bims-proteo Biomed News
on Proteostasis
Issue of 2022‒08‒21
27 papers selected by
Eric Chevet

  1. J Biol Chem. 2022 Aug 12. pii: S0021-9258(22)00816-X. [Epub ahead of print] 102373
      Misfolded proteins in the lumen of the endoplasmic reticulum (ER) are retro-translocated into the cytosol and degraded by the ubiquitin-proteasome system, a pathway termed luminal ER-associated protein degradation (ERAD-L). Retro-translocation is mediated by a conserved protein complex, consisting of the ubiquitin ligase Hrd1 and four associated proteins (Der1, Usa1, Hrd3, and Yos9). Photo-crosslinking experiments provided preliminary evidence for the polypeptide path through the membrane, but did not reveal specific interactions between amino acids in the substrate and Hrd1 complex. Here, we have used site-specific disulfide crosslinking to map the interactions of a glycosylated model substrate with the Hrd1 complex in live S. cerevisiae cells. Together with available electron cryo-microscopy (cryo-EM) structures, the results show that the substrate interacts on the luminal side with both a groove in Hrd3 and the lectin domain of Yos9, and inserts a loop into the membrane, with one side of the loop interacting with the lateral gate of Der1, and the other with the lateral gate of Hrd1. Our disulfide crosslinking experiments also show that two Hrd1 molecules can interact through their lateral gates, and that Hrd1 auto-ubiquitination is required for the disassembly of these Hrd1 dimers. Taken together, these data define the path of a polypeptide through the ER membrane and suggest that auto-ubiquitination of inactive Hrd1 dimers is required to generate active Hrd1 monomers.
    Keywords:  Der1; ERAD; Hrd1; Hrd3; Usa1; Yos9; crosslinking; disulfide; retrotranslocation
  2. Trends Cell Biol. 2022 Aug 16. pii: S0962-8924(22)00188-X. [Epub ahead of print]
      Ribosome-associated protein quality control (RQC) is a protein surveillance mechanism that eliminates defective nascent polypeptides. The E3 ubiquitin ligase, Ltn1, is a key regulator of RQC that targets substrates for ubiquitination. Argonaute proteins (AGOs) are central players in miRNA-mediated gene silencing and have recently been shown to also regulate RQC by facilitating Ltn1. Therefore, AGOs directly coordinate post-transcriptional gene silencing and RQC, ensuring efficient gene silencing. We summarize the principles of RQC and the functions of AGOs in miRNA-mediated gene silencing, and discuss how AGOs associate with the endoplasmic reticulum (ER) to assist Ltn1 in controlling RQC. We highlight that RQC not only eliminates defective nascent polypeptides but also removes unwanted protein products when AGOs participate.
    Keywords:  Ltn1; RNA-induced gene silencing; VCP; ribosome stalling; ubiquitin
  3. EMBO Rep. 2022 Aug 18. e54859
      The hexameric AAA-ATPase valosin-containing protein (VCP) is essential for mitochondrial protein quality control. How VCP is recruited to mammalian mitochondria remains obscure. Here we report that UBXD8, an ER- and lipid droplet-localized VCP adaptor, also localizes to mitochondria and locally recruits VCP. UBXD8 associates with mitochondrial and ER ubiquitin E3 ligases and targets their substrates for degradation. Remarkably, both mitochondria- and ER-localized UBXD8 can degrade mitochondrial and ER substrates in cis and in trans. UBXD8 also associates with the TOM complex but is dispensable for translocation-associated degradation. UBXD8 knockout impairs the degradation of the pro-survival protein Mcl1 but surprisingly sensitizes cells to apoptosis and mitochondrial stresses. UBXD8 knockout also hyperactivates mitophagy. We identify pro-apoptotic BH3-only proteins Noxa, Bik, and Bnip3 as novel UBXD8 substrates and determine that UBXD8 inhibits apoptosis via degrading Noxa and restrains mitophagy via degrading Bnip3. Collectively, our characterizations reveal UBXD8 as the major mitochondrial adaptor of VCP and unveil its role in apoptosis and mitophagy regulation.
    Keywords:  UBXD8; VCP; apoptosis; mitochondria-associated degradation; mitophagy
  4. Trends Biochem Sci. 2022 Aug 10. pii: S0968-0004(22)00189-X. [Epub ahead of print]
      Covalent modification by the small protein ubiquitin can target proteins for destruction by the proteasome, but the ubiquitin signal itself is recycled. Surprisingly, proteasomes contain three different deubiquitinating enzymes (DUBs). Recent work by Zhang and Zou et al. reveals how one of these enzymes, Usp14, regulates, and is regulated by, the proteasome.
    Keywords:  Rpn11; Ubp6; Usp14; proteasome; ubiquitin
  5. Chem Soc Rev. 2022 Aug 19.
      The von Hippel-Lindau (VHL) Cullin RING E3 ligase is an essential enzyme in the ubiquitin-proteasome system that recruits substrates such as the hypoxia inducible factor for ubiquitination and subsequent proteasomal degradation. The ubiquitin-proteasome pathway can be hijacked toward non-native neo-substrate proteins using proteolysis targeting chimeras (PROTACs), bifunctional molecules designed to simultaneously bind to an E3 ligase and a target protein to induce target ubiquitination and degradation. The availability of high-quality small-molecule ligands with good binding affinity for E3 ligases is fundamental for PROTAC development. Lack of good E3 ligase ligands as starting points to develop PROTAC degraders was initially a stumbling block to the development of the field. Herein, the journey towards the design of small-molecule ligands binding to VHL is presented. We cover the structure-based design of VHL ligands, their application as inhibitors in their own right, and their implementation into rationally designed, potent PROTAC degraders of various target proteins. We highlight the key findings and learnings that have provided strong foundations for the remarkable development of targeted protein degradation, and that offer a blueprint for designing new ligands for E3 ligases beyond VHL.
  6. Mol Carcinog. 2022 Aug 17.
      Transforming Growth Factor β1 (TGFβ1) is a critical regulator of tumor progression in response to HRas. Recently, TGFβ1 has been shown to trigger ER stress in many disease models; however, its role in oncogene-induced ER stress is unclear. Oncogenic HRas induces the unfolded protein response (UPR) predominantly via the Inositol-requiring enzyme 1α (IRE1α) pathway to initiate the adaptative responses to ER stress, with importance for both proliferation and senescence. Here, we show a role of the UPR sensor proteins IRE1α and (PKR)-like endoplasmic reticulum kinase (PERK) to mediate the tumor-suppressive roles of TGFβ1 in mouse keratinocytes expressing mutant forms of HRas. TGFβ1 suppressed IRE1α phosphorylation and activation by HRas both in in vitro and in vivo models while simultaneously activating the PERK pathway. However, the increase in ER stress indicated an uncoupling of ER stress and IRE1α activation by TGFβ1. Pharmacological and genetic approaches demonstrated that TGFβ1-dependent dephosphorylation of IRE1α was mediated by PERK through RNA Polymerase II Associated Protein 2 (RPAP2), a PERK-dependent IRE1α phosphatase. In addition, TGFβ1-mediated growth arrest in oncogenic HRas keratinocytes was partially dependent on PERK-induced IRE1α dephosphorylation and inactivation. Together, these results demonstrate a critical cross-talk between UPR proteins that is important for TGFβ1-mediated tumor suppressive responses.
    Keywords:  ER stress; HRas; IRE1α; PERK; TGFβ1; proliferation; unfolded protein response
  7. EMBO J. 2022 Aug 15. e110398
      Autophagy depends on the repopulation of lysosomes to degrade intracellular components and recycle nutrients. How cells co-ordinate lysosome repopulation during basal autophagy, which occurs constitutively under nutrient-rich conditions, is unknown. Here, we identify an endosome-dependent phosphoinositide pathway that links PI3Kα signaling to lysosome repopulation during basal autophagy. We show that PI3Kα-derived PI(3)P generated by INPP4B on late endosomes was required for basal but not starvation-induced autophagic degradation. PI(3)P signals were maintained as late endosomes matured into endolysosomes, and served as the substrate for the 5-kinase, PIKfyve, to generate PI(3,5)P2 . The SNX-BAR protein, SNX2, was recruited to endolysosomes by PI(3,5)P2 and promoted lysosome reformation. Inhibition of INPP4B/PIKfyve-dependent lysosome reformation reduced autophagic clearance of protein aggregates during proteotoxic stress leading to increased cytotoxicity. Therefore under nutrient-rich conditions, PI3Kα, INPP4B, and PIKfyve sequentially contribute to basal autophagic degradation and protection from proteotoxic stress via PI(3,5)P2 -dependent lysosome reformation from endolysosomes. These findings reveal that endosome maturation couples PI3Kα signaling to lysosome reformation during basal autophagy.
    Keywords:  INPP4B; PI3Kα; PIKfyve; autophagy; lysosome
  8. Biochim Biophys Acta Mol Cell Biol Lipids. 2022 Aug 15. pii: S1388-1981(22)00109-3. [Epub ahead of print] 159219
      The endoplasmic reticulum (ER) is an organelle that performs several key functions such as protein synthesis and folding, lipid metabolism and calcium homeostasis. When these functions are disrupted, such as upon protein misfolding, ER stress occurs. ER stress can trigger adaptive responses to restore proper functioning such as activation of the unfolded protein response (UPR). In certain cells, the free fatty acid palmitate has been shown to induce the UPR. Here, we examined the effects of palmitate on UPR gene expression in a human neuronal cell line and compared it with thapsigargin, a known depletor of ER calcium and trigger of the UPR. We used a Gaussia luciferase-based reporter to assess how palmitate treatment affects ER proteostasis and calcium homeostasis in the cells. We also investigated how ER calcium depletion by thapsigargin affects lipid membrane composition by performing mass spectrometry on subcellular fractions and compared this to palmitate. Surprisingly, palmitate treatment did not activate UPR despite prominent changes to membrane phospholipids. Conversely, thapsigargin induced a strong UPR, but did not significantly change the membrane lipid composition in subcellular fractions. In summary, our data demonstrate that changes in membrane lipid composition and disturbances in ER calcium homeostasis have a minimal influence on each other in neuronal cells. These data provide new insight into the adaptive interplay of lipid homeostasis and proteostasis in the cell.
    Keywords:  Brain lipids; Cell signaling; ER calcium; Endoplasmic reticulum; Exodosis; Lipidomics; Lipids; Palmitate; Phospholipids; Thapsigargin; UPR; Unfolded protein response
  9. Bioessays. 2022 Aug 19. e2200064
      An entirely different mechanism and localization were recently proposed for the COPII coat complex, challenging its well-accepted function to select and concentrate cargo into small COPII-coated spherical transport vesicles. Instead, the COPII complex is suggested to form a dynamic yet stationary collar that forms a boundary between the ER and the ER export membrane domain. This membrane domain, the ER exit site (ERES), is the site of COPII-mediated sorting and concentration of transport competent proteins. Subsequently, the ERES is implicated to mature and bud to form a sizeable pleiomorphic transport carrier that translocate on microtubules to fuse with the Golgi apparatus. Despite this drastic mechanistic dogma shift, most of the underlying protein-protein and protein-membrane interactions remain unchanged. Here, we attempt to provide a detailed description of the newly proposed model of how ER to Golgi transport works by describing the role of several essential proteins of the transport machinery.
    Keywords:  COPII; ER exit site; cargo protein; early secretory pathway; endoplasmic reticulum; transport carriers; transport vesicles
  10. Nat Commun. 2022 Aug 15. 13(1): 4789
      The covalent modification of target proteins with ubiquitin or ubiquitin-like modifiers is initiated by E1 activating enzymes, which typically transfer a single modifier onto cognate conjugating enzymes. UBA6 is an unusual E1 since it activates two highly distinct modifiers, ubiquitin and FAT10. Here, we report crystal structures of UBA6 in complex with either ATP or FAT10. In the UBA6-FAT10 complex, the C-terminal domain of FAT10 binds to where ubiquitin resides in the UBA1-ubiquitin complex, however, a switch element ensures the alternate recruitment of either modifier. Simultaneously, the N-terminal domain of FAT10 interacts with the 3-helix bundle of UBA6. Site-directed mutagenesis identifies residues permitting the selective activation of either ubiquitin or FAT10. These results pave the way for studies investigating the activation of either modifier by UBA6 in physiological and pathophysiological settings.
  11. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01012-9. [Epub ahead of print]40(7): 111195
      ATG9A is a highly conserved membrane protein required for autophagy initiation. It is trafficked from the trans-Golgi network (TGN) to the phagophore to act as a membrane source for autophagosome expansion. Here, we show that ATG9A is not just a passenger protein in the TGN but rather works in concert with GRASP55, a stacking factor for Golgi structure, to organize Golgi dynamics and integrity. Upon heat stress, the E3 ubiquitin ligase MARCH9 is promoted to ubiquitinate ATG9A in the form of K63 conjugation, and the nondegradable ubiquitinated ATG9A disperses from the Golgi apparatus to the cytoplasm more intensely, accompanied by inhibiting GRASP55 oligomerization, further resulting in Golgi fragmentation. Knockout of ATG9A or MARCH9 largely prevents Golgi fragmentation and protects Golgi functions under heat and other Golgi stresses. Our results reveal a noncanonical function of ATG9A for Golgi dynamics and suggest the pathway for sensing Golgi stress via the MARCH9/ATG9A axis.
    Keywords:  ATG9A; CP: Molecular biology; Golgi dynamics; Golgi stress response; ubiquitination
  12. Nat Struct Mol Biol. 2022 Aug 18.
      Most cullin-RING ubiquitin ligases (CRLs) form homologous assemblies between a neddylated cullin-RING catalytic module and a variable substrate-binding receptor (for example, an F-box protein). However, the vertebrate-specific CRL7FBXW8 is of interest because it eludes existing models, yet its constituent cullin CUL7 and F-box protein FBXW8 are essential for development, and CUL7 mutations cause 3M syndrome. In this study, cryo-EM and biochemical analyses reveal the CRL7FBXW8 assembly. CUL7's exclusivity for FBXW8 among all F-box proteins is explained by its unique F-box-independent binding mode. In CRL7FBXW8, the RBX1 (also known as ROC1) RING domain is constrained in an orientation incompatible with binding E2~NEDD8 or E2~ubiquitin intermediates. Accordingly, purified recombinant CRL7FBXW8 lacks auto-neddylation and ubiquitination activities. Instead, our data indicate that CRL7 serves as a substrate receptor linked via SKP1-FBXW8 to a neddylated CUL1-RBX1 catalytic module mediating ubiquitination. The structure reveals a distinctive CRL-CRL partnership, and provides a framework for understanding CUL7 assemblies safeguarding human health.
  13. Immunometabolism (Cobham). 2022 Jul;4(3): e00007
      The endoplasmic reticulum (ER) is a specialized organelle that participates in multiple cellular functions including protein folding, maturation, trafficking, and degradation to maintain homeostasis. However, hostile conditions in the tumor microenvironment (TME) disturb ER homeostasis. To overcome these conditions, cells activate ER stress response pathways, which are shown to augment the suppressive phenotypes of immune cells; however, the molecular mechanisms underpinning this process remain elusive. Here, we discuss a recent study by Raines et al, that suggests the role of the helper T-cell 2 (TH2) cytokine interleukin-4 (IL-4), and the TME in facilitating a protein kinase RNA-like ER kinase (PERK)-signaling cascade in macrophages, which promotes immunosuppressive M2 macrophage activation and proliferation. Further, the authors showed that PERK signaling promotes both mitochondrial respirations to fulfill cellular energy requirements and signaling through ATF4, which regulate phosphoserine aminotransferase 1 (PSAT1) activity to mediate the serine biosynthesis pathway. These results highlight a previously uncharacterized role for PERK in cellular metabolism and epigenetic modification in M2 macrophages, and thus offers a new therapeutic strategy for overcoming the immunosuppressive effects in the TME.
    Keywords:  ER stress; M2 macrophage; endoplasmic reticulum; myeloid cell-derived suppressor cells; protein kinase RNA-like ER kinase; tumor-associated macrophages
  14. J Cell Biol. 2022 Sep 05. pii: e202112107. [Epub ahead of print]221(9):
      Lipid droplets (LDs) are the primary organelles of lipid storage, buffering energy fluctuations of the cell. They store neutral lipids in their core that is surrounded by a protein-decorated phospholipid monolayer. LDs arise from the endoplasmic reticulum (ER). The ER protein seipin, localizing at ER-LD junctions, controls LD nucleation and growth. However, how LD biogenesis is spatially and temporally coordinated remains elusive. Here, we show that the lipid transfer proteins ORP5 and ORP8 control LD biogenesis at mitochondria-associated ER membrane (MAM) subdomains, enriched in phosphatidic acid. We found that ORP5/8 regulates seipin recruitment to these MAM-LD contacts, and their loss impairs LD biogenesis. Importantly, the integrity of ER-mitochondria contact sites is crucial for ORP5/8 function in regulating seipin-mediated LD biogenesis. Our study uncovers an unprecedented ORP5/8 role in orchestrating LD biogenesis and maturation at MAMs and brings novel insights into the metabolic crosstalk between mitochondria, ER, and LDs at the membrane contact sites.
  15. Cell Biosci. 2022 Aug 14. 12(1): 129
      The endoplasmic reticulum (ER) is a multi-layered organelle that is essential for the synthesis, folding, and structural maturation of almost one-third of the cellular proteome. It houses several resident proteins for these functions including the 21 members of the protein disulfide isomerase (PDI) family. The signature of proteins belonging to this family is the presence of the thioredoxin domain which mediates the formation, and rearrangement of disulfide bonds of substrate proteins in the ER. This process is crucial not only for the proper folding of ER substrates but also for maintaining a balanced ER proteostasis. The inclusion of new PDI members with a wide variety of structural determinants, size and enzymatic activity has brought additional epitomes of how PDI functions. Notably, some of them do not carry the thioredoxin domain and others have roles outside the ER. This also reflects that PDIs may have specialized functions and their functions are not limited within the ER. Large-scale expression datasets of human clinical samples have identified that the expression of PDI members is elevated in pathophysiological states like cancer. Subsequent functional interrogations using structural, molecular, cellular, and animal models suggest that some PDI members support the survival, progression, and metastasis of several cancer types. Herein, we review recent research advances on PDIs, vis-à-vis their expression, functions, and molecular mechanisms in supporting cancer growth with special emphasis on the anterior gradient (AGR) subfamily. Last, we posit the relevance and therapeutic strategies in targeting the PDIs in cancer.
  16. Nature. 2022 Aug 17.
      Compelling evidence shows that brown and beige adipose tissue are protective against metabolic diseases1,2. PR domain-containing 16 (PRDM16) is a dominant activator of the biogenesis of beige adipocytes by forming a complex with transcriptional and epigenetic factors and is therefore an attractive target for improving metabolic health3-8. However, a lack of knowledge surrounding the regulation of PRDM16 protein expression hampered us from selectively targeting this transcriptional pathway. Here we identify CUL2-APPBP2 as the ubiquitin E3 ligase that determines PRDM16 protein stability by catalysing its polyubiquitination. Inhibition of CUL2-APPBP2 sufficiently extended the half-life of PRDM16 protein and promoted beige adipocyte biogenesis. By contrast, elevated CUL2-APPBP2 expression was found in aged adipose tissues and repressed adipocyte thermogenesis by degrading PRDM16 protein. Importantly, extended PRDM16 protein stability by adipocyte-specific deletion of CUL2-APPBP2 counteracted diet-induced obesity, glucose intolerance, insulin resistance and dyslipidaemia in mice. These results offer a cell-autonomous route to selectively activate the PRDM16 pathway in adipose tissues.
  17. Nat Chem Biol. 2022 Aug 18.
      The E3 ligase TRIM7 has emerged as a critical player in viral infection and pathogenesis. However, the mechanism governing the TRIM7-substrate association remains to be defined. Here we report the crystal structures of TRIM7 in complex with 2C peptides of human enterovirus. Structure-guided studies reveal the C-terminal glutamine residue of 2C as the primary determinant for TRIM7 binding. Leveraged by this finding, we identify norovirus and SARS-CoV-2 proteins, and physiological proteins, as new TRIM7 substrates. Crystal structures of TRIM7 in complex with multiple peptides derived from SARS-CoV-2 proteins display the same glutamine-end recognition mode. Furthermore, TRIM7 could trigger the ubiquitination and degradation of these substrates, possibly representing a new Gln/C-degron pathway. Together, these findings unveil a common recognition mode by TRIM7, providing the foundation for further mechanistic characterization of antiviral and cellular functions of TRIM7.
  18. Cell Death Dis. 2022 Aug 16. 13(8): 712
      Recent studies suggest that Forkhead box D1 (FOXD1) plays an indispensable role in maintaining the mesenchymal (MES) properties of glioblastoma (GBM) stem cells (GSCs). Thus, understanding the mechanisms that control FOXD1 protein expression is critical for guiding GBM treatment, particularly in patients with therapy-resistant MES subtypes. In this study, we identify the ubiquitin-specific peptidase 21 (USP21) as a critical FOXD1 deubiquitinase in MES GSCs. We find that USP21 directly interacts with and stabilizes FOXD1 by reverting its proteolytic ubiquitination. Silencing of USP21 enhances polyubiquitination of FOXD1, promotes its proteasomal degradation, and ultimately attenuates MES identity in GSCs, while these effects could be largely restored by reintroduction of FOXD1. Remarkably, we show that disulfiram, a repurposed drug that could block the enzymatic activities of USP21, suppresses GSC tumorigenicity in MES GSC-derived GBM xenograft model. Additionally, we demonstrate that USP21 is overexpressed and positively correlated with FOXD1 protein levels in GBM tissues, and its expression is inversely correlated with patient survival. Collectively, our work reveals that USP21 maintains MES identity by antagonizing FOXD1 ubiquitination and degradation, suggesting that USP21 is a potential therapeutic target for the MES subtype of GBM.
  19. PLoS Pathog. 2022 Aug 15. 18(8): e1010543
      Although picornaviruses are conventionally considered 'nonenveloped', members of multiple picornaviral genera are released nonlytically from infected cells in extracellular vesicles. The mechanisms underlying this process are poorly understood. Here, we describe interactions of the hepatitis A virus (HAV) capsid with components of host endosomal sorting complexes required for transport (ESCRT) that play an essential role in release. We show release of quasi-enveloped virus (eHAV) in exosome-like vesicles requires a conserved export signal located within the 8 kDa C-terminal VP1 pX extension that functions in a manner analogous to late domains of canonical enveloped viruses. Fusing pX to a self-assembling engineered protein nanocage (EPN-pX) resulted in its ESCRT-dependent release in extracellular vesicles. Mutational analysis identified a 24 amino acid peptide sequence located within the center of pX that was both necessary and sufficient for nanocage release. Deleting a YxxL motif within this sequence ablated eHAV release, resulting in virus accumulating intracellularly. The pX export signal is conserved in non-human hepatoviruses from a wide range of mammalian species, and functional in pX sequences from bat hepatoviruses when fused to the nanocage protein, suggesting these viruses are released as quasi-enveloped virions. Quantitative proteomics identified multiple ESCRT-related proteins associating with EPN-pX, including ALG2-interacting protein X (ALIX), and its paralog, tyrosine-protein phosphatase non-receptor type 23 (HD-PTP), a second Bro1 domain protein linked to sorting of ubiquitylated cargo into multivesicular endosomes. RNAi-mediated depletion of either Bro1 domain protein impeded eHAV release. Super-resolution fluorescence microscopy demonstrated colocalization of viral capsids with endogenous ALIX and HD-PTP. Co-immunoprecipitation assays using biotin-tagged peptides and recombinant proteins revealed pX interacts directly through the export signal with N-terminal Bro1 domains of both HD-PTP and ALIX. Our study identifies an exceptionally potent viral export signal mediating extracellular release of virus-sized protein assemblies and shows release requires non-redundant activities of both HD-PTP and ALIX.
  20. iScience. 2022 Aug 19. 25(8): 104780
      The role of tripartite motif (TRIM) 38, a ubiquitin E3 ligase regulating various pathophysiological processes, in cardiac fibrosis remains unclear. Here, a model of angiotensin II and myocardial infarction (MI)-induced fibrosis was established to explore its role in cardiac fibrosis and its underlying mechanisms. Cardiac fibrosis in the mouse MI model was mitigated by TRIM38 overexpression, but aggravated by its depletion. Consistently, in vitro overexpression or knockdown of TRIM38 ameliorated or aggravated the proliferation and secretion of cardiac fibroblasts (CFs) exposed to fibrotic stimulation, respectively. Mechanistically, TRIM38 suppressed cardiac fibrosis progression by attenuating TAK1/MAPK signaling. Inhibiting TAK1/MAPK signaling with a pharmacological inhibitor greatly reversed the effects of TRIM38 knockdown on CF secretion. Specifically, TRIM38 interacted with and "targeted" TAB2 and TAB3 for degradation, subsequently inhibiting TAK1 phosphorylation and negatively regulating MAPK signaling. These findings can help develop therapeutic strategies to treat and prevent cardiac fibrosis.
    Keywords:  cardiovascular medicine; cell biology; functional aspects of cell biology; pathophysiology
  21. J Microsc. 2022 Aug 19.
      The actin cytoskeleton is the driver of gross ER remodelling and the movement and positioning of other membrane-bound organelles such as Golgi bodies. Rapid ER membrane remodelling is a feature of most plant cells and is important for normal cellular processes, including targeted secretion, immunity and signalling. Modifications to the actin cytoskeleton, through pharmacological agents such as Latrunculin B and phalloidin, or disruption of normal myosin function also affect ER structure and/or dynamics. Here, we investigate the impact of changes in the actin cytoskeleton on structure and dynamics on the ER as well as in return the impact of modified ER structure on the architecture of the actin cytoskeleton. By expressing actin markers that affect actin dynamics, or expressing of ER-shaping proteins that influence ER architecture, we found that the structure of ER-actin networks is closely inter-related; affecting one component is likely to have a direct effect on the other. Therefore, our results indicate that a complicated regulatory machinery and cross-talk between these two structures must exist in plants to co-ordinate the function of ER-actin network during multiple subcellular processes. In addition, when considering organelle structure and dynamics, the choice of actin marker is essential in preventing off-target organelle structure and dynamics modifications. This article is protected by copyright. All rights reserved.
    Keywords:  ActinCb; FABD2; Lifeact; actin; actin marker; endoplasmic reticulum; structure analysis
  22. Cell Rep. 2022 Aug 16. pii: S2211-1247(22)01022-1. [Epub ahead of print]40(7): 111205
      Despite its crucial role in initiation of cytotoxic immune responses, the molecular pathways underlying antigen cross-presentation remain incompletely understood. The mechanism of antigen exit from endocytic compartments into the cytosol is a long-standing matter of controversy, confronting two main models: transfer through specific channels/transporters or rupture of endocytic membranes and leakage of luminal content. By monitoring the occurrence of intracellular damage in conventional dendritic cells (cDCs), we show that cross-presenting cDC1s display more frequent endomembrane injuries and increased recruitment of endosomal sorting complex required for transport (ESCRT)-III, the main repair system for intracellular membranes, relative to cDC2s. Silencing of CHMP2a or CHMP4b, two effector subunits of ESCRT-III, enhances cytosolic antigen export and cross-presentation. This phenotype is partially reversed by chemical inhibition of RIPK3, suggesting that endocytic damage is related to basal activation of the necroptosis pathway. Membrane repair therefore proves crucial in containing antigen export to the cytosol and cross-presentation in cDCs.
    Keywords:  CP: Cell biology; CP: Immunology; ESCRT-III; cross-presentation; dendritic cells; endomembrane disruption; membrane repair; necroptosis
  23. J Mol Biol. 2022 Aug 12. pii: S0022-2836(22)00398-9. [Epub ahead of print] 167790
      Sec secretory proteins are distinguished from cytoplasmic ones by N-terminal signal peptides with multiple roles during post-translational translocation. They contribute to preprotein targeting to the translocase by slowing down folding, bind receptors and trigger secretion. While signal peptides get cleaved after translocation, mature domains traffic further and/or fold into functional states. How signal peptides delay folding temporarily, to keep mature domains translocation-competent, remains unclear. We previously reported that the foldon landscape of the periplasmic prolyl-peptidyl isomerase is altered by its signal peptide and mature domain features. Here, we reveal that the dynamics of signal peptides and mature domains crosstalk. This involves the signal peptide's hydrophobic helical core, the short unstructured connector to the mature domain and the flexible rheostat at the mature domain N-terminus. Through this cis mechanism a signal peptide delays the formation of early initial foldons thus, altering their hierarchy and delaying mature domain folding. We propose that sequence elements outside a protein's native core exploit their structural dynamics to influence the folding landscape.
    Keywords:  Secretion; Signal peptide; foldon; mature domain; non-folding; rheostat
  24. Nucleic Acids Res. 2022 Aug 18. pii: gkac709. [Epub ahead of print]
      Failure to prevent accumulation of the non-canonical nucleotide inosine triphosphate (ITP) by inosine triphosphate pyrophosphatase (ITPase) during nucleotide synthesis results in misincorporation of inosine into RNA and can cause severe and fatal developmental anomalies in humans. While the biochemical activity of ITPase is well understood, the pathogenic basis of ITPase deficiency and the molecular and cellular consequences of ITP misincorporation into RNA remain cryptic. Here, we demonstrate that excess ITP in the nucleotide pool during in vitro transcription results in T7 polymerase-mediated inosine misincorporation in luciferase RNA. In vitro translation of inosine-containing luciferase RNA reduces resulting luciferase activity, which is only partly explained by reduced abundance of the luciferase protein produced. Using Oxford Nanopore Direct RNA sequencing, we reveal inosine misincorporation to be stochastic but biased largely towards misincorporation in place of guanosine, with evidence for misincorporation also in place of cytidine, adenosine and uridine. Inosine misincorporation into RNA is also detected in Itpa-null mouse embryonic heart tissue as an increase in relative variants compared with the wild type using Illumina RNA sequencing. By generating CRISPR/Cas9 rat H9c2 Itpa-null cardiomyoblast cells, we validate a translation defect in cells that accumulate inosine within endogenous RNA. Furthermore, we observe hindered cellular translation of transfected luciferase RNA containing misincorporated inosine in both wild-type and Itpa-null cells. We therefore conclude that inosine misincorporation into RNA perturbs translation, thus providing mechanistic insight linking ITPase deficiency, inosine accumulation and pathogenesis.
  25. Autophagy. 2022 Aug 13. 1-3
      The hallmark of cellular events observed upon macroautophagic/autophagic induction is the conjugation of LC3B, one of the mammalian Atg8 homologs, with phosphatidylethanolamine. This conversion from LC3B-I (an unconjugated form) to LC3B-II (a conjugated form) is essential for phagophore expansion and formation of autophagosomes. Our recent study revealed that LC3B binds to RNAs with a preference for the consensus AAUAAA motif and recruits the CCR4-NOT deadenylase complex. Consequently, LC3B elicits rapid degradation of mRNAs, which we have termed as LC3B-mediated mRNA decay (LMD). LMD requires the conversion of LC3B-I to LC3B-II and occurs before the formation of autolysosomes. Furthermore, we identified PRMT1 mRNA, which encodes a protein that functions as a negative regulator of autophagy, as an LMD substrate. A failure of rapid degradation of PRMT1 mRNA via LMD results in inefficient autophagy. Thus, our study unravels an important role of LC3B in autophagy as an RNA-binding protein for efficient mRNA decay.
    Keywords:  ATG8; CCR4-NOT deadenylase; LC3B; PRMT1; autophagy; mRNA decay
  26. J Biol Chem. 2022 Aug 10. pii: S0021-9258(22)00811-0. [Epub ahead of print] 102368
      During translation initiation, the underlying mechanism by which the eukaryotic initiation factor (eIF) 4E, eIF4A, and eIF4G components of eIF4F coordinate their binding activities to regulate eIF4F binding to mRNA is poorly defined. Here, we used fluorescence anisotropy to generate thermodynamic and kinetic frameworks for the interaction of uncapped RNA with human eIF4F. We demonstrate the binding of eIF4E to an autoinhibitory domain in eIF4G generates a high-affinity binding conformation of the eIF4F complex for RNA. Additionally, we show the nucleotide-bound state of the eIF4A component further regulates uncapped RNA binding by eIF4F, with a four-fold decrease in the equilibrium dissociation constant observed in the presence versus absence of ATP. By monitoring uncapped RNA dissociation in real time, we reveal ATP reduces the dissociation rate constant of RNA for eIF4F by ∼4-orders of magnitude. Thus, release of ATP from eIF4A places eIF4F in a highly dynamic state that has very fast association and dissociation rates from RNA. Monitoring the kinetic framework for eIF4A binding to eIF4G revealed two different rate constants that likely reflect two conformational states of the eIF4F complex. Furthermore, we determined the eIF4G autoinhibitory domain promotes a more stable, less dynamic, eIF4A binding state, which is overcome by eIF4E binding. Overall, our data support a model whereby eIF4E binding to eIF4G/4A stabilizes a high-affinity RNA-binding state of eIF4F and enables eIF4A to adopt a more dynamic interaction with eIF4G. This dynamic conformation may contribute to the ability of eIF4F to rapidly bind and release mRNA during scanning.
    Keywords:  RNA; cooperativity; eIF4A; eIF4E; eIF4F; eIF4G; translation initiation
  27. Antioxid Redox Signal. 2022 Aug 18.
      Aims: Oxidative modifications of cysteine thiols regulate various physiological processes including inflammatory responses. The thioredoxin system plays a key role in thiol redox control. The aim of this study was to characterize the dynamic cysteine proteome of human macrophages upon activation by the prototypical pro-inflammatory agent, bacterial lipopolysaccharide (LPS), and/or perturbation of the thioredoxin system. Results: Herein, we profiled the cellular and redox proteome of human THP-1-derived macrophages during the early phase of LPS activation and/or inhibition of thioredoxin system activity by auranofin, by employing a peptide-centric, resin-assisted capture, redox proteomic workflow. Among 4200 identified cysteines, the oxidation of nearly 10% was selectively affected by LPS or auranofin treatments. Notably, the proteomic analysis uncovered a subset of ~100 thiols, mapped to proteins involved in diverse processes, whose oxidation is antagonistically regulated by LPS and thioredoxin. Compared to the redox proteome, the cellular proteome was largely unchanged, highlighting the importance of redox modifications as a mechanism that allows for rapid modulation of macrophage activities in response to a pro-inflammatory or a pro-oxidant insult. Structural-functional analyses provided mechanistic insights into redox regulation of selected proteins, including the glutathione synthesizing enzyme, glutamate cysteine ligase and the autophagy adaptor SQSTM1/p62, suggesting mechanisms by which macrophages adapt and fine-tune their responses according to a changing inflammatory and redox environment. Innovation: This study provides a rich resource for further characterization of redox mechanisms that regulate macrophage inflammatory activities. Conclusion: The dynamic thiol redox proteome allows macrophages to efficiently respond and adapt to redox and inflammatory challenges.