bims-proteo Biomed News
on Proteostasis
Issue of 2022‒07‒24
thirty papers selected by
Eric Chevet

  1. MicroPubl Biol. 2022 ;2022
      Protein folding and quality control is tightly regulated at the endoplasmic reticulum (ER), and its disruption is associated with many diseases. In eukaryotes, the accumulation of unfolded protein in the ER is sensed by the three sensors, IRE1, PERK, and ATF6 to activate the unfolded protein response (UPR) to restore ER homeostasis. However, uncoupling the sensing of each sensor and their respective downstream pathways has been challenging as the absence of one is compensated by the remaining two sensors. Here, we report a fully functional human PERK (hPERK) chimeric protein expressed in Saccharomyces cerevisiae that could be used for high throughput screen to identify new PERK inhibitory or activating compounds as well as to characterize the PERK stress sensing mechanisms.
  2. Autophagy. 2022 Jul 20. 1-16
      Endoplasmic reticulum stress is an emerging significant player in the molecular pathology of connective tissue disorders. In response to endoplasmic reticulum stress, cells can upregulate macroautophagy/autophagy, a fundamental cellular homeostatic process used by cells to degrade and recycle proteins or remove damaged organelles. In these scenarios, autophagy activation can support cell survival. Here we demonstrated by in vitro and in vivo approaches that megakaryocytes derived from col6a1-⁄- (collagen, type VI, alpha 1) null mice display increased intracellular retention of COL6 polypeptides, endoplasmic reticulum stress and apoptosis. The unfolded protein response is activated in col6a1-⁄- megakaryocytes, as evidenced by the upregulation of molecular chaperones, by the increased splicing of Xbp1 mRNA and by the higher level of the pro-apoptotic regulator DDIT3/CHOP. Despite the endoplasmic reticulum stress, basal autophagy is impaired in col6a1-⁄- megakaryocytes, which show lower BECN1 levels and reduced autophagosome maturation. Starvation and rapamycin treatment rescue the autophagic flux in col6a1-⁄- megakaryocytes, leading to a decrease in intracellular COL6 polypeptide retention, endoplasmic reticulum stress and apoptosis. Furthermore, megakaryocytes cultured from peripheral blood hematopoietic progenitors of patients affected by Bethlem myopathy and Ullrich congenital muscular dystrophy, two COL6-related disorders, displayed increased apoptosis, endoplasmic reticulum stress and impaired autophagy. These data demonstrate that genetic disorders of collagens, endoplasmic reticulum stress and autophagy regulation in megakaryocytes may be interrelated.Abbreviations: 7-AAD: 7-amino-actinomycin D; ATF: activating transcriptional factor; BAX: BCL2 associated X protein; BCL2: B cell leukemia/lymphoma 2; BCL2L1/Bcl-xL: BCL2-like 1; BM: bone marrow; COL6: collagen, type VI; col6a1-⁄-: mice that are null for Col6a1; DDIT3/CHOP/GADD153: DNA-damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; reticulophagy: endoplasmic reticulum-selective autophagy; HSPA5/Bip: heat shock protein 5; HSP90B1/GRP94: heat shock protein 90, beta (Grp94), member 1; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; Mk: megakaryocytes; MTOR: mechanistic target of rapamycin kinase; NIMV: noninvasive mechanical ventilation; PI3K: phosphoinositide 3-kinase; PPP1R15A/GADD34: protein phosphatase 1, regulatory subunit 15A; RT-qPCR: reverse transcription-quantitative real-time PCR; ROS: reactive oxygen species; SERPINH1/HSP47: serine (or cysteine) peptidase inhibitor, clade H, member 1; sh-RNA: short hairpin RNA; SOCE: store operated calcium entry; UCMD: Ullrich congenital muscular dystrophy; UPR: unfolded protein response; WIPI2: WD repeat domain, phosphoinositide-interacting 2; WT: wild type; XBP1: X-box binding protein 1.
    Keywords:  Apoptosis; autophagy; collagen VI; endoplasmic reticulum stress; megakaryocytes; rapamycin; unfolded protein response
  3. Proc Natl Acad Sci U S A. 2022 Jul 26. 119(30): e2203218119
      The exposed N-terminal or C-terminal residues of proteins can act, in cognate sequence contexts, as degradation signals (degrons) that are targeted by specific E3 ubiquitin ligases for proteasome-dependent degradation by N-degron or C-degron pathways. Here, we discovered a distinct C-degron pathway, termed the Gln/C-degron pathway, in which the B30.2 domain of E3 ubiquitin ligase TRIM7 (TRIM7B30.2) mediates the recognition of proteins bearing a C-terminal glutamine. By determining crystal structures of TRIM7B30.2 in complexes with various peptides, we show that TRIM7B30.2 forms a positively charged binding pocket to engage the "U"-shaped Gln/C-degron. The four C-terminal residues of a substrate play an important role in C-degron recognition, with C-terminal glutamine as the principal determinant. In vitro biochemical and cellular experiments were used to further analyze the substrate specificity and selective degradation of the Gln/C-degron by TRIM7.
    Keywords:  E3 ubiquitin ligase; TRIM7; crystal structure; degron; protein degradation
  4. PLoS Biol. 2022 Jul;20(7): e3001710
      Gustatory Receptor 64 (Gr64) genes are a cluster of 6 neuronally expressed receptors involved in sweet taste sensation in Drosophila melanogaster. Gr64s modulate calcium signalling and excitatory responses to several different sugars. Here, we discover an unexpected nonneuronal function of Gr64 receptors and show that they promote proteostasis in epithelial cells affected by proteotoxic stress. Using heterozygous mutations in ribosome proteins (Rp), which have recently been shown to induce proteotoxic stress and protein aggregates in cells, we show that Rp/+ cells in Drosophila imaginal discs up-regulate expression of the entire Gr64 cluster and depend on these receptors for survival. We further show that loss of Gr64 in Rp/+ cells exacerbates stress pathway activation and proteotoxic stress by negatively affecting autophagy and proteasome function. This work identifies a noncanonical role in proteostasis maintenance for a family of gustatory receptors known for their function in neuronal sensation.
  5. Chem Biol Drug Des. 2022 Jul 20.
      Proteolysis-targeting chimeras (PROTACs) are novel therapeutics for the treatment of human disease. They exploit the enormous potential of the E3 ligases, a class of proteins that mark a target protein for degradation via the ubiquitin-proteasome system. Despite the existence of several E3 ligase-related databases, the choice of the functioning ligase is limited to only 1.6% of those available, probably due to the fragmentary understanding of their structures and their known ligands; in fact, none of the existing databases report detailed studies covering their 3D structure or their pockets. Here we report ELIOT (E3 LIgase pocketOme navigaTor), an accurate and complete platform containing the E3 ligase pocketome to enable navigation and selection of new E3 ligases and new ligands for the design of new PROTACs. All E3 ligase pockets were characterized with innovative 3D descriptors including their PROTAC-ability score and similarities analysis between E3 pockets are presented. Tissue specificity and their degree of involvement in patients with specific cancer types are also annotated for each E3 ligase, enabling appropriate selection for design of a PROTAC with improved specificity. All data are available at
    Keywords:  Cancer; E3 ligase; PROTACs; Protein Degradation; Ubiquitin-Proteasome System (UPS)
  6. Angew Chem Int Ed Engl. 2022 Jul 22.
      Targeted protein degradation via proteasomal and lysosomal pathways has become a promising therapeutic approach, and proteins in cytoplasm or on the cell membrane can be easily contacted and have become the major targets. However, degradation of disease-related proteins that exist in membrane-bound organelles (MBO) such as the endoplasmic reticulum (ER) remains unsolved due to the membrane limits. Here we describe a DNA nanodevice that shows ER targeting capacity and undergoes new intracellular degradation via the autophagy-dependent pathway. Then the DNA nanostructure functionalized with specific ligands are used to selectively catch ER-localized proteins and then transport them to the lysosome for degradation. Through this technique, the degradation of both exogenous ER-resident protein (ER-eGFP) and endogenous overexpressed molecular chaperone (glucose-regulated protein 78) in cancer cells has been successfully executed with high efficiency. This strategy expands the range of currently proposed protein degradation platforms and may contribute to the development of targeted cancer therapeutics.
    Keywords:  Autophagy; DNA nanostructure; Organelle targeting; Targeted protein degradation
  7. Sci Signal. 2022 Jul 05. 15(741): eabm7524
      The endoplasmic reticulum (ER) is the largest organelle of the cell and participates in multiple essential functions, including the production of secretory proteins, lipid synthesis, and calcium storage. Sustaining proteostasis requires an intimate coupling with energy production. Mitochondrial respiration evolved to be functionally connected to ER physiology through a physical interface between both organelles known as mitochondria-associated membranes. This quasi-synaptic structure acts as a signaling hub that tunes the function of both organelles in a bidirectional manner and controls proteostasis, cell death pathways, and mitochondrial bioenergetics. Here, we discuss the main signaling mechanisms governing interorganellar communication and their putative role in diseases including cancer and neurodegeneration.
  8. Cell Chem Biol. 2022 Jul 14. pii: S2451-9456(22)00242-2. [Epub ahead of print]
      Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first mapped proteasome-associated genetic co-dependencies. We identified heat shock protein 70 (HSP70) chaperones as potential targets, consistent with proposed mechanisms of myeloma cells overcoming PI-induced stress. We therefore explored allosteric HSP70 inhibitors (JG compounds) as myeloma therapeutics. JG compounds exhibited increased efficacy against acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Shotgun and pulsed SILAC mass spectrometry demonstrated that JGs unexpectedly impact myeloma proteostasis by destabilizing the 55S mitoribosome. Our data suggest JGs have the most pronounced anti-myeloma effect not through inhibiting cytosolic HSP70 proteins but instead through mitochondrial-localized HSP70, HSPA9/mortalin. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize myeloma proteostasis networks under therapeutic pressure while motivating further investigation of HSPA9 as a specific vulnerability in PI-resistant disease.
    Keywords:  HSP70; bortezomib; mitochondria; mitoribosome; myeloma; proteasome inhibitor; proteomics; proteostasis; resistance
  9. J Cell Physiol. 2022 Jul 19.
      Selective autophagy is the lysosomal degradation of specific intracellular components sequestered into autophagosomes, late endosomes, or lysosomes through the activity of selective autophagy receptors. CALCOCO family proteins are the newly found selective autophagy receptors, which include calcium binding and coiled-coil domain 1 (CALCOCO1), calcium binding and coiled-coil domain 2/nuclear domain 10 protein 52 (CALCOCO2/NDP52), and calcium binding and coiled-coil domain 3/Tax1-binding protein 1 (CALCOCO3/TAX1BP1). Specifically, CALCOCO1 can be recruited to endoplasmic reticulum (ER) and Golgi to mediate selective ER-phagy and Golgiphagy. CALCOCO2 and CALCOCO3, which are two essential cargo receptors, can mediate mitophagy and xenophagy through interacting with autophagy-related-8/microtubule-associated protein 1 light chain 3 (ATG8/LC3) on the growing autophagosome, and binding ubiquitin for cargo recruitment. Considering the significance of these proteins in selective autophagy, we review the structures, distribution, posttranslational modifications, and phylogenetic analysis of CALCOCO family proteins and their roles in different selective autophagy.
    Keywords:  CALCOCO1; CALCOCO2; CALCOCO3; ER-phagy; mitophagy; selective autophagy
  10. mBio. 2022 Jul 18. e0070322
      The insect immune deficiency (IMD) pathway is a defense mechanism that senses and responds to Gram-negative bacteria. Ticks lack genes encoding upstream components that initiate the IMD pathway. Despite this deficiency, core signaling molecules are present and functionally restrict tick-borne pathogens. The molecular events preceding activation remain undefined. Here, we show that the unfolded-protein response (UPR) initiates the IMD network. The endoplasmic reticulum (ER) stress receptor IRE1α is phosphorylated in response to tick-borne bacteria but does not splice the mRNA encoding XBP1. Instead, through protein modeling and reciprocal pulldowns, we show that Ixodes IRE1α complexes with TRAF2. Disrupting IRE1α-TRAF2 signaling blocks IMD pathway activation and diminishes the production of reactive oxygen species. Through in vitro, in vivo, and ex vivo techniques, we demonstrate that the UPR-IMD pathway circuitry limits the Lyme disease-causing spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale (anaplasmosis). Altogether, our study uncovers a novel linkage between the UPR and the IMD pathway in arthropods. IMPORTANCE The ability of an arthropod to harbor and transmit pathogens is termed "vector competency." Many factors influence vector competency, including how arthropod immune processes respond to the microbe. Divergences in innate immunity between arthropods are increasingly being reported. For instance, although ticks lack genes encoding key upstream molecules of the immune deficiency (IMD) pathway, it is still functional and restricts causative agents of Lyme disease (Borrelia burgdorferi) and anaplasmosis (Anaplasma phagocytophilum). How the IMD pathway is activated in ticks without classically defined pathway initiators is not known. Here, we found that a cellular stress response network, the unfolded-protein response (UPR), functions upstream to induce the IMD pathway and restrict transmissible pathogens. Collectively, this explains how the IMD pathway can be activated in the absence of canonical pathway initiators. Given that the UPR is highly conserved, UPR-initiated immunity may be a fundamental principle impacting vector competency across arthropods.
    Keywords:  Anaplasma phagocytophilum; Borrelia burgdorferi; Ixodes scapularis; immune deficiency pathway; tick-borne disease; unfolded-protein response; vector immunity
  11. Front Physiol. 2022 ;13 913063
      E3s comprise a structurally diverse group of at least 800 members, most of which target multiple substrates through specific and regulated protein-protein interactions. These interactions typically rely on short linear motifs (SLiMs), called "degrons", in an intrinsically disordered region (IDR) of the substrate, with variable rules of engagement governing different E3-docking events. These rules of engagement are of importance to the field of targeted protein degradation (TPD), where substrate ubiquitination and destruction require tools to effectively harness ubiquitin ligases (E3s). Substrates are often found to contain multiple degrons, or multiple copies of a degron, contributing to the affinity and selectivity of the substrate for its E3. One important paradigm for E3-substrate docking is presented by the Anaphase-Promoting Complex/Cyclosome (APC/C), a multi-subunit E3 ligase that targets hundreds of proteins for destruction during mitotic exit. APC/C substrate targeting takes place in an ordered manner thought to depend on tightly regulated interactions of substrates, with docking sites provided by the substoichiometric APC/C substrate adaptors and coactivators, Cdc20 or Cdh1/FZR1. Both structural and functional studies of individual APC/C substrates indicate that productive ubiquitination usually requires more than one degron, and that degrons are of different types docking to distinct sites on the coactivators. However, the dynamic nature of APC/C substrate recruitment, and the influence of multiple degrons, remains poorly understood. Here we review the significance of multiple degrons in a number of E3-substrate interactions that have been studied in detail, illustrating distinct kinetic effects of multivalency and allovalency, before addressing the role of multiple degrons in APC/C substrates, key to understanding ordered substrate destruction by APC/C. Lastly, we consider how lessons learnt from these studies can be applied in the design of TPD tools.
    Keywords:  E3-substrate interaction; SLiM; degron; multivalency; targeted protein degradation; ubiquitin ligase
  12. J Biol Chem. 2022 Jul 19. pii: S0021-9258(22)00731-1. [Epub ahead of print] 102289
      The protein product of the CDKN1A gene, p21, has been extensively characterized as a negative regulator of the cell cycle. Nevertheless, it is clear that p21 has manifold complex and context-dependent roles that can be either tumor suppressive or oncogenic. Most well-studied as a transcriptional target of the p53 tumor suppressor protein, there are other means by which p21 levels can be regulated. In this study we show that pharmacological inhibition or siRNA mediated reduction of O-GlcNAc transferase (OGT), the enzyme responsible for glycosylation of intracellular proteins, increases expression of p21 in both p53-dependent and -independent manners in non-transformed and cancer cells. In cells harboring wild-type p53, we demonstrate that inhibition of OGT leads to p53-mediated transactivation of CDKN1A, while in cells that do not express p53, inhibiting OGT leads to increased p21 protein stabilization. p21 is normally degraded by the ubiquitin-proteasome system following ubiquitination by, among others, the E3 ligase Skp-Cullin-F-box (SCF) complex; however, in this case, we show blocking OGT causes impairment of the SCF ubiquitin complex as a result of disruption of the FoxM1 transcription factor-mediated induction of Skp2 expression. In either setting, we conclude that p21 levels induced by OGT inhibition correlate with cell cycle arrest and decreased cancer cell proliferation.
    Keywords:  O-GlcNAcylation; OGT; cell cycle; p21; p53; protein degradation
  13. Cell Rep. 2022 Jul 19. pii: S2211-1247(22)00894-4. [Epub ahead of print]40(3): 111092
      The integrated stress response (ISR) plays a pivotal role in adaptation of translation machinery to cellular stress. Here, we demonstrate an ISR-independent osmoadaptation mechanism involving reprogramming of translation via coordinated but independent actions of mTOR and plasma membrane amino acid transporter SNAT2. This biphasic response entails reduced global protein synthesis and mTOR signaling followed by translation of SNAT2. Induction of SNAT2 leads to accumulation of amino acids and reactivation of mTOR and global protein synthesis, paralleled by partial reversal of the early-phase, stress-induced translatome. We propose SNAT2 functions as a molecular switch between inhibition of protein synthesis and establishment of an osmoadaptive translation program involving the formation of cytoplasmic condensates of SNAT2-regulated RNA-binding proteins DDX3X and FUS. In summary, we define key roles of SNAT2 in osmotolerance.
    Keywords:  CP: Molecular biology; amino acids; cytoplasmic condensates; hypertonic stress; mTOR signaling; osmolytes; osmotolerance; translation
  14. Cell Rep. 2022 Jul 19. pii: S2211-1247(22)00898-1. [Epub ahead of print]40(3): 111096
      Accurate and efficient folding of nascent protein sequences into their native states requires support from the protein homeostasis network. Herein we probe which newly translated proteins are thermo-sensitive, making them susceptible to misfolding and aggregation under heat stress using pulse-SILAC mass spectrometry. We find a distinct group of proteins that is highly sensitive to this perturbation when newly synthesized but not once matured. These proteins are abundant and highly structured. Notably, they display a tendency to form β sheet secondary structures, have more complex folding topology, and are enriched for chaperone-binding motifs, suggesting a higher demand for chaperone-assisted folding. These polypeptides are also more often components of stable protein complexes in comparison with other proteins. Combining these findings suggests the existence of a specific subset of proteins in the cell that is particularly vulnerable to misfolding and aggregation following synthesis before reaching the native state.
    Keywords:  CP: Molecular biology; heat stress; limited proteolysis; misfolding; protein aggregation; protein folding; protein mass spectrometry; proteomics; proteostasis; pulse SILAC; translation
  15. Proc Natl Acad Sci U S A. 2022 Jul 26. 119(30): e2122495119
      Regulation of catalytic activity of E3 ubiquitin ligases is critical for their cellular functions. We identified an unexpected mode of regulation of E3 catalytic activity by ions and osmolarity; enzymatic activity of the HECT family E3 Nedd4-2/Nedd4L is enhanced by increased intracellular Na+ ([Na+]i) and by hyperosmolarity. This stimulated activity is mediated by activation of p38-MAPK and is inhibited by WNKs. Moreover, protease (Furin)-mediated activation of the epithelial Na+ channel ENaC (a bona fide Nedd4-2 substrate), which leads to increased [Na+]i and osmolarity, results in enhanced Nedd4-2 catalytic activity. This enhancement is inhibited by a Furin inhibitor, by a protease-resistant ENaC mutant, or by treatment with the ENaC inhibitor amiloride. Moreover, WNK inhibition, which stimulates catalytic activity of Nedd4-2, leads to reduced levels of cell-surface ENaC and reduced channel activity. ENaC activity does not affect Nedd4-2:ENaC binding. Therefore, these results demonstrate activation of a ubiquitin ligase by Na+ and osmotic changes. Importantly, they reveal a negative feedback loop in which active ENaC leads to stimulation of catalytic activity of its own suppressor, Nedd4-2, to protect cells from excessive Na+ loading and hyperosmotic stress and to protect the animal from hypertension.
    Keywords:  ENaC; HECT; Nedd4L; hyperosmolarity; sodium
  16. EMBO Mol Med. 2022 Jul 21. e15855
      FBXW7 is one of the most frequently mutated tumor suppressors, deficiency of which has been associated with resistance to some anticancer therapies. Through bioinformatics and genome-wide CRISPR screens, we here reveal that FBXW7 deficiency leads to multidrug resistance (MDR). Proteomic analyses found an upregulation of mitochondrial factors as a hallmark of FBXW7 deficiency, which has been previously linked to chemotherapy resistance. Despite this increased expression of mitochondrial factors, functional analyses revealed that mitochondria are under stress, and genetic or chemical targeting of mitochondria is preferentially toxic for FBXW7-deficient cells. Mechanistically, the toxicity of therapies targeting mitochondrial translation such as the antibiotic tigecycline relates to the activation of the integrated stress response (ISR) in a GCN2 kinase-dependent manner. Furthermore, the discovery of additional drugs that are toxic for FBXW7-deficient cells showed that all of them unexpectedly activate a GCN2-dependent ISR regardless of their accepted mechanism of action. Our study reveals that while one of the most frequent mutations in cancer reduces the sensitivity to the vast majority of available therapies, it renders cells vulnerable to ISR-activating drugs.
    Keywords:  FBXW7; GCN2; ISR; drug resistance; mitochondria
  17. Nat Commun. 2022 Jul 16. 13(1): 4146
      Enhancing the removal of aggregate-prone toxic proteins is a rational therapeutic strategy for a number of neurodegenerative diseases, especially Huntington's disease and various spinocerebellar ataxias. Ideally, such approaches should preferentially clear the mutant/misfolded species, while having minimal impact on the stability of wild-type/normally-folded proteins. Furthermore, activation of both ubiquitin-proteasome and autophagy-lysosome routes may be advantageous, as this would allow effective clearance of both monomeric and oligomeric species, the latter which are inaccessible to the proteasome. Here we find that compounds that activate the D1 ATPase activity of VCP/p97 fulfill these requirements. Such effects are seen with small molecule VCP activators like SMER28, which activate autophagosome biogenesis by enhancing interactions of PI3K complex components to increase PI(3)P production, and also accelerate VCP-dependent proteasomal clearance of such substrates. Thus, this mode of VCP activation may be a very attractive target for many neurodegenerative diseases.
  18. Nat Cell Biol. 2022 Jul 18.
      Cellular senescence plays a causal role in ageing and, in mice, depletion of p16INK4a-expressing senescent cells delays ageing-associated disorders1,2. Adenosine deaminases acting on RNA (ADARs) are RNA-editing enzymes that are also implicated as important regulators of human ageing, and ADAR inactivation causes age-associated pathologies such as neurodegeneration in model organisms3,4. However, the role, if any, of ADARs in cellular senescence is unknown. Here we show that ADAR1 is post-transcriptionally downregulated by autophagic degradation to promote senescence through p16INK4a upregulation. The ADAR1 downregulation is sufficient to drive senescence in both in vitro and in vivo models. Senescence induced by ADAR1 downregulation is p16INK4a-dependent and independent of its RNA-editing function. Mechanistically, ADAR1 promotes SIRT1 expression by affecting its RNA stability through HuR, an RNA-binding protein that increases the half-life and steady-state levels of its target mRNAs. SIRT1 in turn antagonizes translation of mRNA encoding p16INK4a. Hence, downregulation of ADAR1 and SIRT1 mediates p16INK4a upregulation by enhancing its mRNA translation. Finally, Adar1 is downregulated during ageing of mouse tissues such as brain, ovary and intestine, and Adar1 expression correlates with Sirt1 expression in these tissues in mice. Together, our study reveals an RNA-editing-independent role for ADAR1 in the regulation of senescence by post-transcriptionally controlling p16INK4a expression.
  19. Mol Cell. 2022 Jul 12. pii: S1097-2765(22)00607-4. [Epub ahead of print]
      Aberrant phase separation of globular proteins is associated with many diseases. Here, we use a model protein system to understand how the unfolded states of globular proteins drive phase separation and the formation of unfolded protein deposits (UPODs). We find that for UPODs to form, the concentrations of unfolded molecules must be above a threshold value. Additionally, unfolded molecules must possess appropriate sequence grammars to drive phase separation. While UPODs recruit molecular chaperones, their compositional profiles are also influenced by synergistic physicochemical interactions governed by the sequence grammars of unfolded proteins and cellular proteins. Overall, the driving forces for phase separation and the compositional profiles of UPODs are governed by the sequence grammars of unfolded proteins. Our studies highlight the need for uncovering the sequence grammars of unfolded proteins that drive UPOD formation and cause gain-of-function interactions whereby proteins are aberrantly recruited into UPODs.
    Keywords:  Cry2; SOD1; TRiC; barnase; chaperonin-containing T-complex; molecular condensate; protein deposit; protein folding; protein misfolding; protein quality control; proteostasis; superoxide dismutase 1
  20. J Am Chem Soc. 2022 Jul 18.
      Asparaginyl endopeptidases (AEPs) have recently been widely utilized for peptide and protein modification. Labeling is however restricted to protein termini, severely limiting flexibility and scope in creating diverse conjugates as needed for therapeutic and diagnostic applications. Here, we use genetic code expansion to site-specifically modify target proteins with an isopeptide-linked glycylglycine moiety that serves as an acceptor nucleophile in AEP-mediated transpeptidation with various probes containing a tripeptidic recognition motif. Our approach allows simple and flexible labeling of recombinant proteins at any internal site and leaves a minimal, entirely peptidic footprint (NGG) in the conjugation product. We show site-specific labeling of diverse target proteins with various biophysical probes, including dual labeling at an internal site and the N-terminus. Furthermore, we harness AEP-mediated transpeptidation for generation of ubiquitin- and ubiquitin-like-modifier conjugates bearing a native isopeptide bond and only one point mutation in the linker region.
  21. Proc Natl Acad Sci U S A. 2022 Jul 26. 119(30): e2120339119
      During translation initiation, eIF4G1 dynamically interacts with eIF4E and eIF1. While the role of eIF4E-eIF4G1 is well established, the regulatory functions of eIF4G1-eIF1 are poorly understood. Here, we report the identification of the eIF4G1-eIF1 inhibitors i14G1-10 and i14G1-12. i14G1s directly bind eIF4G1 and inhibit translation in vitro and in the cell, and their effects on translation are dependent on eIF4G1 levels. Translatome analyses revealed that i14G1s mimic eIF1 and eIF4G1 perturbations on the stringency of start codon selection and the opposing roles of eIF1-eIF4G1 in scanning-dependent and scanning-independent short 5' untranslated region (UTR) translation. Remarkably, i14G1s activate ER/unfolded protein response (UPR) stress-response genes via enhanced ribosome loading, elevated 5'UTR translation at near-cognate AUGs, and unexpected concomitant up-regulation of coding-region translation. These effects are, at least in part, independent of eIF2α-phosphorylation. Interestingly, eIF4G1-eIF1 interaction itself is negatively regulated by ER stress and mTOR inhibition. Thus, i14G1s uncover an unknown mechanism of ER/UPR translational stress response and are valuable research tools and potential drugs against diseases exhibiting dysregulated translation.
    Keywords:  eIF1; eIF4G1; i14G1-10; i14G1-12; translation
  22. Autophagy. 2022 Jul 22.
      Small 30-nm vesicles containing the integral membrane protein Atg9 provide the initial membrane source for autophagy in yeast. Atg23, is an Atg9 binding protein that is required for Atg9 vesicle trafficking but whose exact function is unknown. In our recent paper, we explored the function of Atg23 using an approach combining cellular biology and biochemistry on purified protein. We determined that Atg23 is an elongated dimer spanning 320 Å in length. We also demonstrated that Atg23 is a membrane-binding and -tethering protein. Furthermore, we identified a series of amino acids residing in a putative coiled- coil region that when mutated prevent Atg23 dimer formation resulting in a stable Atg23 monomer. Last, we demonstrated that when monomeric Atg23 is expressed in yeast lacking Atg23, this leads to a loss of Atg23 puncta, a reduction in Atg9 puncta, a reduction in non-selective autophagy and a complete block in the cytoplasm-to-vacuole targeting (Cvt) pathway.
    Keywords:  Autophagy; cytoplasm-to-vacuole targeting; membrane binding protein; vacuole; yeast
  23. Brain Commun. 2022 ;4(4): fcac176
      Mutations in p97/VCP cause two motor neuron diseases: inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia and familial amyotrophic lateral sclerosis. How p97 mutations lead to motor neuron degeneration is, however, unknown. Here we used patient-derived induced pluripotent stem cells to generate p97 mutant motor neurons. We reduced the genetic background variation by comparing mutant motor neurons to its isogenic wild type lines. Proteomic analysis reveals that p97R155H/+ motor neurons upregulate several cell cycle proteins at Day 14, but this effect diminishes by Day 20. Molecular changes linked to delayed cell cycle exit are observed in p97 mutant motor neurons. We also find that two p97 inhibitors, CB-5083 and NMS-873, restore some dysregulated protein levels. In addition, two p97 inhibitors and a food and drug administration-approved cyclin-dependent kinase 4/6 inhibitor, Abemaciclib, can rescue motor neuron death. Overall, we successfully used iPSC-derived motor neurons, identified dysregulated proteome and transcriptome and showed that p97 inhibitors rescue phenotypes in this disease model.
    Keywords:  amyotrophic lateral sclerosis; cell cycle; inclusion body myopathy associated with paget disease of bone and frontotemporal dementia; motor neuron disease; p97 inhibitor
  24. Oncogenesis. 2022 Jul 20. 11(1): 40
      Ras-related C3 botulinum toxin substrate 1 (RAC1) overexpressiosn and hyperactivation are correlated with aggressive growth and other malignant characteristics in a wide variety of cancers including hepatocellular carcinoma (HCC). However, the regulatory mechanism of RAC1 expression and activation in HCC is not fully understood. Here, we demonstrated that E3 ubiquitin ligase MG53 (also known as tripartite motif 72, TRIM72) acted as a direct inhibitor of RAC1, and it catalyzed the ubiquitination of RAC1 and further inhibited RAC1 activity in HCC cells. Mechanistically, MG53 directly bound with RAC1 through its coiled-coil domain and suppressed RAC1 activity by catalyzing the Lys48 (K48)-linked polyubiquitination of RAC1 at Lys5 residue in HCC cells. We further demonstrated that MG53 significantly suppressed the malignant behaviors of HCC cells and enhanced the chemosensitivity of HCC cells to sorafenib treatment by inhibiting RAC1-MAPK signaling axis. In summary, we identified MG53 as a novel RAC1 inhibitor and tumor suppressor in HCC, and it suppressed HCC progression by inducing K48-linked polyubiquitination of RAC1 and further inhibiting the RAC1-MAPK signaling. Altogether, our investigation provided a new therapeutic strategy for RAC1 overactivated tumors by modulating MG53.
  25. Nat Commun. 2022 Jul 22. 13(1): 4243
      Co-translational folding is a fundamental process for the efficient biosynthesis of nascent polypeptides that emerge through the ribosome exit tunnel. To understand how this process is modulated by the shape and surface of the narrow tunnel, we have rationally engineered three exit tunnel protein loops (uL22, uL23 and uL24) of the 70S ribosome by CRISPR/Cas9 gene editing, and studied the co-translational folding of an immunoglobulin-like filamin domain (FLN5). Our thermodynamics measurements employing 19F/15N/methyl-TROSY NMR spectroscopy together with cryo-EM and molecular dynamics simulations reveal how the variations in the lengths of the loops present across species exert their distinct effects on the free energy of FLN5 folding. A concerted interplay of the uL23 and uL24 loops is sufficient to alter co-translational folding energetics, which we highlight by the opposite folding outcomes resulting from their extensions. These subtle modulations occur through a combination of the steric effects relating to the shape of the tunnel, the dynamic interactions between the ribosome surface and the unfolded nascent chain, and its altered exit pathway within the vestibule. These results illustrate the role of the exit tunnel structure in co-translational folding, and provide principles for how to remodel it to elicit a desired folding outcome.
  26. EMBO J. 2022 Jul 17. e110698
      The Arf GTPase family is involved in a wide range of cellular regulation including membrane trafficking and organelle-structure assembly. Here, we have generated a proximity interaction network for the Arf family using the miniTurboID approach combined with TMT-based quantitative mass spectrometry. Our interactome confirmed known interactions and identified many novel interactors that provide leads for defining Arf pathway cell biological functions. We explored the unexpected finding that phospholipase D1 (PLD1) preferentially interacts with two closely related but poorly studied Arf family GTPases, ARL11 and ARL14, showing that PLD1 is activated by ARL11/14 and may recruit these GTPases to membrane vesicles, and that PLD1 and ARL11 collaborate to promote macrophage phagocytosis. Moreover, ARL5A and ARL5B were found to interact with and recruit phosphatidylinositol 4-kinase beta (PI4KB) at trans-Golgi, thus promoting PI4KB's function in PI4P synthesis and protein secretion.
    Keywords:  ARL11; ARL5; PI4KB; PLD1; Phagocytosis
  27. Nat Chem Biol. 2022 Jul 21.
      Reversible protein phosphorylation is an important mechanism for regulating (dis)assembly of biomolecular condensates. However, condensate-specific phosphosites remain largely unknown, thereby limiting our understanding of the underlying mechanisms. Here, we combine solubility proteome profiling with phosphoproteomics to quantitatively map several hundred phosphosites enriched in either soluble or condensate-bound protein subpopulations, including a subset of phosphosites modulating protein-RNA interactions. We show that multi-phosphorylation of the C-terminal disordered segment of heteronuclear ribonucleoprotein A1 (HNRNPA1), a key RNA-splicing factor, reduces its ability to locate to nuclear clusters. For nucleophosmin 1 (NPM1), an essential nucleolar protein, we show that phosphorylation of S254 and S260 is crucial for lowering its partitioning to the nucleolus and additional phosphorylation of distal sites enhances its retention in the nucleoplasm. These phosphorylation events decrease RNA and protein interactions of NPM1 to regulate its condensation. Our dataset is a rich resource for systematically uncovering the phosphoregulation of biomolecular condensates.
  28. Nucleic Acids Res. 2022 Jul 15. pii: gkac605. [Epub ahead of print]
      RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of a non-tumoral versus a metastatic cell line revealed prevalent changes in RNA-binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression, map its RNA-binding domain, and find that RNA binding is required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells.
  29. Nat Chem Biol. 2022 Jul 21.
      Drugs that directly impede the function of driver oncogenes offer exceptional efficacy and a therapeutic window. The recently approved mutant selective small-molecule cysteine-reactive covalent inhibitor of the G12C mutant of K-Ras, sotorasib, provides a case in point. KRAS is the most frequently mutated proto-oncogene in human cancer, yet despite success targeting the G12C allele, targeted therapy for other hotspot mutants of KRAS has not been described. Here we report the discovery of small molecules that covalently target a G12S somatic mutation in K-Ras and suppress its oncogenic signaling. We show that these molecules are active in cells expressing K-Ras(G12S) but spare the wild-type protein. Our results provide a path to targeting a second somatic mutation in the oncogene KRAS by overcoming the weak nucleophilicity of an acquired serine residue. The chemistry we describe may serve as a basis for the selective targeting of other unactivated serines.
  30. Proc Natl Acad Sci U S A. 2022 Jul 12. 119(28): e2113465119
      The role of autophagy in cancer is complex. Both tumor-promoting and tumor-suppressive effects are reported, with tumor type, stage and specific genetic lesions dictating the role. This calls for analysis in models that best recapitulate each tumor type, from initiation to metastatic disease, to specifically understand the contribution of autophagy in each context. Here, we report the effects of deleting the essential autophagy gene Atg7 in a model of pancreatic ductal adenocarcinoma (PDAC), in which mutant KrasG12D and mutant Trp53172H are induced in adult tissue leading to metastatic PDAC. This revealed that Atg7 loss in the presence of KrasG12D/+ and Trp53172H/+ was tumor promoting, similar to previous observations in tumors driven by embryonic KrasG12D/+ and deletion of Trp53. However, Atg7 hemizygosity also enhanced tumor initiation and progression, even though this did not ablate autophagy. Moreover, despite this enhanced progression, fewer Atg7 hemizygous mice had metastases compared with animals wild type for this allele, indicating that ATG7 is a promoter of metastasis. We show, in addition, that Atg7+/- tumors have comparatively lower levels of succinate, and that cells derived from Atg7+/- tumors are also less invasive than those from Atg7+/+ tumors. This effect on invasion can be rescued by ectopic expression of Atg7 in Atg7+/- cells, without affecting the autophagic capacity of the cells, or by treatment with a cell-permeable analog of succinate. These findings therefore show that ATG7 has roles in invasion and metastasis that are not related to the role of the protein in the regulation of autophagy.
    Keywords:  ATG7; autophagy; metastasis; pancreatic cancer