylation is constitutively enhanced in and promotes proliferation of melanoma cells. Our results provide mechanisms for HSF1 phosphorylation-dependent establishment of an active chromatin status, which is important for tumorigenesis.
  18. J Extracell Vesicles. 2022 Jul;11(7): e12244
      We characterized the in vivo interstitial fluid (IF) content of extracellular vesicles (EVs) using the GFP-4T1 syngeneic murine cancer model to study EVs in-transit to the draining lymph node. GFP labelling confirmed the IF EV tumour cell origin. Molecular analysis revealed an abundance of IF EV-associated proteins specifically involved in mitophagy and secretory autophagy. A set of proteins required for sequential steps of fission-induced mitophagy preferentially populated the CD81+/PD-L1+ IF EVs; PINK1, TOM20, and ARIH1 E3 ubiquitin ligase (required for Parkin-independent mitophagy), DRP1 and FIS1 (mitochondrial peripheral fission), VDAC-1 (ubiquitination state triggers mitophagy away from apoptosis), VPS35, SEC22b, and Rab33b (vacuolar sorting). Comparing in vivo IF EVs to in vitro EVs revealed 40% concordance, with an elevation of mitophagy proteins in the CD81+ EVs for both murine and human cell lines subjected to metabolic stress. The export of cellular mitochondria proteins to CD81+ EVs was confirmed by density gradient isolation from the bulk EV isolate followed by anti-CD81 immunoprecipitation, molecular sieve chromatography, and MitoTracker export into CD81+ EVs. We propose the 4T1 in vivo model as a versatile tool to functionally characterize IF EVs. IF EV export of fission mitophagy proteins has broad implications for mitochondrial function and cellular immunology.
    Keywords:  autophagosome; autophagy; breast cancer; extracellular vesicle; mitochondria; mitophagy
  19. Protein Sci. 2022 Aug;31(8): e4378
      Molecular chaperones are essential to maintain proteostasis. While the functions of intracellular molecular chaperones that oversee protein synthesis, folding and aggregation, are established, those specialized to work in the extracellular environment are less understood. Extracellular proteins reside in a considerably more oxidizing milieu than cytoplasmic proteins and are stabilized by abundant disulfide bonds. Hence, extracellular proteins are potentially destabilized and sensitive to aggregation under reducing conditions. We combine biochemical and mass spectrometry experiments and elucidate that the molecular chaperone functions of the extracellular protein domain Bri2 BRICHOS only appear under reducing conditions, through the assembly of monomers into large polydisperse oligomers by an intra- to intermolecular disulfide bond relay mechanism. Chaperone-active assemblies of the Bri2 BRICHOS domain are efficiently generated by physiological thiol-containing compounds and proteins, and appear in parallel with reduction-induced aggregation of extracellular proteins. Our results give insights into how potent chaperone activity can be generated from inactive precursors under conditions that are destabilizing to most extracellular proteins and thereby support protein stability/folding in the extracellular space. SIGNIFICANCE: Chaperones are essential to cells as they counteract toxic consequences of protein misfolding particularly under stress conditions. Our work describes a novel activation mechanism of an extracellular molecular chaperone domain, called Bri2 BRICHOS. This mechanism is based on reducing conditions that initiate small subunits to assemble into large oligomers via a disulfide relay mechanism. Activated Bri2 BRICHOS inhibits reduction-induced aggregation of extracellular proteins and could be a means to boost proteostasis in the extracellular environment upon reductive stress.
    Keywords:  ATP-independent molecular chaperone; BRICHOS domain; Bri2 BRICHOS; disulfide bond formation; extracellular protein aggregation
  20. Cell Death Differ. 2022 Jul 23.
      The precise regulation of STING homeostasis is essential for its antiviral function. Post-translational modification, especially ubiquitination, is important for the regulation of STING homeostasis. Previous studies have focused on how STING is degraded, but little is known about its maintenance. Here, we show that UFM1 specific ligase UFL1 promotes innate immune response by maintaining STING expression independent of UFMylation. Mechanistically, UFL1 inhibits TRIM29 to interact with STING, thereby reducing its ubiquitination at K338/K347/K370 and subsequent proteasomal degradation. DNA virus infection reduces the UFL1 expression, which may promote STING degradation and facilitate viral expansion. Our study identifies UFL1 as a crucial regulator for the maintenance of STING stability and antiviral function, and provides novel insights into the mechanistic explanation for the immunological escape of DNA virus.
  21. Biomolecules. 2022 Jun 29. pii: 911. [Epub ahead of print]12(7):
      "Extracellular" Heat Shock Protein-90 (Hsp90) was initially reported in the 1970s but was not formally recognized until 2008 at the 4th International Conference on The Hsp90 Chaperone Machine (Monastery Seeon, Germany). Studies presented under the topic of "extracellular Hsp90 (eHsp90)" at the conference provided direct evidence for eHsp90's involvement in cancer invasion and skin wound healing. Over the past 15 years, studies have focused on the secretion, action, biological function, therapeutic targeting, preclinical evaluations, and clinical utility of eHsp90 using wound healing, tissue fibrosis, and tumour models both in vitro and in vivo. eHsp90 has emerged as a critical stress-responding molecule targeting each of the pathophysiological conditions. Despite the studies, our current understanding of several fundamental questions remains little beyond speculation. Does eHsp90 indeed originate from purposeful live cell secretion or rather from accidental dead cell leakage? Why did evolution create an intracellular chaperone that also functions as a secreted factor with reported extracellular duties that might be (easily) fulfilled by conventional secreted molecules? Is eHsp90 a safer and more optimal drug target than intracellular Hsp90 chaperone? In this review, we summarize how much we have learned about eHsp90, provide our conceptual views of the findings, and make recommendations on the future studies of eHsp90 for clinical relevance.
    Keywords:  extracellular Hsp90; mechanism of action; stress; wound healing and cancer
  22. Nat Commun. 2022 Jul 25. 13(1): 4273
      3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is the rate-limiting enzyme in cholesterol synthesis and target of cholesterol-lowering statin drugs. Accumulation of sterols in endoplasmic reticulum (ER) membranes accelerates degradation of HMGCR, slowing the synthesis of cholesterol. Degradation of HMGCR is inhibited by its binding to UBIAD1 (UbiA prenyltransferase domain-containing protein-1). This inhibition contributes to statin-induced accumulation of HMGCR, which limits their cholesterol-lowering effects. Here, we report cryo-electron microscopy structures of the HMGCR-UBIAD1 complex, which is maintained by interactions between transmembrane helix (TM) 7 of HMGCR and TMs 2-4 of UBIAD1. Disrupting this interface by mutagenesis prevents complex formation, enhancing HMGCR degradation. TMs 2-6 of HMGCR contain a 170-amino acid sterol sensing domain (SSD), which exists in two conformations-one of which is essential for degradation. Thus, our data supports a model that rearrangement of the TMs in the SSD permits recruitment of proteins that initate HMGCR degradation, a key reaction in the regulatory system that governs cholesterol synthesis.
  23. Nat Commun. 2022 Jul 29. 13(1): 4412
      Gonadal sexual fate in mammals is determined during embryonic development and must be actively maintained in adulthood. In the mouse ovary, oestrogen receptors and FOXL2 protect ovarian granulosa cells from transdifferentiation into Sertoli cells, their testicular counterpart. However, the mechanism underlying their protective effect is unknown. Here, we show that TRIM28 is required to prevent female-to-male sex reversal of the mouse ovary after birth. We found that upon loss of Trim28, ovarian granulosa cells transdifferentiate to Sertoli cells through an intermediate cell type, different from gonadal embryonic progenitors. TRIM28 is recruited on chromatin in the proximity of FOXL2 to maintain the ovarian pathway and to repress testicular-specific genes. The role of TRIM28 in ovarian maintenance depends on its E3-SUMO ligase activity that regulates the sex-specific SUMOylation profile of ovarian-specific genes. Our study identifies TRIM28 as a key factor in protecting the adult ovary from the testicular pathway.
  24. Cancer Res. 2022 Jul 27. pii: CAN-21-3882. [Epub ahead of print]
      Glioblastoma (GBM) is the most common type of primary adult brain tumor. Glioma stem cell (GSC) residence and Temozolomide (TMZ) resistance in GBM both contribute to poor patient outcome. TRAF4 is a scaffold protein with E3 ubiquitin ligase activity that has recently been discovered to promote invasion and metastasis in several malignancies, but the effects and functions of TRAF4 in GBM remain to be determined. Here, we report that TRAF4 is preferentially overexpressed in GSCs and is required for stem-like properties as well as TMZ sensitivity in GBM cells. TRAF4 specifically interacted with the N-terminal tail of Caveolin-1 (CAV1), an important contributor to the tumorigenicity of GBM cells. TRAF4 regulated CAV1 stability by preventing ZNRF1-mediated ubiquitination and facilitating USP7-mediated deubiquitination independently of its E3 ubiquitin ligase catalytic activity. TRAF4-mediated stabilization of CAV1 activated protumorigenic AKT/ERK1/2 signaling, and disruption of this axis resulted in defects in stemness maintenance. In addition, expression of TRAF4 and CAV1 was positively correlated and predicted poor prognosis in human GBM samples. Screening of common nervous system drugs identified Risperidone interaction with TRAF4, and Risperidone treatment resulted in the dissociation of TRAF4 and CAV1. Importantly, pharmacological inhibition of TRAF4 with Risperidone potently inhibited self-renewal, abrogated tumorigenicity, and reversed TMZ resistance in GBM. Overall, TRAF4-mediated stabilization of CAV1 promotes stemness and TMZ resistance in GBM, providing a therapeutic strategy that could improve patient outcomes.
  25. Elife. 2022 Jul 28. pii: e79602. [Epub ahead of print]11
      Assembly pathways of protein complexes should be precise and efficient to minimise misfolding and unwanted interactions with other proteins in the cell. One way to achieve this efficiency is by seeding assembly pathways during translation via the cotranslational assembly of subunits. While recent evidence suggests that such cotranslational assembly is widespread, little is known about the properties of protein complexes associated with the phenomenon. Here, using a combination of proteome-specific protein complex structures and publicly available ribosome profiling data, we show that cotranslational assembly is particularly common between subunits that form large intermolecular interfaces. To test whether large interfaces have evolved to promote cotranslational assembly, as opposed to cotranslational assembly being a non-adaptive consequence of large interfaces, we compared the sizes of first and last translated interfaces of heteromeric subunits in bacterial, yeast, and human complexes. When considering all together, we observe the N-terminal interface to be larger than the C-terminal interface 54% of the time, increasing to 64% when we exclude subunits with only small interfaces, which are unlikely to cotranslationally assemble. This strongly suggests that large interfaces have evolved as a means to maximise the chance of successful cotranslational subunit binding.
    Keywords:  E. coli; S. cerevisiae; computational biology; evolutionary biology; human; systems biology
  26. Cell Signal. 2022 Jul 25. pii: S0898-6568(22)00176-0. [Epub ahead of print] 110414
      Impairing autophagy disrupts transforming growth factor beta 1 (TGFβ1) signalling and epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC). Since autophagy and proteasome-mediated degradation are interdependent, we investigated how prolonged downregulation of proteasomal catalytic activity affected TGFβ1-dependent signalling and EMT. Proteasome-dependent degradation was inhibited in A549 and H1299 NSCLC cells using MG132 and lactacystin, which are reversible and irreversible proteasome inhibitors, respectively. We observed that inhibiting proteasomal activity for 24 h decreased TGFβ-dependent nuclear accumulation of Smad2/3. Time course studies were then carried out to characterize the time frame of this observation. Short-term (< 8 h) proteasome inhibition resulted in increased receptor regulated Smad (R-Smad) phosphorylation and steady-state TGFβ receptor type II (TGFβRII) levels. However, prolonged (8-24 h) proteasome inhibition decreased TGFβ1-dependent R-Smad phosphorylation and steady-state TGFβRI and TGFβRII levels. Furthermore, proteasome inhibition blunted TGFβ-dependent E- to N-Cadherin shift, stress fiber formation, and increased cellular apoptosis via the TAK-1-TRAF6-p38 MAPK pathway. Interestingly, proteasome inhibition also increased autophagic flux, steady-state microtubule-associated protein light chain 3B-II and active uncoordinated 51-like autophagy activating kinase 1 levels, and co-localization of lysosomes with autophagy cargo proteins and autophagy-related proteins. Finally, we observed that proteasome inhibition increased TGFβRII endocytosis and trafficking to lysosomes and we conclude that prolonged proteasome inhibition disrupts TGFβ signalling outcomes through altered TGFβ receptor trafficking.
    Keywords:  Autophagy; Endocytosis; Epithelial-mesenchymal transition; Lysosome; Proteasome; Transforming growth factor beta; p62/SQSTM1
  27. Sci Adv. 2022 Jul 29. 8(30): eabo0340
      Mitochondrial quality in skeletal muscle is crucial for maintaining energy homeostasis during metabolic stresses. However, how muscle mitochondrial quality is controlled and its physiological impacts remain unclear. Here, we demonstrate that mitoprotease LONP1 is essential for preserving muscle mitochondrial proteostasis and systemic metabolic homeostasis. Skeletal muscle-specific deletion of Lon protease homolog, mitochondrial (LONP1) impaired mitochondrial protein turnover, leading to muscle mitochondrial proteostasis stress. A benefit of this adaptive response was the complete resistance to diet-induced obesity. These favorable metabolic phenotypes were recapitulated in mice overexpressing LONP1 substrate ΔOTC in muscle mitochondria. Mechanistically, mitochondrial proteostasis imbalance elicits an unfolded protein response (UPRmt) in muscle that acts distally to modulate adipose tissue and liver metabolism. Unexpectedly, contrary to its previously proposed role, ATF4 is dispensable for the long-range protective response of skeletal muscle. Thus, these findings reveal a pivotal role of LONP1-dependent mitochondrial proteostasis in directing muscle UPRmt to regulate systemic metabolism.
  28. Cell Chem Biol. 2022 Jul 20. pii: S2451-9456(22)00245-8. [Epub ahead of print]
      The mitochondrial caseinolytic protease P (ClpP) is a target candidate for treating leukemia; however, the effects of ClpP modulation on solid tumors have not been adequately explored. Here, we report a potent activator of ClpP with the therapeutic potential for pancreatic ductal adenocarcinoma (PDAC). We first validated that aberrant ClpP activation leads to growth arrest of PDAC cells and tumors. We then performed high-throughput screening and synthetic optimization, from which we identified ZG111, a potent activator of ClpP. ZG111 binds to ClpP and promotes the ClpP-mediated degradation of respiratory chain complexes. This degradation activates the JNK/c-Jun pathway, induces the endoplasmic reticulum stress response, and consequently causes the growth arrest of PDAC cells. ZG111 also produces inhibitory effects on tumor growth in cell line-derived and patient-derived xenograft mouse models. Altogether, our data demonstrate a promising therapeutic strategy for PDAC suppression through the chemical activation of ClpP.
    Keywords:  ClpP activator; cancer therapy; mitochondrial proteome homeostasis; oxidative phosphorylation; pancreatic ductal adenocarcinoma; respiratory chain complexes; target validation