bims-proteo Biomed News
on Proteostasis
Issue of 2022‒06‒26
25 papers selected by
Eric Chevet

  1. Autophagy. 2022 Jun 19. 1-27
      Neurons and astrocytes face unique demands on their proteome to enable proper function and survival of the nervous system. Consequently, both cell types are critically dependent on robust quality control pathways such as macroautophagy (hereafter referred to as autophagy) and the ubiquitin-proteasome system (UPS). We previously reported that autophagy is differentially regulated in astrocytes and neurons in the context of metabolic stress, but less is understood in the context of proteotoxic stress induced by inhibition of the UPS. Dysfunction of the proteasome or autophagy has been linked to the progression of various neurodegenerative diseases. Therefore, in this study, we explored the connection between autophagy and the proteasome in primary astrocytes and neurons. Prior studies largely in non-neural models report a compensatory relationship whereby inhibition of the UPS stimulates autophagy. To our surprise, inhibition of the proteasome did not robustly upregulate autophagy in astrocytes or neurons. In fact, the effects on autophagy are modest particularly in comparison to paradigms of metabolic stress. Rather, we find that UPS inhibition in astrocytes induces formation of Ub-positive aggregates that harbor the selective autophagy receptor, SQSTM1/p62, but these structures were not productive substrates for autophagy. By contrast, we observed a significant increase in lysosomal degradation in astrocytes in response to UPS inhibition, but this stimulation was not sufficient to reduce total SQSTM1 levels. Last, UPS inhibition was more toxic in neurons compared to astrocytes, suggesting a cell type-specific vulnerability to proteotoxic stress.Abbreviations: Baf A1: bafilomycin A1; CQ: chloroquine; Epox: epoxomicin; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; p-ULK1: phospho-ULK1; SQSTM1/p62: sequestosome 1; Ub: ubiquitin; ULK1: unc-51 like kinase 1; UPS: ubiquitin-proteasome system.
    Keywords:  Astrocytes; LC3; SQSTM1; autophagy; lysosomes; neurons; proteasome; ubiquitin
  2. Elife. 2022 Jun 22. pii: e75580. [Epub ahead of print]11
      Endoplasmic reticulum (ER) to nucleus homeostatic signaling, known as the unfolded protein response (UPR), relies on the non-canonical splicing of XBP1 mRNA. The molecular switch that initiates splicing is the oligomerization of the ER stress sensor and UPR endonuclease IRE1α (inositol-requiring enzyme 1 alpha). While IRE1α can form large clusters that have been proposed to function as XBP1 processing centers on the ER, the actual oligomeric state of active IRE1α complexes as well as the targeting mechanism that recruits XBP1 to IRE1α oligomers remains unknown. Here, we have developed a single-molecule imaging approach to monitor the recruitment of individual XBP1 transcripts to the ER surface. Using this methodology, we confirmed that stable ER association of unspliced XBP1 mRNA is established through HR2 (hydrophobic region 2)-dependent targeting and relies on active translation. In addition, we show that IRE1α-catalyzed splicing mobilizes XBP1 mRNA from the ER membrane in response to ER stress. Surprisingly, we find that XBP1 transcripts are not recruited into large IRE1α clusters, which are only observed upon overexpression of fluorescently tagged IRE1α during ER stress. Our findings support a model where ribosome-engaged, immobilized XBP1 mRNA is processed by small IRE1α assemblies that could be dynamically recruited for processing of mRNA transcripts on the ER.
    Keywords:  ER; IRE1; XBP1; cell biology; human; imaging; single-molecule; unfolded protein response
  3. Cancers (Basel). 2022 Jun 15. pii: 2949. [Epub ahead of print]14(12):
      Protein quality control mechanisms play an important role in cancer progression by providing adaptive responses and morphologic stability against genome-wide copy number alterations, aneuploidy, and conformation-altering somatic mutations. This dependency on protein quality control mechanisms creates a vulnerability that may be exploited for therapeutic benefits by targeting components of the protein quality control mechanism. Recently, valosin-containing protein (VCP), also known at p97 AAA-ATPase, has emerged as a druggable target in cancer cells to affect their dependency on protein quality control. Here, we show that VCP inhibitors induce cytotoxicity in several ovarian cancer cell lines and these compounds act synergistically with mifepristone, a drug previously shown to induce an atypical unfolded protein response. Although mifepristone at a clinically achievable dose induces a weak unfolded protein response, it enhances the cytotoxic effects of VCP inhibitor CB-5083. Mechanistically, mifepristone blocks the cytoprotective effect of ATF6 in response to endoplasmic reticulum (ER) stress while activating the cytotoxic effects of ATF4 and CHOP through the HRI (EIF2AK1)-mediated signal transduction pathway. In contrast, CB-5083 activates ATF4 and CHOP through the PERK (EIF2AK3)-mediated signaling pathway. This combination activates ATF4 and CHOP while blocking the adaptive response provided by ATF6, resulting in increased cytotoxic effects and synergistic drug interaction.
    Keywords:  VCP inhibitors; cancer therapy; mifepristone; ovarian cancer; proteostasis
  4. Elife. 2022 Jun 22. pii: e74342. [Epub ahead of print]11
      Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remain unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1's lumenal domain. Phosphorylation of IRE1's kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enables trans-autophosphorylation, which in turn drives IRE1's RNase activity.
    Keywords:  IRE1; UPR; cell biology; endoplasmic reticulum; human; molecular biophysics; single-molecule; stress signaling; structural biology
  5. J Biol Chem. 2022 Jun 17. pii: S0021-9258(22)00600-7. [Epub ahead of print] 102158
      Chaperones and other quality control machinery guard proteins from inappropriate aggregation, which is a hallmark of neurodegenerative diseases. However, how the systems that regulate the 'foldedness' of the proteome remain buffered under stress conditions and in different cellular compartments remains incompletely understood. In this study, we applied a FRET-based strategy to explore how well quality control machinery protects against the misfolding and aggregation of "bait" biosensor proteins, made from the prokaryotic ribonuclease barnase, in the nucleus and cytosol of HEK293T cells. We found those barnase biosensors prone to misfolding, were less engaged by quality control machinery and more prone to inappropriate aggregation in the nucleus as compared to the cytosol, and that these effects could be regulated by chaperone Hsp70-related machinery. Furthermore, aggregation of mutant huntingtin exon 1 protein (Httex1) in the cytosol appeared to outcompete and thus prevented the engagement of quality control machinery with the biosensor in the cytosol. This effect correlated with reduced levels of DNAJB1 and HSPA1A chaperones in the cell outside those sequestered to the aggregates, particularly in the nucleus. Unexpectedly, we found Httex1 aggregation also increased the apparent engagement of the barnase biosensor with quality control machinery in the nucleus suggesting an independent implementation of 'holdase' activity of chaperones other than DNAJB1 and HSPA1A. Collectively these results suggest that proteostasis stress can trigger a rebalancing of chaperone abundance in different subcellular compartments through a dynamic network involving different chaperone-client interactions.
    Keywords:  Hsp40; Hsp70; chaperone DnaJ (DnaJ); chaperone DnaK (DnaK); flow cytometry; fluorescence resonance energy transfer (FRET); protein aggregation; protein folding; protein quality control; proteostasis
  6. Autophagy. 2022 Jun 19.
      Targeted protein degradation allows targeting undruggable proteins for therapeutic applications as well as eliminating proteins of interest for research purposes. While several types of degraders that harness the proteasome or the lysosome have been developed, a technology that simultaneously degrades targets and accelerates cellular autophagic flux remains unavailable. In this study, we developed a general chemical tool by which given intracellular proteins are targeted to macroautophagy for lysosomal degradation. This platform technology, termed AUTOTAC (AUTOphagy-TArgeting Chimera), employs bifunctional molecules composed of target-binding ligands (TBLs) linked to autophagy-targeting ligands (ATLs). Upon binding to targets via the TBL, the ATL binds the ZZ domain of the otherwise dormant autophagy receptor SQSTM1/p62 (sequestosome 1), which activates SQSTM1 associated with targets and sequesters them into oligomeric species for autophagic targeting and lysosomal degradation. AUTOTACs were used to degrade various oncoproteins or aggregation-prone proteins in neurodegeneration both in vitro and/or in vivo. We suggest that AUTOTAC provides a platform for selective proteolysis as a research tool and in drug development.
    Keywords:  N-degron pathway; N-terminal arginylation; SQSTM1/p62; chemical tools; neurodegeneration; protein quality control; proteinopathy; proteolysis; selective autophagy; targeted protein degradation (TPD)
  7. FEBS Lett. 2022 Jun 23.
      Much remains to be determined about the participation of ubiquitin receptors in proteasomal degradation and their potential as therapeutic targets. Suppression of the ubiquitin receptor S5A/PSMD4/hRpn10 alone stabilises p53/TP53 but not the key p53 repressor MDM2. Here, we observed S5A and the ubiquitin receptors ADRM1/PSMD16/hRpn13 and RAD23A and B functionally overlap in MDM2 degradation. We provide further evidence that degradation of only a subset of ubiquitinated proteins is sensitive to S5A knockdown because ubiquitin receptor redundancy is commonplace. p53 can be upregulated by S5A modulation while degradation of substrates with redundant receptors is maintained. Our observations and analysis of Cancer Dependency Map (DepMap) screens show S5A depletion/loss substantially reduces cancer cell line viability. This and selective S5A dependency of proteasomal substrates make S5A a target of interest for cancer therapy.
    Keywords:  ADRM1/PSMD16/hRpn13; MDM2; RAD23A; RAD23B; S5A/PSMD4/hRpn10; cancer; p53/TP53; proteasome; redundancy; ubiquitin receptor
  8. J Biol Chem. 2022 Jun 20. pii: S0021-9258(22)00612-3. [Epub ahead of print] 102170
      In Saccharomyces cerevisiae, proteins destined for secretion utilize the post-translational translocon machinery to gain entry into the endoplasmic reticulum (ER). These proteins then mature by undergoing a number of post-translational modifications in different compartments of the secretory pathway. While these modifications have been well established for many proteins, to date only a few studies have been conducted regarding the conditions and factors affecting maturation of these proteins before entering into the ER. Here, using immunoblotting, microscopy and spot test assays, we show that excess copper inhibits the Sec61 translocon function and causes accumulation of two well-known post-translationally translocated (PTT) proteins, Gas1 and CPY, in the cytosol. We further show the copper-sensitive phenotype of sec61 deficient yeast cells is ameliorated by restoring the levels of SEC61 through plasmid transformation. Further, screening of translocation-defective Sec61 mutants revealed that sec61-22, bearing L80M, V134I, M248V, and L342S mutations, is resistant to copper, suggesting that copper might be inflicting toxicity through one of these residues. In conclusion, these findings imply that copper mediated accumulation of PTT proteins is due to the inhibition of Sec61.
    Keywords:  cell biology; copper; endoplasmic reticulum (ER); protein processing; protein translocation
  9. Curr Opin Pharmacol. 2022 Jun 21. pii: S1471-4892(22)00085-6. [Epub ahead of print]65 102258
      Excessive and chronic airway inflammation associated with increased morbidity and mortality is a hallmark of cystic fibrosis (CF) airway disease. Previous studies underscored the role of endoplasmic reticulum (ER) signaling in CF airway inflammatory responses. In this review we discuss 1) how airway inflammation induces ER stress-triggered activation of the unfolded protein response and 2) the functional importance of the ER stress transducer inositol requiring enzyme 1α (IRE1α) in CF airway epithelial inflammatory responses. We also briefly review the current understanding of IRE1α activation and the development of small molecules aimed at modulating IRE1α kinase and RNase activities. Inhibition of IRE1α kinase and RNase may be considered as a novel therapeutic strategy to ameliorate the robust inflammatory status of CF airways.
  10. DNA Repair (Amst). 2022 Jun 10. pii: S1568-7864(22)00089-1. [Epub ahead of print]116 103356
      Multiple eukaryotic SWI2/SNF2 DNA translocases safeguard genome integrity, mostly by remodelling nucleosomes, but also by fine-tuning mechanisms of DNA repair, such as homologous recombination. Among this large family there is a unique class of Rad5/16-like enzymes, including Saccharomyces cerevisiae Uls1 and its Schizosaccharomyces pombe orthologues Rrp1 and Rrp2, that have both translocase and E3 ubiquitin ligase activities, and are often directed towards their substrates by SUMOylation. Here we summarize recent advances in understanding how different activities of these yeast proteins jointly contribute to their important roles in replication stress response particularly at centromeres and telomeres. This extends the possible range of functions performed by this class of SNF2 enzymes in human cells involving both their translocase and ubiquitin ligase activities and related to SUMOylation pathways within the nucleus.
    Keywords:  DNA repair; Replication stress; SUMOylation; Translocase; Ubiquitin ligase; Yeast
  11. Nat Cell Biol. 2022 Jun 23.
      When cells are stressed, bulk translation is often downregulated to reduce energy demands while stress-response proteins are simultaneously upregulated. To promote proteasome assembly and activity and maintain cell viability upon TORC1 inhibition, 19S regulatory-particle assembly chaperones (RPACs) are selectively translated. However, the molecular mechanism for such selective translational upregulation is unclear. Here, using yeast, we discover that remodelling of the actin cytoskeleton is important for RPAC translation following TORC1 inhibition. mRNA of the RPAC ADC17 is associated with actin cables and is enriched at cortical actin patches under stress, dependent upon the early endocytic protein Ede1. ede1∆ cells failed to induce RPACs and proteasome assembly upon TORC1 inhibition. Conversely, artificially tethering ADC17 mRNA to cortical actin patches enhanced its translation upon stress. These findings suggest that actin-dense structures such as cortical actin patches may serve as a translation platform for a subset of stress-induced mRNAs including regulators of proteasome homeostasis.
  12. Structure. 2022 Jun 09. pii: S0969-2126(22)00224-6. [Epub ahead of print]
      RING-between-RING (RBR) E3 ligases mediate ubiquitin transfer through an obligate E3-ubiquitin thioester intermediate prior to substrate ubiquitination. Although RBRs share a conserved catalytic module, substrate recruitment mechanisms remain enigmatic, and the relevant domains have yet to be identified for any member of the class. Here we characterize the interaction between the auto-inhibited RBR, HHARI (AriH1), and its target protein, 4EHP, using a combination of XL-MS, HDX-MS, NMR, and biochemical studies. The results show that (1) a di-aromatic surface on the catalytic HHARI Rcat domain forms a binding platform for substrates and (2) a phosphomimetic mutation on the auto-inhibitory Ariadne domain of HHARI promotes release and reorientation of Rcat for transthiolation and substrate modification. The findings identify a direct binding interaction between a RING-between-RING ligase and its substrate and suggest a general model for RBR substrate recognition.
    Keywords:  4EHP; AriH1; HDX-MS; HHARI; RING-between-RING; XL-MS; mono-ubiquitination; ubiquitin
  13. Nat Rev Mol Cell Biol. 2022 Jun 24.
      Sumoylation is an essential post-translational modification that is catalysed by a small number of modifying enzymes but regulates thousands of target proteins in a dynamic manner. Small ubiquitin-like modifiers (SUMOs) can be attached to target proteins as one or more monomers or in the form of polymers of different types. Non-covalent readers recognize SUMO-modified proteins via SUMO interaction motifs. SUMO simultaneously modifies groups of functionally related proteins to regulate predominantly nuclear processes, including gene expression, the DNA damage response, RNA processing, cell cycle progression and proteostasis. Recent progress has increased our understanding of the cellular and pathophysiological roles of SUMO modifications, extending their functions to the regulation of immunity, pluripotency and nuclear body assembly in response to oxidative stress, which partly occurs through the recently characterized mechanism of liquid-liquid phase separation. Such progress in understanding the roles and regulation of sumoylation opens new avenues for the targeting of SUMO to treat disease, and indeed the first drug blocking sumoylation is currently under investigation in clinical trials as a possible anticancer agent.
  14. Trends Cell Biol. 2022 Jun 18. pii: S0962-8924(22)00135-0. [Epub ahead of print]
      The J-domain proteins (JDP) form the largest protein family among cellular chaperones. In cooperation with the Hsp70 chaperone system, these co-chaperones orchestrate a plethora of distinct functions, including those that help maintain cellular proteostasis and development. JDPs evolved largely through the fusion of a J-domain with other protein subdomains. The highly conserved J-domain facilitates the binding and activation of Hsp70s. How JDPs (re)wire Hsp70 chaperone circuits and promote functional diversity remains insufficiently explained. Here, we discuss recent advances in our understanding of the JDP family with a focus on the regulation built around J-domains to ensure correct pairing and assembly of JDP-Hsp70 machineries that operate on different clientele under various cellular growth conditions.
    Keywords:  Hsp40; Hsp70; J-domain; J-domain proteins; protein conformational diseases; protein homeostasis
  15. Proc Natl Acad Sci U S A. 2022 Jun 28. 119(26): e2111506119
      Macroautophagy promotes cellular homeostasis by delivering cytoplasmic constituents to lysosomes for degradation [Mizushima, Nat. Cell Biol. 20, 521-527 (2018)]. However, while most studies have focused on the mechanisms of protein degradation during this process, we report here that macroautophagy also depends on glycan degradation via the glycosidase, α-l-fucosidase 1 (FUCA1), which removes fucose from glycans. We show that cells lacking FUCA1 accumulate lysosomal glycans, which is associated with impaired autophagic flux. Moreover, in a mouse model of fucosidosis-a disease characterized by inactivating mutations in FUCA1 [Stepien et al., Genes (Basel) 11, E1383 (2020)]-glycan and autophagosome/autolysosome accumulation accompanies tissue destruction. Mechanistically, using lectin capture and mass spectrometry, we identified several lysosomal enzymes with altered fucosylation in FUCA1-null cells. Moreover, we show that the activity of some of these enzymes in the absence of FUCA1 can no longer be induced upon autophagy stimulation, causing retardation of autophagic flux, which involves impaired autophagosome-lysosome fusion. These findings therefore show that dysregulated glycan degradation leads to defective autophagy, which is likely a contributing factor in the etiology of fucosidosis.
    Keywords:  fucosidosis; lysosomes; macroautophagy; α-l-fucosidase 1
  16. Chem Soc Rev. 2022 Jun 20.
      Protein-protein interactions (PPIs) govern all biological processes. Some small molecules modulate PPIs through induced protein proximity. In particular, molecular glue degraders are monovalent compounds that orchestrate interactions between a target protein and an E3 ubiquitin ligase, prompting the proteasomal degradation of the former. This and other pharmacological strategies of targeted protein degradation (e.g. proteolysis-targeting chimeras - PROTACs) overcome some limitations of traditional occupancy-based therapeutics. Here, we provide an overview of the "molecular glue" concept, with a special focus on natural and synthetic inducers of proximity to E3s. We then briefly highlight the serendipitous discoveries of some clinical and preclinical molecular glue degraders, and discuss the first examples of intentional discoveries. Specifically, we outline the different screening strategies reported in this rapidly evolving arena and our thoughts on future perspectives. By mastering the ability to influence PPIs, molecular glue degraders can induce the degradation of unligandable proteins, thus providing an exciting path forward to broaden the targetable proteome.
  17. Elife. 2022 Jun 23. pii: e72879. [Epub ahead of print]11
      Deubiquitinating enzymes (DUBs), ~100 of which are found in human cells, are proteases that remove ubiquitin conjugates from proteins, thereby regulating protein turnover. They are involved in a wide range of cellular activities and are emerging therapeutic targets for cancer and other diseases. Drugs targeting USP1 and USP30 are in clinical development for cancer and kidney disease respectively. However, the majority of substrates and pathways regulated by DUBs remain unknown, impeding efforts to prioritize specific enzymes for research and drug development. To assemble a knowledgebase of DUB activities, co-dependent genes, and substrates, we combined targeted experiments using CRISPR libraries and inhibitors with systematic mining of functional genomic databases. Analysis of the Dependency Map, Connectivity Map, Cancer Cell Line Encyclopedia, and multiple protein-protein interaction databases yielded specific hypotheses about DUB function, a subset of which were confirmed in follow-on experiments. The data in this paper are browsable online in a newly developed DUB Portal and promise to improve understanding of DUBs as a family as well as the activities of incompletely characterized DUBs (e.g. USPL1 and USP32) and those already targeted with investigational cancer therapeutics (e.g. USP14, UCHL5, and USP7).
    Keywords:  CRISPR; Cancer Dependency Map; biochemistry; bioinformatics; cancer; chemical biology; computational biology; deubiquitinating enzyme; human; inhibitor; systems biology
  18. Nat Chem. 2022 Jun 20.
      The translation of messenger RNA (mRNA) is a fundamental process in gene expression, and control of translation is important to regulate protein synthesis in cells. The primary hallmark of eukaryotic mRNAs is their 5' cap, whose molecular contacts to the eukaryotic translation initiation factor eIF4E govern the initiation of translation. Here we report 5' cap analogues with photo-cleavable groups (FlashCaps) that prohibit binding to eIF4E and resist cleavage by decapping enzymes. These compounds are compatible with the general and efficient production of mRNAs by in vitro transcription. In FlashCap-mRNAs, the single photocaging group abrogates translation in vitro and in mammalian cells without increasing immunogenicity. Irradiation restores the native cap, triggering efficient translation. FlashCaps overcome the problem of remaining sequence or structure changes in mRNA after irradiation that limited previous designs. Together, these results demonstrate that FlashCaps offer a route to regulate the expression of any given mRNA and to dose mRNA therapeutics with spatio-temporal control.
  19. Adv Sci (Weinh). 2022 Jun 24. e2105320
      Under ER stress conditions, the ER form of transmembrane proteins can reach the plasma membrane via a Golgi-independent unconventional protein secretion (UPS) pathway. However, the targeting mechanisms of membrane proteins for UPS are unknown. Here, this study reports that TMED proteins play a critical role in the ER stress-associated UPS of transmembrane proteins. The gene silencing results reveal that TMED2, TMED3, TMED9 and TMED10 are involved in the UPS of transmembrane proteins, such as CFTR, pendrin and SARS-CoV-2 Spike. Subsequent mechanistic analyses indicate that TMED3 recognizes the ER core-glycosylated protein cargos and that the heteromeric TMED2/3/9/10 complex mediates their UPS. Co-expression of all four TMEDs improves, while each single expression reduces, the UPS and ion transport function of trafficking-deficient ΔF508-CFTR and p.H723R-pendrin, which cause cystic fibrosis and Pendred syndrome, respectively. In contrast, TMED2/3/9/10 silencing reduces SARS-CoV-2 viral release. These results provide evidence for a common role of TMED3 and related TMEDs in the ER stress-associated, Golgi-independent secretion of transmembrane proteins.
    Keywords:  CFTR; SARS-CoV-2 spike; TMED; UPS; pendrin
  20. Nat Commun. 2022 Jun 20. 13(1): 3542
      Mucin domains are densely O-glycosylated modular protein domains found in various extracellular and transmembrane proteins. Mucin-domain glycoproteins play important roles in many human diseases, such as cancer and cystic fibrosis, but the scope of the mucinome remains poorly defined. Recently, we characterized a bacterial O-glycoprotease, StcE, and demonstrated that an inactive point mutant retains binding selectivity for mucin-domain glycoproteins. In this work, we leverage inactive StcE to selectively enrich and identify mucin-domain glycoproteins from complex samples like cell lysate and crude ovarian cancer patient ascites fluid. Our enrichment strategy is further aided by an algorithm to assign confidence to mucin-domain glycoprotein identifications. This mucinomics platform facilitates detection of hundreds of glycopeptides from mucin domains and highly overlapping populations of mucin-domain glycoproteins from ovarian cancer patients. Ultimately, we demonstrate our mucinomics approach can reveal key molecular signatures of cancer from in vitro and ex vivo sources.
  21. J Biol Chem. 2022 Jun 15. pii: S0021-9258(22)00587-7. [Epub ahead of print] 102145
      Class I WW domains are present in many proteins of various functions and mediate protein interactions by binding to short linear PPxY motifs. Tandem WW domains often bind peptides with multiple PPxY motifs, but the interplay of WW/peptide interactions is not always intuitive. The WW domain-containing oxidoreductase (WWOX) harbors two WW domains: an unstable WW1 capable of PPxY binding, and stable WW2 that cannot bind PPxY. The WW2 domain has been suggested to act as a WW1-domain chaperone, but the underlying mechanism of its chaperone activity remains to be revealed. Here we combined nuclear magnetic resonance (NMR), isothermal calorimetry (ITC), and structural modeling to elucidate the roles of both WW domains in WWOX binding to its PPxY-containing substrate ErbB4. Using NMR, we identified an interaction surface between these two domains that supports a WWOX conformation compatible with peptide substrate binding. ITC and NMR measurements also indicated that while binding affinity to a single PPxY motif is marginally increased in the presence of WW2, affinity to a dual-motif peptide increases tenfold. Furthermore, we found WW2 can directly bind double-motif peptides using its canonical binding site. Finally, differential binding of peptides in mutagenesis experiments was consistent with a parallel N- to C-terminal PPxY tandem motif orientation in binding to the WW1-WW2 tandem domain, validating structural models of the interaction. Taken together, our results reveal the complex nature of tandem WW-domain organization and substrate binding, highlighting the contribution of WWOX WW2 to both protein stability and target binding.
    Keywords:  Peptide interaction; WW domains; WWOX; binding of tandem motifs; binding via tandem domains; computer modeling; isothermal titration calorimetry (ITC); nuclear magnetic resonance (NMR); peptide docking; protein dynamic; protein-protein interaction
  22. Nat Commun. 2022 Jun 24. 13(1): 3624
      The precise regulation of RNA Polymerase II (Pol II) transcription after genotoxic stress is crucial for proper execution of the DNA damage-induced stress response. While stalling of Pol II on transcription-blocking lesions (TBLs) blocks transcript elongation and initiates DNA repair in cis, TBLs additionally elicit a response in trans that regulates transcription genome-wide. Here we uncover that, after an initial elongation block in cis, TBLs trigger the genome-wide VCP-mediated proteasomal degradation of promoter-bound, P-Ser5-modified Pol II in trans. This degradation is mechanistically distinct from processing of TBL-stalled Pol II, is signaled via GSK3, and contributes to the TBL-induced transcription block, even in transcription-coupled repair-deficient cells. Thus, our data reveal the targeted degradation of promoter-bound Pol II as a critical pathway that allows cells to cope with DNA damage-induced transcription stress and enables the genome-wide adaptation of transcription to genotoxic stress.
  23. Nat Cell Biol. 2022 Jun 23.
      Neutrophils migrating towards chemoattractant gradients amplify their recruitment range by releasing the secondary chemoattractant leukotriene B4 (LTB4) refs. 1,2. We previously demonstrated that LTB4 and its synthesizing enzymes, 5-lipoxygenase (5-LO), 5-LO activating protein (FLAP) and leukotriene A4 hydrolase, are packaged and released in exosomes3. Here we report that the biogenesis of the LTB4-containing exosomes originates at the nuclear envelope (NE) of activated neutrophils. We show that the neutral sphingomyelinase 1 (nSMase1)-mediated generation of ceramide-enriched lipid-ordered microdomains initiates the clustering of the LTB4-synthesizing enzymes on the NE. We isolated and analysed exosomes from activated neutrophils and established that the FLAP/5-LO-positive exosome population is distinct from that of the CD63-positive exosome population. Furthermore, we observed a strong co-localization between ALIX and FLAP at the periphery of nuclei and within cytosolic vesicles. We propose that the initiation of NE curvature and bud formation is mediated by nSMase1-dependent ceramide generation, which leads to FLAP and ALIX recruitment. Together, these observations elucidate the mechanism for LTB4 secretion and identify a non-conventional pathway for exosome generation.
  24. Nature. 2022 Jun 22.
      Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.
  25. J Clin Invest. 2022 Jun 21. pii: e153519. [Epub ahead of print]
      Epithelial cells lining mucosal surfaces of the gastrointestinal and respiratory tracts uniquely express ERN2/IRE1β, a paralogue of the most evolutionarily conserved endoplasmic reticulum stress sensor ERN1/IRE1α. How ERN2 functions at the host-environment interface and why a second paralogue evolved remain incompletely understood. Using conventionally raised and germ-free Ern2-/- mice, we found that ERN2 was required for microbiota-induced goblet cell maturation and mucus barrier assembly in the colon. This occurred only after colonization of the alimentary tract with normal gut microflora, which induced Ern2 expression. ERN2 acted by splicing Xbp1 mRNA to expand ER function and prevent ER stress in goblet cells. Although ERN1 can also splice Xbp1 mRNA, it did not act redundantly to ERN2 in this context. By regulating assembly of the colon mucus layer, ERN2 further shaped the composition of the gut microbiota. Mice lacking Ern2 had a dysbiotic microbial community that failed to induce goblet cell development and increased susceptibility to colitis when transferred into germ-free wild type mice. These results show that ERN2 evolved at mucosal surfaces to mediate crosstalk between gut microbes and the colonic epithelium required for normal homeostasis and host defense.
    Keywords:  Cell stress; Gastroenterology; Inflammatory bowel disease