bims-proteo Biomed News
on Proteostasis
Issue of 2022‒04‒10
35 papers selected by
Eric Chevet

  1. Autophagy. 2022 Apr 07. 1-15
      The mammalian Atg18 ortholog WIPI2 is a key regulator of LC3 lipidation to promote autophagosome biogenesis during nonselective macroautophagy, while its functions in selective autophagy such as mitophagy remain largely unexplored. In this study, we explored the role of WIPI2 in PINK1-PRKN/parkin-mediated mitophagy. First, we found that WIPI2 is recruited to damaged mitochondria upon mitophagy induction. Second, loss of WIPI2 impedes mitochondrial damaging agents-induced mitophagy. Third, at molecular level, WIPI2 binds to and promotes AAA-ATPase VCP/p97 (valosin containing protein) to damaged mitochondria; and WIPI2 depletion blunts the recruitment of VCP to damaged mitochondria, leading to reduction in degradation of outer mitochondrial membrane (OMM) proteins and mitophagy. Finally, WIPI2 is implicated in cell fate decision as cells deficient in WIPI2 are largely resistant to cell death induced by mitochondrial damage. In summary, our study reveals a critical regulatory role of WIPI2 in mitochondrial recruitment of VCP to promote OMM protein degradation and eventual mitophagy.Abbreviations: ATG, autophagy related; CALCOCO2/NDP52, calcium binding and coiled-coil domain 2; CCCP, carbonyl cyanide chlorophenylhydrazone; CYCS, cytochrome c, somatic; HSPD1/HSP60, heat shock protein family D (Hsp60) member 1; IMM, inner mitochondrial membrane; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; NPLOC4, NPL4 homolog, ubiquitin recognition factor; OMM, outer mitochondrial membrane; OPTN, optineurin; PtdIns3P, phosphatidylinositol-3-phosphate; PINK1, PTEN induced kinase 1; PRKN/Parkin, parkin RBR E3 ubiquitin protein ligase; UBXN6/UBXD1, UBX domain protein 6; UFD1, ubiquitin recognition factor in ER associated degradation 1; VCP/p97, valosin containing protein; WIPI2, WD repeat domain, phosphoinositide interacting 2.
    Keywords:  Autophagy; PINK1; PRKN; VCP; WIPI2; cell death; mitophagy
  2. Sci Adv. 2022 Apr 08. 8(14): eabm2094
      Misfolding of secretory proteins in the endoplasmic reticulum (ER) features in many human diseases. In α1-antitrypsin deficiency, the pathogenic Z variant aberrantly assembles into polymers in the hepatocyte ER, leading to cirrhosis. We show that α1-antitrypsin polymers undergo a liquid:solid phase transition, forming a protein matrix that retards mobility of ER proteins by size-dependent molecular filtration. The Z-α1-antitrypsin phase transition is promoted during ER stress by an ATF6-mediated unfolded protein response. Furthermore, the ER chaperone calreticulin promotes Z-α1-antitrypsin solidification and increases protein matrix stiffness. Single-particle tracking reveals that solidification initiates in cells with normal ER morphology, previously assumed to represent a healthy pool. We show that Z-α1-antitrypsin-induced hypersensitivity to ER stress can be explained by immobilization of ER chaperones within the polymer matrix. This previously unidentified mechanism of ER dysfunction provides a template for understanding a diverse group of related proteinopathies and identifies ER chaperones as potential therapeutic targets.
  3. Hum Mol Genet. 2022 Apr 04. pii: ddac076. [Epub ahead of print]
      Amyotrophic Lateral Sclerosis (ALS) is a fatal, late-onset, progressive motor neurodegenerative disorder. A key pathological feature of the disease is the presence of heavily ubiquitinated protein inclusions. Both the Unfolded Protein Response (UPR) and the Ubiquitin Proteasome System (UPS) appear significantly impaired in patients and animal models of ALS. We have studied cellular and molecular mechanisms involved in ALS using a vesicle-associated membrane protein-associated protein B (VAPB/ALS8) Drosophila model (Moustaqim-Barrette et al., 2014), which mimics many systemic aspects of the human disease. Here, we show that VAPB, located on the cytoplasmic face of the ER membrane, interacts with Caspar, an ortholog of human fas associated factor 1 (FAF1). Caspar, in turn, interacts with transitional endoplasmic reticulum ATPase (TER94), a fly ortholog of ALS14 (VCP/p97, Valosin-containing protein). Caspar overexpression in the glia extends lifespan and also slows the progression of motor dysfunction in the ALS8 disease model, a phenomenon that we ascribe to its ability to restrain age-dependant inflammation, which is modulated by Relish/NFκB signalling. Caspar binds to VAPB via an FFAT motif, and we find that Caspar's ability to negatively regulate NFκB signalling is not dependant on the VAPB:Caspar interaction. We hypothesize that Caspar is a key molecule in the pathogenesis of ALS. The VAPB:Caspar:TER94 complex appears to be a candidate for regulating both protein homeostasis and NFκB signalling, with our study highlighting a role for Caspar in glial inflammation. We project human FAF1 as an important protein target to alleviate the progression of motor neuron disease.
  4. J Proteome Res. 2022 Apr 07.
      Eukaryotic cells respond to heat shock through several regulatory processes including upregulation of stress responsive chaperones and reversible shutdown of cellular activities through formation of protein assemblies. However, the underlying regulatory mechanisms of the recovery of these heat-induced protein assemblies remain largely elusive. Here, we measured the proteome abundance and solubility changes during recovery from heat shock in the mouse Neuro2a cell line. We found that prefoldins and translation machinery are rapidly down-regulated as the first step in the heat shock response. Analysis of proteome solubility reveals that a rapid mobilization of protein quality control machineries, along with changes in cellular energy metabolism, translational activity, and actin cytoskeleton are fundamental to the early stress responses. In contrast, longer term adaptation to stress involves renewal of core cellular components. Inhibition of the Hsp70 family, pivotal for the heat shock response, selectively and negatively affects the ribosomal machinery and delays the solubility recovery of many nuclear proteins. ProteomeXchange: PXD030069.
    Keywords:  chaperone; disaggregation; heat shock; heat shock protein 70; protein solubility; quantitative proteomics; recovery
  5. Cell. 2022 Mar 27. pii: S0092-8674(22)00265-3. [Epub ahead of print]
      Protein aggregation is a hallmark of multiple human pathologies. Autophagy selectively degrades protein aggregates via aggrephagy. How selectivity is achieved has been elusive. Here, we identify the chaperonin subunit CCT2 as an autophagy receptor regulating the clearance of aggregation-prone proteins in the cell and the mouse brain. CCT2 associates with aggregation-prone proteins independent of cargo ubiquitination and interacts with autophagosome marker ATG8s through a non-classical VLIR motif. In addition, CCT2 regulates aggrephagy independently of the ubiquitin-binding receptors (P62, NBR1, and TAX1BP1) or chaperone-mediated autophagy. Unlike P62, NBR1, and TAX1BP1, which facilitate the clearance of protein condensates with liquidity, CCT2 specifically promotes the autophagic degradation of protein aggregates with little liquidity (solid aggregates). Furthermore, aggregation-prone protein accumulation induces the functional switch of CCT2 from a chaperone subunit to an autophagy receptor by promoting CCT2 monomer formation, which exposes the VLIR to ATG8s interaction and, therefore, enables the autophagic function.
    Keywords:  CCT2; FUS; GABARAP; Huntington’s disease; LC3; NBR1; P62; SOD1; TAX1BP1; TRiC; aggrephagy; autophagy; chaperone; chaperonin; huntingtin; neurodegeneration; phase separation; protein aggregates; protein aggregation; tau
  6. Proc Natl Acad Sci U S A. 2022 Apr 12. 119(15): e2104309119
      SignificanceThe endoplasmic reticulum (ER) and the microtubule (MT) cytoskeleton form a coextensive, dynamic system that pervades eukaryotic cells. The shape of the ER is generated by a set of evolutionarily conserved membrane proteins that are able to control ER morphology and dynamics independently of MTs. Here we uncover that the molecular machinery that determines ER network dynamics can influence the subcellular distribution of MTs. We show that active control of local ER tubule junction density by ER tethering and fusion is important for the spatial organization of the combined ER-MT system. Our work suggests that cells might alter ER junction dynamics to drive formation of MT bundles, which are important structures, e.g., in migrating cells or in neuronal axons.
    Keywords:  cellular organization; endoplasmic reticulum; microtubule bundles; microtubules
  7. PLoS One. 2022 ;17(4): e0266478
      Acute myeloid leukemia (AML) is a heterogeneous malignancy characterized by the accumulation of undifferentiated white blood cells (blasts) in the bone marrow. Valosin-containing protein (VCP) is an abundant molecular chaperone that extracts ubiquitylated substrates from protein complexes and cellular compartments prior to their degradation by the proteasome. We found that treatment of AML cell lines with the VCP inhibitor CB-5083 leads to an accumulation of ubiquitylated proteins, activation of unfolded protein response (UPR) and apoptosis. Using quantitative mass spectrometry-based proteomics we assessed the effects of VCP inhibition on the cellular ubiquitin-modified proteome. We could further show that CB-5083 decreases the survival of the AML cell lines THP-1 and MV4-11 in a concentration-dependent manner, and acts synergistically with the antimetabolite cytarabine and the BH3-mimetic venetoclax. Finally, we showed that prolonged treatment of AML cells with CB-5083 leads to development of resistance mediated by mutations in VCP. Taken together, inhibition of VCP leads to a lethal unfolded protein response in AML cells and might be a relevant therapeutic strategy for treatment of AML, particularly when combined with other drugs. The toxicity and development of resistance possibly limit the utility of VCP inhibitors and have to be further explored in animal models and clinical trials.
  8. J Immunol. 2022 Apr 04. pii: ji2100466. [Epub ahead of print]
      The mechanism regulating the life span of short-lived plasma cells (SLPCs) remains poorly understood. Here we demonstrated that the EP4-mediated activation of AKT by PGE2 was required for the proper control of inositol-requiring transmembrane kinase endoribonuclease-1α (IRE1α) hyperactivation and hence the endoplasmic reticulum (ER) homeostasis in IgM-producing SLPCs. Disruption of the PGE2-EP4-AKT signaling pathway resulted in IRE1α-induced activation of JNK, leading to accelerated death of SLPCs. Consequently, Ptger4-deficient mice (C57BL/6) exhibited a markedly impaired IgM response to T-independent Ags and increased susceptibility to Streptococcus pneumoniae infection. This study reveals a highly selective impact of the PGE2-EP4 signal on the humoral immunity and provides a link between ER stress response and the life span of SLPCs.
  9. Nat Commun. 2022 Apr 05. 13(1): 1819
      Post-translational protein modifications by ubiquitin and ubiquitin-like modifiers regulate many major pathways in the cell. These modifications can be reversed by de-ubiquitinating enzymes such as ubiquitin-specific proteases (USPs). Proteolytic activity towards ubiquitin-modified substrates is common to all USP family members except for USPL1, which shows a unique preference for the ubiquitin-like modifier SUMO. Here, we present the crystal structure of USPL1 bound to SUMO2, defining the key structural elements for the unusual deSUMOylase activity of USPL1. We identify specific contacts between SUMO2 and the USPL1 subdomains, including a unique hydrogen bond network of the SUMO2 C-terminal tail. In addition, we find that USPL1 lacks major structural elements present in all canonical USPs members such as the so-called blocking loops, which facilitates SUMO binding. Our data give insight into how a structural protein scaffold designed to bind ubiquitin has evolved to bind SUMO, providing an example of divergent evolution in the USP family.
  10. Cell Death Dis. 2022 Apr 07. 13(4): 316
      ULK1 is crucial for initiating autophagosome formation and its activity is tightly regulated by post-translational modifications and protein-protein interactions. In the present study, we demonstrate that TMEM189 (Transmembrane protein 189), also known as plasmanylethanolamine desaturase 1 (PEDS1), negatively regulates the proteostasis of ULK1 and autophagy activity. In TMEM189-overexpressed cells, the formation of autophagesome is impaired, while TMEM189 knockdown increases cell autophagy. Further investigation reveals that TMEM189 interacts with and increases the instability of ULK1, as well as decreases its kinase activities. The TMEM189 N-terminal domain is required for the interaction with ULK1. Additionally, TMEM189 overexpression can disrupt the interaction between ULK1 and TRAF6, profoundly impairs K63-linked polyubiquitination of ULK1 and self-association, leading to the decrease of ULK1 stability. Moreover, in vitro and in vivo experiments suggest that TMEM189 deficiency results in the inhibition of tumorigenicity of gastric cancer. Our findings provide a new insight into the molecular regulation of autophagy and laboratory evidence for investigating the physiological and pathological roles of TMEM189.
  11. Nat Commun. 2022 Apr 05. 13(1): 1821
      BiP co-chaperones ERdj4, ERdj5, and GRP170 associate in cells with peptides predicted to be aggregation prone. Here, extending these findings to a full-length protein, we examine two Interstitial Lung Disease-associated mutants (ILD) of surfactant protein C (SP-C). The TANGO algorithm, which identifies sequences prone to formation of β strand aggregates, found three such regions in SP-C: the N-terminal transmembrane (TM) domain and two sites in the intermolecular chaperone BRICHOS domain. We show the ILD mutants disrupt di-sulfide bond formation in the BRICHOS domain and expose the aggregation-prone peptides leading to binding of ERdj4, ERdj5, and GRP170. The destabilized mutant BRICHOS domain fails to properly insert its TM region in the ER membrane, exposing part of the N-terminal TM domain site. Our studies with ILD-associated mutant proteins provide insights into the specificity of ERdj4, ERdj5, and GRP170, identify context-dependent differences in their binding, and reveal molecular consequences of disease-associated mutants on folding.
  12. J Biochem. 2022 Apr 08. pii: mvac031. [Epub ahead of print]
      Ubiquitination is a post-translational modification system essential for regulating a wide variety of biological processes in eukaryotes. Ubiquitin (Ub) itself undergoes post-translational modifications, including ubiquitination. All seven lysine residues and one N-terminal amino group of Ub can act as acceptors for further ubiquitination, producing eight types of Ub chains. Ub chains of different linkage types have different cellular functions and are referred to as the 'ubiquitin code'. Decoder molecules that contain linkage-specific Ub-binding domains (UBDs) recognize the Ub chains to regulate different cellular functions. On the other hand, deubiquitinases (DUBs) cleave Ub chains to reverse ubiquitin signals. This review discusses the molecular mechanisms of linkage-specific recognitions of Ub chains by UBDs and DUBs, which have been revealed by structural studies.
    Keywords:  deubiquitinase; linkage specificity; ubiquitin binding domain; ubiquitin chain; ubiquitin code
  13. Sci Adv. 2022 Apr 08. 8(14): eabl5765
      Ubiquitination-mediated protein degradation in both the 26S proteasome and vacuole is an important process in abscisic acid (ABA) signaling. However, the role of deubiquitination in this process remains elusive. Here, we demonstrate that two deubiquitinating enzymes (DUBs), ubiquitin-specific protease 12 (UBP12) and UBP13, modulate ABA signaling and drought tolerance by deubiquitinating and stabilizing the endosomal sorting complex required for transport-I (ESCRT-I) component vacuolar protein sorting 23A (VPS23A) and thereby affect the stability of ABA receptors in Arabidopsis thaliana. Genetic analysis showed that VPS23A overexpression could rescue the ABA hypersensitive and drought tolerance phenotypes of ubp12-2w or ubp13-1. In addition to the direct regulation of VPS23A, we found that UBP12 and UBP13 also stabilized the E3 ligase XB3 ortholog 5 in A. thaliana (XBAT35.2) in response to ABA treatment. Hence, we demonstrated that UBP12 and UBP13 are previously unidentified rheostatic regulators of ABA signaling and revealed a mechanism by which deubiquitination precisely monitors the XBAT35/VPS23A ubiquitination module in the ABA response.
  14. Genetics. 2022 Apr 04. pii: iyac051. [Epub ahead of print]
      The ubiquitin-mediated degradation of oocyte translational regulatory proteins is a conserved feature of the oocyte-to-embryo transition (OET). In the nematode Caenorhabditis elegans, multiple translational regulatory proteins, including the TRIM-NHL RNA-binding protein LIN-41/Trim71 and the Pumilio-family RNA-binding proteins PUF-3 and PUF-11, are degraded during the OET. Degradation of each protein requires activation of the M-phase cyclin-dependent kinase CDK-1, is largely complete by the end of the first meiotic division and does not require the anaphase promoting complex (APC). However, only LIN-41 degradation requires the F-box protein SEL-10/FBW7/Cdc4p, the substrate recognition subunit of an SCF-type E3 ubiquitin ligase. This finding suggests that PUF-3 and PUF-11, which localize to LIN-41-containing ribonucleoprotein particles (RNPs), are independently degraded through the action of other factors and that the oocyte RNPs are disassembled in a concerted fashion during the OET. We develop and test the hypothesis that PUF-3 and PUF-11 are targeted for degradation by the proteasome-associated HECT-type ubiquitin ligase ETC-1/UBE3C/Hul5, which is broadly expressed in C. elegans. We find that several GFP-tagged fusion proteins that are degraded during the OET, including fusions with PUF-3, PUF-11, LIN-41, IFY-1/Securin and CYB-1/Cyclin B, are incompletely degraded when ETC-1 function is compromised. However, it is the fused GFP moiety that appears to be the critical determinant of this proteolysis defect. These findings are consistent with a conserved role for ETC-1 in promoting proteasome processivity and suggest that proteasomal processivity is an important element of the OET during which many key oocyte regulatory proteins are rapidly targeted for degradation.
    Keywords:  RNA-binding proteins; oocyte meiotic maturation; oocyte-to-embryo transition; translational regulation; ubiquitin-mediated protein degradation
  15. STAR Protoc. 2022 Jun 17. 3(2): 101247
      The neuroprotective E3-ubiquitin ligase CHIP is linked to healthy aging. Here, we present a protocol using a patient-derived iPSC line with a triplication of the α-synuclein gene to produce gene-edited cells isogenic for CHIP. We describe iPSC differentiation into cortical neurons and their identity validation. We then detail mass spectrometry-based approaches (SWATH-MS) to identify dominant changes in the steady state proteome generated by loss of CHIP function. This protocol can be adapted to other proteins that impact proteostasis in neurons. For complete details on the use and execution of this protocol, please refer to Dias et al. (2021).
    Keywords:  CRISPR; Cell Biology; Cell Differentiation; Cell culture; Mass Spectrometry; Neuroscience; Proteomics; Stem Cells
  16. Proc Natl Acad Sci U S A. 2022 Apr 12. 119(15): e2119531119
      SignificanceThe viral RNA sensor RIG-I initiates the antiviral innate immune response by activating a signaling cascade that induces interferon. Activation of the RIG-I signaling pathway is highly regulated to quickly mount a protective immune response while preventing dysregulation that can lead to excessive inflammation or autoimmune disorders. Here, we characterize one such mechanism of regulation. We describe that UFL1, an E3 ligase for the ubiquitin-like modifier conjugation system called ufmylation, is important to promote RIG-I signaling. Using molecular approaches, we show that ufmylation promotes RIG-I interaction with the membrane-targeting protein 14-3-3ε. As such, ufmylation positively regulates RIG-I recruitment to its signaling adaptor protein MAVS for induction of interferon in response to RNA virus infection.
    Keywords:  RLR signaling; mitochondrial-associated ER membranes; retinoic acid-inducible gene I; ubiquitin-like modifications
  17. Mol Biol Cell. 2022 Apr 07. mbcE21110578
      Pharmacological chaperones represent a class of therapeutic compounds for treating protein misfolding diseases. One of the most prominent examples is the FDA-approved pharmacological chaperone lumacaftor (VX-809), which has transformed cystic fibrosis (CF) therapy. CF is a fatal disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). VX-809 corrects folding of F508del CFTR, the most common patient mutation, yet F508del exhibits only mild VX-809 response. In contrast, rarer mutations P67L and L206W are hyper-responsive to VX-809, while G85E is non-responsive. Despite the clinical success of VX-809, the mechanistic origin for the distinct susceptibility of mutants remains unclear. Here, we use interactomics to characterize the impact of VX-809 on proteostasis interactions of P67L and L206W and compare these to F508del and G85E. We determine hyper-responsive mutations P67L and L206W exhibit decreased interactions with proteasomal, and autophagy degradation machinery compared to F508del and G85E. We then show inhibiting the proteasome attenuates P67L and L206W VX-809 response. Our data suggests a previously unidentified but required role for protein degradation in VX-809 correction. Furthermore, we present an approach for identifying proteostasis characteristics of mutant-specific therapeutic response to pharmacological chaperones.
  18. J Cell Biol. 2022 Jun 06. pii: e202110044. [Epub ahead of print]221(6):
      Membrane contact sites between organelles are organized by protein bridges. Among the components of these contacts, the VAP family comprises ER-anchored proteins, such as MOSPD2, that function as major ER-organelle tethers. MOSPD2 distinguishes itself from the other members of the VAP family by the presence of a CRAL-TRIO domain. In this study, we show that MOSPD2 forms ER-lipid droplet (LD) contacts, thanks to its CRAL-TRIO domain. MOSPD2 ensures the attachment of the ER to LDs through a direct protein-membrane interaction. The attachment mechanism involves an amphipathic helix that has an affinity for lipid packing defects present at the surface of LDs. Remarkably, the absence of MOSPD2 markedly disturbs the assembly of lipid droplets. These data show that MOSPD2, in addition to being a general ER receptor for inter-organelle contacts, possesses an additional tethering activity and is specifically implicated in the biology of LDs via its CRAL-TRIO domain.
  19. Nat Commun. 2022 Apr 08. 13(1): 1923
      The cytokine IFNγ differentially impacts on tumors upon immune checkpoint blockade (ICB). Despite our understanding of downstream signaling events, less is known about regulation of its receptor (IFNγ-R1). With an unbiased genome-wide CRISPR/Cas9 screen for critical regulators of IFNγ-R1 cell surface abundance, we identify STUB1 as an E3 ubiquitin ligase for IFNγ-R1 in complex with its signal-relaying kinase JAK1. STUB1 mediates ubiquitination-dependent proteasomal degradation of IFNγ-R1/JAK1 complex through IFNγ-R1K285 and JAK1K249. Conversely, STUB1 inactivation amplifies IFNγ signaling, sensitizing tumor cells to cytotoxic T cells in vitro. This is corroborated by an anticorrelation between STUB1 expression and IFNγ response in ICB-treated patients. Consistent with the context-dependent effects of IFNγ in vivo, anti-PD-1 response is increased in heterogenous tumors comprising both wildtype and STUB1-deficient cells, but not full STUB1 knockout tumors. These results uncover STUB1 as a critical regulator of IFNγ-R1, and highlight the context-dependency of STUB1-regulated IFNγ signaling for ICB outcome.
  20. Acta Neuropathol. 2022 Apr 07.
      Selective neuronal vulnerability to protein aggregation is found in many neurodegenerative diseases including Alzheimer's disease (AD). Understanding the molecular origins of this selective vulnerability is, therefore, of fundamental importance. Tau protein aggregates have been found in Wolframin (WFS1)-expressing excitatory neurons in the entorhinal cortex, one of the earliest affected regions in AD. The role of WFS1 in Tauopathies and its levels in tau pathology-associated neurodegeneration, however, is largely unknown. Here we report that WFS1 deficiency is associated with increased tau pathology and neurodegeneration, whereas overexpression of WFS1 reduces those changes. We also find that WFS1 interacts with tau protein and controls the susceptibility to tau pathology. Furthermore, chronic ER stress and autophagy-lysosome pathway (ALP)-associated genes are enriched in WFS1-high excitatory neurons in human AD at early Braak stages. The protein levels of ER stress and autophagy-lysosome pathway (ALP)-associated proteins are changed in tau transgenic mice with WFS1 deficiency, while overexpression of WFS1 reverses those changes. This work demonstrates a possible role for WFS1 in the regulation of tau pathology and neurodegeneration via chronic ER stress and the downstream ALP. Our findings provide insights into mechanisms that underpin selective neuronal vulnerability, and for developing new therapeutics to protect vulnerable neurons in AD.
    Keywords:  Alzheimer’s disease; Autophagy-lysosome pathway; ER stress; Entorhinal cortex; Neurodegeneration; Neuronal vulnerability; Tau pathology; WFS1; Wolframin
  21. Sci Adv. 2022 Apr 08. 8(14): eabm8501
      In contrast to prokaryotes wherein GUG and UUG are permissive start codons, initiation frequencies from non-AUG codons are generally low in eukaryotes, with CUG being considered as strongest. Here, we report that combined 5-cytosine methylation (5mC) and pseudouridylation (Ψ) of near-cognate non-AUG start codons convert GUG and UUG initiation strongly favored over CUG initiation in eukaryotic translation under a certain context. This prokaryotic-like preference is attributed to enhanced NUG initiation by Ψ in the second base and reduced CUG initiation by 5mC in the first base. Molecular dynamics simulation analysis of tRNAiMet anticodon base pairing to the modified codons demonstrates that Ψ universally raises the affinity of codon:anticodon pairing within the ribosomal preinitiation complex through partially mitigating discrimination against non-AUG codons imposed by eukaryotic initiation factor 1. We propose that translational control by chemical modifications of start codon bases can offer a new layer of proteome diversity regulation and therapeutic mRNA technology.
  22. Proc Natl Acad Sci U S A. 2022 Apr 12. 119(15): e2119076119
      SignificanceOne of the key unresolved questions in the field of molecular chaperones is how they can actively unfold proteins. In this study, we discovered that the Hsp70/Hsp40 chaperone system completely unfolds a native soluble substrate protein, the ligand-binding domain of the glucocorticoid receptor, in a concerted action. Our high-resolution optical tweezers data show in real time how the substrate is attacked by the chaperone machinery. As soon as the hormone has left the binding pocket, up to five Hsp70/Hsp40 complexes bind and unfold the protein in a stepwise manner. This finding constitutes direct evidence that the chaperone machinery can bind to the folded core of the receptor, thus providing a mechanism for Hsp70-induced protein unfolding.
    Keywords:  Hsp70; glucocorticoid receptor; optical tweezers; protein folding; single-molecule
  23. Chem Soc Rev. 2022 Apr 08.
      In recent years, proteolysis-targeting chimeras (PROTACs), capable of achieving targeted protein degradation, have proven their great therapeutic potential and usefulness as molecular biology tools. These heterobifunctional compounds are comprised of a protein-targeting ligand, an appropriate linker, and a ligand binding to the E3 ligase of choice. A successful PROTAC induces the formation of a ternary complex, leading to the E3 ligase-mediated ubiquitination of the targeted protein and its proteasomal degradation. In over 20 years since the concept was first demonstrated, the field has grown substantially, mainly due to the advancements in the discovery of non-peptidic E3 ligase ligands. Development of small-molecule E3 binders with favourable physicochemical profiles aided the design of PROTACs, which are known for breaking the rules of established guidelines for discovering small molecules. Synthetic accessibility of the ligands and numerous successful applications led to the prevalent use of cereblon and von Hippel-Lindau as the hijacked E3 ligase. However, the pool of over 600 human E3 ligases is full of untapped potential, which is why expanding the artillery of E3 ligands could contribute to broadening the scope of targeted protein degradation. In this comprehensive review, we focus on the chemistry aspect of the PROTAC design process by providing an overview of liganded E3 ligases, their chemistries, appropriate derivatisation, and synthetic approaches towards their incorporation into heterobifunctional degraders. By covering syntheses of both established and underexploited E3 ligases, this review can serve as a chemistry blueprint for PROTAC researchers during their future ventures into the complex field of targeted protein degradation.
  24. Autophagy. 2022 Apr 07. 1-19
      MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) is widely used as marker of autophagic compartments at different stages of maturation. Electron microscopy (EM) combined with immunolabeling is the only technique that can reveal the ultrastructural identity of LC3-labeled compartments. However, immuno-EM of endogenous LC3 proteins has proven difficult. Here, we test a panel of commercially available antibodies and apply different labeling conditions to present an optimized procedure for LC3 immuno-EM. Using ultrathin cryosections and protein A-colloidal gold or gold enhancement labeling, we localize endogenous LC3 in starved cells or tissues in the presence or absence of the proton pump inhibitor bafilomycin A1. We localize LC3 to early and late stage autophagic compartments that can be classified by their morphology. By on-section correlative light-electron microscopy (CLEM) we show that comparable fluorescent LC3-positive puncta can represent different autophagic intermediates. We also show that our approach is sufficiently robust to label endogenous LC3 simultaneously with other lysosomal and autophagy markers, LAMP1 or SQSTM1/p62, and can be used for quantitative approaches. Thus, we demonstrate that bafilomycin A1 treatment from 2.5 up to 24 h does not inhibit fusion between autophagosomes and lysosomes, but leads to the accumulation of LC3-positive material inside autolysosomes. Together, this is the first study presenting an extensive overview of endogenous LC3 localization at ultrastructural resolution without the need for cell permeabilization and using a commercially available antibody. This provides researchers with a tool to study canonical and non-canonical roles of LC3 in native conditions.
    Keywords:  Autophagy; CLEM; LC3; bafilomycin A1; immuno-electron microscopy; ultrathin cryosections
  25. Biochemistry. 2022 Apr 05.
      The physiological consequences of varying in vivo CO2 levels point to a general mechanism for CO2 to influence cellular homeostasis beyond regulating pH. Aside from a few instances where CO2 has been observed to cause post-translational protein modification, by forming long-lived carbamates, little is known about how transitory and ubiquitous carbamylation events could induce a physiological response. Ubiquitin is a versatile protein involved in a multitude of cellular signaling pathways as polymeric chains of various lengths formed through one of the seven lysines or N-terminal amine. Unique polyubiquitin (polyUb) compositions present recognition signals for specific ubiquitin-receptors which enables this one protein to be involved in many different cellular processes. Advances in proteomic methods have allowed the capture and identification of protein carbamates in vivo, and Ub was found carbamylated at lysines K48 and K33. This was shown to negatively regulate ubiquitin-mediated signaling by inhibiting polyUb chain formation. Here, we expand upon these observations by characterizing the carbamylation susceptibility for all Ub amines simultaneously. Using NMR methods which directly probe 15N resonances, we determined carbamylation rates under various environmental conditions and related them to the intrinsic pKas. Our results show that the relatively low pKas for half of the Ub amines are correlated with enhanced susceptibility to carbamylation under physiological conditions. Two of these carbamylated amines, not observed by chemical capture, appear to be physiologically relevant post-translational modifications. These findings point to a mechanism for varying the levels of CO2 due to intracellular localization, cellular stresses, and metabolism to affect certain polyUb-mediated signaling pathways.
  26. Autophagy. 2022 Apr 05.
      High levels of reactive oxygen species (ROS) result in oxidative stress, which damages cells and leads to the development of many diseases. Macroautophagy/autophagy plays an important role in protecting cells from diverse stress stimuli including oxidative stress. However, the molecular mechanisms of autophagy activation in response to oxidative stress remain largely unclear. In this study, we showed that TRAF6 mediates oxidative stress-induced ATG9A ubiquitination at two C-terminal lysine residues (K581 and K838). ATG9A ubiquitination promotes its association with BECN1, BECN1-PIK3C3/VPS34-UVRAG complex assembly and PIK3C3/VPS34 activation, thereby activating autophagy and endocytic trafficking. We also identified TNFAIP3/A20 as a negative regulator of oxidative-induced autophagy by counteracting TRAF6-mediated ATG9A ubiquitination. Moreover, ATG9A depletion attenuates LPS-induced autophagy and causes aberrant TLR4 signaling and inflammatory responses. Our findings revealed a critical role of ATG9A ubiquitination in oxidative-induced autophagy, endocytic trafficking and innate immunity.
    Keywords:  ATG9A; BECN1; PIK3C3/VPS34; TNFAIP3/A20; TRAF6; UVRAG; autophagy; endocytic trafficking; oxidative stress; ubiquitination
  27. Autophagy. 2022 Apr 06. 1-18
      At a time when complex diseases affect globally 280 million people and claim 14 million lives every year, there is an urgent need to rapidly increase our knowledge into their underlying etiologies. Though critical in identifying the people at risk, the causal environmental factors (microbiome and/or pollutants) and the affected pathophysiological mechanisms are not well understood. Herein, we consider the variations of autophagy-related (ATG) genes at the heart of mechanisms of increased susceptibility to environmental stress. A comprehensive autophagy genomic resource is presented with 263 single nucleotide polymorphisms (SNPs) for 69 autophagy-related genes associated with 117 autoimmune, inflammatory, infectious, cardiovascular, neurological, respiratory, and endocrine diseases. We thus propose the term 'autophagopathies' to group together a class of complex human diseases the etiology of which lies in a genetic defect of the autophagy machinery, whether directly related or not to an abnormal flux in autophagy, LC3-associated phagocytosis, or any associated trafficking. The future of precision medicine for common diseases will lie in our ability to exploit these ATG SNP x environment relationships to develop new polygenetic risk scores, new management guidelines, and optimal therapies for afflicted patients.Abbreviations: ATG, autophagy-related; ALS-FTD, amyotrophic lateral sclerosis-frontotemporal dementia; ccRCC, clear cell renal cell carcinoma; CD, Crohn disease; COPD, chronic obstructive pulmonary disease; eQTL, expression quantitative trait loci; HCC, hepatocellular carcinoma; HNSCC, head and neck squamous cell carcinoma; GTEx, genotype-tissue expression; GWAS, genome-wide association studies; LAP, LC3-associated phagocytosis; LC3-II, phosphatidylethanolamine conjugated form of LC3; LD, linkage disequilibrium; LUAD, lung adenocarcinoma; MAF, minor allele frequency; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NSCLC, non-small cell lung cancer; OS, overall survival; PtdIns3K CIII, class III phosphatidylinositol 3 kinase; PtdIns3P, phosphatidylinositol-3-phosphate; SLE, systemic lupus erythematosus; SNPs, single-nucleotide polymorphisms; mQTL, methylation quantitative trait loci; ULK, unc-51 like autophagy activating kinase; UTRs, untranslated regions; WHO, World Health Organization.
    Keywords:  Autophagy; cancers; diseases; eQTL; pollutants/exposomics; polymorphism; prognosis; risk; susceptibility; theragnosis
  28. Nature. 2022 Apr 08.
    Indonesia Schizophrenia Consortium
      Schizophrenia has a heritability of 60-80%1, much of which is attributable to common risk alleles. Here, in a two-stage genome-wide association study of up to 76,755 individuals with schizophrenia and 243,649 control individuals, we report common variant associations at 287 distinct genomic loci. Associations were concentrated in genes that are expressed in excitatory and inhibitory neurons of the central nervous system, but not in other tissues or cell types. Using fine-mapping and functional genomic data, we identify 120 genes (106 protein-coding) that are likely to underpin associations at some of these loci, including 16 genes with credible causal non-synonymous or untranslated region variation. We also implicate fundamental processes related to neuronal function, including synaptic organization, differentiation and transmission. Fine-mapped candidates were enriched for genes associated with rare disruptive coding variants in people with schizophrenia, including the glutamate receptor subunit GRIN2A and transcription factor SP4, and were also enriched for genes implicated by such variants in neurodevelopmental disorders. We identify biological processes relevant to schizophrenia pathophysiology; show convergence of common and rare variant associations in schizophrenia and neurodevelopmental disorders; and provide a resource of prioritized genes and variants to advance mechanistic studies.
  29. EMBO Mol Med. 2022 Apr 04. e13952
      Amyloid beta 42 (Abeta42) is the principal trigger of neurodegeneration during Alzheimer's disease (AD). However, the etiology of its noxious cellular effects remains elusive. In a combinatory genetic and proteomic approach using a yeast model to study aspects of intracellular Abeta42 toxicity, we here identify the HSP40 family member Ydj1, the yeast orthologue of human DnaJA1, as a crucial factor in Abeta42-mediated cell death. We demonstrate that Ydj1/DnaJA1 physically interacts with Abeta42 (in yeast and mouse), stabilizes Abeta42 oligomers, and mediates their translocation to mitochondria. Consequently, deletion of YDJ1 strongly reduces co-purification of Abeta42 with mitochondria and prevents Abeta42-induced mitochondria-dependent cell death. Consistently, purified DnaJ chaperone delays Abeta42 fibrillization in vitro, and heterologous expression of human DnaJA1 induces formation of Abeta42 oligomers and their deleterious translocation to mitochondria in vivo. Finally, downregulation of the Ydj1 fly homologue, Droj2, improves stress resistance, mitochondrial morphology, and memory performance in a Drosophila melanogaster AD model. These data reveal an unexpected and detrimental role for specific HSP40s in promoting hallmarks of Abeta42 toxicity.
    Keywords:  Alzheimer’s disease; HSP40; amyloid beta 42; heat shock proteins; oligomers
  30. J Clin Invest. 2022 Apr 05. pii: e151895. [Epub ahead of print]
      Enhanced de novo lipogenesis mediated by sterol regulatory element-binding proteins (SREBPs) is thought to be involved in nonalcoholic steatohepatitis (NASH) pathogenesis. In this study, we assessed the impact of SREBP inhibition on NASH and liver cancer development in murine models. Unexpectedly, SREBP inhibition via deletion of the SREBP cleavage-activating protein (SCAP) in the liver exacerbated liver injury, fibrosis, and carcinogenesis, despite markedly reduced hepatic steatosis. These phenotypes were ameliorated by restoring SREBP function. Transcriptome and lipidome analyses revealed that SCAP-SREBP pathway inhibition altered the fatty acid (FA) composition of phosphatidylcholines due to both impaired FA synthesis and disorganized FA incorporation into phosphatidylcholine via lysophosphatidylcholine acyltransferase 3 (LPCAT3) downregulation, which led to endoplasmic reticulum (ER) stress and hepatocyte injury. Supplementation of phosphatidylcholines significantly improved liver injury and ER stress induced by SCAP deletion. The activity of SCAP-SREBP-LPCAT3 axis was found inversely associated with liver fibrosis severity in human NASH. SREBP inhibition also cooperated with impaired autophagy to trigger liver injury. Thus, excessively strong and broad lipogenesis inhibition was counterproductive for NASH therapy, which will have important clinical implications in NASH treatment.
    Keywords:  Gastroenterology; Hepatitis
  31. Sci Adv. 2022 Apr 08. 8(14): eabc9061
      The circadian clock controls many aspects of physiology, but it remains undescribed whether extracellular vesicles (EVs), including exosomes, involved in cell-cell communications between tissues are regulated in a circadian pattern. We demonstrate a 24-hour rhythmic abundance of individual proteins in small EVs using liquid chromatography-mass spectrometry in circadian-synchronized tendon fibroblasts. Furthermore, the release of small EVs enriched in RNA binding proteins was temporally separated from those enriched in cytoskeletal and matrix proteins, which peaked during the end of the light phase. Last, we targeted the protein sorting mechanism in the exosome biogenesis pathway and established (by knockdown of circadian-regulated flotillin-1) that matrix metalloproteinase 14 abundance in tendon fibroblast small EVs is under flotillin-1 regulation. In conclusion, we have identified proteomic time signatures for small EVs released by tendon fibroblasts, which supports the view that the circadian clock regulates protein cargo in EVs involved in cell-cell cross-talk.
  32. EMBO Rep. 2022 Apr 05. e54041
      Much cell-to-cell communication is facilitated by cell surface receptor tyrosine kinases (RTKs). These proteins phosphorylate their downstream cytoplasmic substrates in response to stimuli such as growth factors. Despite their central roles, the functions of many RTKs are still poorly understood. To resolve the lack of systematic knowledge, we apply three complementary methods to map the molecular context and substrate profiles of RTKs. We use affinity purification coupled to mass spectrometry (AP-MS) to characterize stable binding partners and RTK-protein complexes, proximity-dependent biotin identification (BioID) to identify transient and proximal interactions, and an in vitro kinase assay to identify RTK substrates. To identify how kinase interactions depend on kinase activity, we also use kinase-deficient mutants. Our data represent a comprehensive, systemic mapping of RTK interactions and substrates. This resource adds information regarding well-studied RTKs, offers insights into the functions of less well-studied RTKs, and highlights RTK-RTK interactions and shared signaling pathways.
    Keywords:  RTK; interaction proteomics; phosphoproteomics; receptor tyrosine kinase; systems biology
  33. J Clin Invest. 2022 Apr 07. pii: e154217. [Epub ahead of print]
      Subendothelial macrophage internalization of modified lipids and foam cell formation are hallmarks of atherosclerosis. Deubiquitinating enzymes (DUBs) are involved in various cellular activities; however, their role in foam cell formation is not fully understood. Here, using a loss-of-function lipid accumulation screening, we identified ubiquitin-specific peptidase 9 X-linked (USP9X) as a factor that suppressed lipid uptake in macrophages. We found that USP9X expression in lesional macrophages was reduced during atherosclerosis development in both humans and rodents. Atherosclerotic lesions from macrophage USP9X-deficient mice showed increased macrophage infiltration, lipid deposition, and necrotic core content than control apolipoprotein E-knockout (Apoe-/-) mice. Additionally, loss-of-function USP9X exacerbated lipid uptake, foam cell formation and inflammatory responses in macrophages. Mechanistically, the class A1 scavenger receptor (SR-A1) was identified as a USP9X substrate that removed the K63 polyubiquitin chain at the K27 site. Genetic or pharmacological inhibition of USP9X increased SR-A1 cell surface internalization following binding of oxidized low-density lipoprotein (ox-LDL). The K27R mutation of SR-A1 dramatically attenuated basal and USP9X knockdown-induced ox-LDL uptake. Moreover, blocking binding of USP9X to SR-A1 with a cell-penetrating peptide exacerbated foam cell formation and atherosclerosis. In this study, we identified macrophage USP9X as a beneficial regulator of atherosclerosis and revealed the specific mechanisms for the development of potential therapeutic strategies for atherosclerosis.
    Keywords:  Atherosclerosis; Cardiology; Immunology; Macrophages; Ubiquitin-proteosome system
  34. Life Sci Alliance. 2022 Jul;pii: e202101174. [Epub ahead of print]5(7):
      The InterFeron-Induced TransMembrane proteins (IFITMs) are members of the dispanin/CD225 family that act as broad viral inhibitors by preventing viral-to-cellular membrane fusion. In this study, we uncover egress from the Golgi as an important step in the biology of IFITM3 by identifying the domain that regulates this process and that similarly controls the egress of the dispanins IFITM1 and PRRT2, protein linked to paroxysmal kinesigenic dyskinesia. In the case of IFITM3, high levels of expression of wild-type, or mutations in the Golgi egress domain, lead to accumulation of IFITM3 in the Golgi and drive generalized glycoprotein trafficking defects. These defects can be relieved upon incubation with Amphotericin B, compound known to relieve IFITM-driven membrane fusion defects, as well as by v-SNARE overexpression, suggesting that IFITM3 interferes with membrane fusion processes important for Golgi functionalities. The comparison of glycoprotein trafficking in WT versus IFITMs-KO cells indicates that the modulation of the secretory pathway is a novel feature of IFITM proteins. Overall, our study defines a novel domain that regulates the egress of several dispanin/CD225 members from the Golgi and identifies a novel modulatory function for IFITM3.
  35. Nature. 2022 Apr 06.
      Stimulator of interferon genes (STING) is an adaptor protein in innate immunity against DNA viruses or bacteria1-5. STING-mediated immunity could be exploited in the development of vaccines or cancer immunotherapies. STING is a transmembrane dimeric protein that is located in the endoplasmic reticulum or in the Golgi apparatus. STING is activated by the binding of its cytoplasmic ligand-binding domain to cyclic dinucleotides that are produced by the DNA sensor cyclic GMP-AMP (cGAMP) synthase or by invading bacteria1,6,7. Cyclic dinucleotides induce a conformational change in the STING ligand-binding domain, which leads to a high-order oligomerization of STING that is essential for triggering the downstream signalling pathways8,9. However, the cGAMP-induced STING oligomers tend to dissociate in solution and have not been resolved to high resolution, which limits our understanding of the activation mechanism. Here we show that a small-molecule agonist, compound 53 (C53)10, promotes the oligomerization and activation of human STING through a mechanism orthogonal to that of cGAMP. We determined a cryo-electron microscopy structure of STING bound to both C53 and cGAMP, revealing a stable oligomer that is formed by side-by-side packing and has a curled overall shape. Notably, C53 binds to a cryptic pocket in the STING transmembrane domain, between the two subunits of the STING dimer. This binding triggers outward shifts of transmembrane helices in the dimer, and induces inter-dimer interactions between these helices to mediate the formation of the high-order oligomer. Our functional analyses show that cGAMP and C53 together induce stronger activation of STING than either ligand alone.