bims-proteo Biomed News
on Proteostasis
Issue of 2021‒10‒03
thirty-six papers selected by
Eric Chevet

  1. Mol Neurobiol. 2021 Sep 29.
      According to cellular demands, ribosomes synthesize and maintain the desired pool of proteins inside the cell. However, sometimes due to defects in ribosomal machinery and faulty mRNAs, these nascent polypeptides are constantly under threat to become non-functional. In such conditions, cells acquire the help of ribosome-associated quality control mechanisms (RQC) to eliminate such aberrant nascent proteins. The primary regulator of RQC is RING domain containing LISTERIN E3 ubiquitin ligase, which is associated with ribosomes and alleviates non-stop proteins-associated stress in cells. Mouse RING finger protein E3 ubiquitin ligase LISTERIN is crucial for embryonic development, and a loss in its function causes neurodegeneration. LISTERIN is overexpressed in the mouse brain and spinal cord regions, and its perturbed functions generate neurological and motor deficits, but the mechanism of the same is unclear. Overall, LISTERIN is crucial for brain health and brain development. The present article systematically describes the detailed nature, molecular functions, and cellular physiological characterization of LISTERIN E3 ubiquitin ligase. Improve comprehension of LISTERIN's neurological roles may uncover pathways linked with neurodegeneration, which in turn might elucidate a promising novel therapeutic intervention against human neurodegenerative diseases.
    Keywords:  E3 ubiquitin ligase; LISTERIN; Misfolded proteins; Neurodegeneration
  2. Elife. 2021 Sep 29. pii: e72328. [Epub ahead of print]10
      Removal of damaged organelles via the process of selective autophagy constitutes a major form of cellular quality control. Damaged organelles are recognized by a dedicated surveillance machinery, leading to the assembly of an autophagosome around the damaged organelle, prior to fusion with the degradative lysosomal compartment. Lysosomes themselves are also prone to damage and are degraded through the process of lysophagy. While early steps involve recognition of ruptured lysosomal membranes by glycan-binding Galectins and ubiquitylation of transmembrane lysosomal proteins, many steps in the process, and their inter-relationships, remain poorly understood, including the role and identity of cargo receptors required for completion of lysophagy. Here, we employ quantitative organelle capture and proximity biotinylation proteomics of autophagy adaptors, cargo receptors, and Galectins in response to acute lysosomal damage, thereby revealing the landscape of lysosome-associated proteome remodeling during lysophagy. Among proteins dynamically recruited to damaged lysosomes were ubiquitin-binding autophagic cargo receptors. Using newly developed lysophagic flux reporters including Lyso-Keima, we demonstrate that TAX1BP1, together with its associated kinase TBK1, are both necessary and sufficient to promote lysophagic flux in both HeLa cells and induced neurons (iNeurons). While the related receptor OPTN can drive damage-dependent lysophagy when overexpressed, cells lacking either OPTN or CALCOCO2 still maintain significant lysophagic flux in HeLa cells. Mechanistically, TAX1BP1-driven lysophagy requires its N-terminal SKICH domain, which binds both TBK1 and the autophagy regulatory factor RB1CC1, and requires upstream ubiquitylation events for efficient recruitment and lysophagic flux. These results identify TAX1BP1 as a central component in the lysophagy pathway and provide a proteomic resource for future studies of the lysophagy process.
    Keywords:  biochemistry; cell biology; chemical biology; human
  3. Cells. 2021 Aug 24. pii: 2178. [Epub ahead of print]10(9):
      During type 1 diabetes mellitus (T1DM) development, beta-cells undergo intense endoplasmic reticulum (ER) stress that could result in apoptosis through the failure of adaptation to the unfolded protein response (UPR). Islet transplantation is considered an attractive alternative among beta-cell replacement therapies for T1DM. To avoid the loss of beta-cells that will jeopardize the transplant's outcome, several strategies are being studied. We have previously shown that prolactin induces protection against proinflammatory cytokines and redox imbalance-induced beta-cell death by increasing heat-shock protein B1 (HSPB1) levels. Since the role of HSPB1 in beta cells has not been deeply studied, we investigated the mechanisms involved in unbalanced protein homeostasis caused by intense ER stress and overload of the proteasomal protein degradation pathway. We tested whether HSPB1-mediated cytoprotective effects involved UPR modulation and improvement of protein degradation via the ubiquitin-proteasome system. We demonstrated that increased levels of HSPB1 attenuated levels of pro-apoptotic proteins such as CHOP and BIM, as well as increased protein ubiquitination and the speed of proteasomal protein degradation. Our data showed that HSPB1 induced resistance to proteotoxic stress and, thus, enhanced cell survival via an increase in beta-cell proteolytic capacity. These results could contribute to generate strategies aimed at the optimization of beta-cell replacement therapies.
    Keywords:  HSPB1; apoptosis; beta-cells; cytoprotection; diabetes mellitus; endoplasmic reticulum stress; heat-shock proteins; proteostasis
  4. Int J Mol Sci. 2021 Sep 21. pii: 10177. [Epub ahead of print]22(18):
      Valosin-containing protein (VCP)/p97, a member of the AAA+ ATPase family, is a molecular chaperone recruited to the endoplasmic reticulum (ER) membrane by binding to membrane adapters (nuclear protein localization protein 4 (NPL4), p47 and ubiquitin regulatory X (UBX) domain-containing protein 1 (UBXD1)), where it is involved in ER-associated protein degradation (ERAD). However, VCP/p97 interacts with many cofactors to participate in different cellular processes that are critical for cancer cell survival and aggressiveness. Indeed, VCP/p97 is reported to be overexpressed in many cancer types and is considered a potential cancer biomarker and therapeutic target. This review summarizes the role of VCP/p97 in different cancers and the advances in the discovery of small-molecule inhibitors with therapeutic potential, focusing on the challenges associated with cancer-related VCP mutations in the mechanisms of resistance to inhibitors.
    Keywords:  AAA+ ATPase; CB-5083; VCP; cancer; p97; prognostic biomarker
  5. EMBO J. 2021 Sep 30. e108008
      The cullin-4-based RING-type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the ˜29 CRL4 substrate receptors reported, the DDB1- and CUL4-associated factor 1 (DCAF1) is essential for cellular survival and growth, and its deregulation has been implicated in tumorigenesis. We carried out biochemical and structural studies to examine the structure and mechanism of the CRL4DCAF1 ligase. In the 8.4 Å cryo-EM map of CRL4DCAF1 , four CUL4-RBX1-DDB1-DCAF1 protomers are organized into two dimeric sub-assemblies. In this arrangement, the WD40 domain of DCAF1 mediates binding with the cullin C-terminal domain (CTD) and the RBX1 subunit of a neighboring CRL4DCAF1 protomer. This renders RBX1, the catalytic subunit of the ligase, inaccessible to the E2 ubiquitin-conjugating enzymes. Upon CRL4DCAF1 activation by neddylation, the interaction between the cullin CTD and the neighboring DCAF1 protomer is broken, and the complex assumes an active dimeric conformation. Accordingly, a tetramerization-deficient CRL4DCAF1 mutant has higher ubiquitin ligase activity compared to the wild-type. This study identifies a novel mechanism by which unneddylated and substrate-free CUL4 ligases can be maintained in an inactive state.
    Keywords:  CRL4/DCAF1; E3 ligases; Oligomerization; Ubiquitin; VprBP
  6. Cells. 2021 Sep 09. pii: 2374. [Epub ahead of print]10(9):
      A hallmark of cancer is dysregulated protein turnover (proteostasis), which involves pathologic ubiquitin-dependent degradation of tumor suppressor proteins, as well as increased oncoprotein stabilization. The latter is due, in part, to mutation within sequences, termed degrons, which are required for oncoprotein recognition by the substrate-recognition enzyme, E3 ubiquitin ligase. Stabilization may also result from the inactivation of the enzymatic machinery that mediates the degradation of oncoproteins. Importantly, inactivation in cancer of E3 enzymes that regulates the physiological degradation of oncoproteins, results in tumor cells that accumulate multiple active oncoproteins with prolonged half-lives, leading to the development of "degradation-resistant" cancer cells. In addition, specific sequences may enable ubiquitinated proteins to evade degradation at the 26S proteasome. While the ubiquitin-proteasome pathway was originally discovered as central for protein degradation, in cancer cells a ubiquitin-dependent protein stabilization pathway actively translates transient mitogenic signals into long-lasting protein stabilization and enhances the activity of key oncoproteins. A central enzyme in this pathway is the ubiquitin ligase RNF4. An intimate link connects protein stabilization with tumorigenesis in experimental models as well as in the clinic, suggesting that pharmacological inhibition of protein stabilization has potential for personalized medicine in cancer. In this review, we highlight old observations and recent advances in our knowledge regarding protein stabilization.
    Keywords:  E3 ubiquitin ligases; RNF4; STUbL; cancer; degradation; degradation-resistant tumors; degron; heterotypic-Ub chains; oncoproteins; proteasome; protein-stabilization; ubiquitin
  7. Cell. 2021 Sep 21. pii: S0092-8674(21)01049-7. [Epub ahead of print]
      Although oxidative phosphorylation is best known for producing ATP, it also yields reactive oxygen species (ROS) as invariant byproducts. Depletion of ROS below their physiological levels, a phenomenon known as reductive stress, impedes cellular signaling and has been linked to cancer, diabetes, and cardiomyopathy. Cells alleviate reductive stress by ubiquitylating and degrading the mitochondrial gatekeeper FNIP1, yet it is unknown how the responsible E3 ligase CUL2FEM1B can bind its target based on redox state and how this is adjusted to changing cellular environments. Here, we show that CUL2FEM1B relies on zinc as a molecular glue to selectively recruit reduced FNIP1 during reductive stress. FNIP1 ubiquitylation is gated by pseudosubstrate inhibitors of the BEX family, which prevent premature FNIP1 degradation to protect cells from unwarranted ROS accumulation. FEM1B gain-of-function mutation and BEX deletion elicit similar developmental syndromes, showing that the zinc-dependent reductive stress response must be tightly regulated to maintain cellular and organismal homeostasis.
    Keywords:  BEX2; BEX3; CUL2; FEM1B; mitochondria; oxidative phosphorylation; reactive oxygen species; reductive stress; ubiquitin
  8. Proc Natl Acad Sci U S A. 2021 Oct 05. pii: e2111391118. [Epub ahead of print]118(40):
      Targeted protein degradation by the ubiquitin-proteasome system represents a new strategy to destroy pathogenic proteins in human diseases, including cancer and neurodegenerative diseases. The immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide have revolutionized the treatment of patients with multiple myeloma (MM) and other hematologic malignancies, but almost all patients eventually develop resistance to IMiDs. CRBN, a substrate receptor of CUL4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase, is a direct target for thalidomide teratogenicity and antitumor activity of IMiDs (now known as Cereblon E3 ligase modulators: CELMoDs). Despite recent advances in developing potent CELMoDs and CRBN-based proteolysis-targeting chimeras (PROTACs), many questions apart from clinical efficacy remain unanswered. CRBN is required for the action of IMiDs, but its protein expression levels do not correlate with intrinsic resistance to IMiDs in MM cells, suggesting other factors involved in regulating resistance to IMiDs. Our recent work revealed that the CRL4CRBN-p97 pathway is required for degradation of natural substrate glutamine synthetase (GS) and neosubstrates. Here, I show that USP15 is a key regulator of the CRL4CRBN-p97 pathway to control stability of GS and neosubstrates IKZF1, IKZF3, CK1-α, RNF166, GSPT1, and BRD4, all of which are crucial drug targets in different types of cancer. USP15 antagonizes ubiquitylation of CRL4CRBN target proteins, thereby preventing their degradation. Notably, USP15 is highly expressed in IMiD-resistant cells, and depletion of USP15 sensitizes these cells to lenalidomide. Inhibition of USP15 represents a valuable therapeutic opportunity to potentiate CELMoD and CRBN-based PROTAC therapies for the treatment of cancer.
    Keywords:  CRBN; IMiDs; PROTACs; USP15; ubiquitin
  9. Elife. 2021 Sep 30. pii: e70885. [Epub ahead of print]10
      Zinc and ring finger 3 (ZNRF3) is a transmembrane E3 ubiquitin ligase that targets Wnt receptors for ubiquitination and lysosomal degradation. Previously we showed that dephosphorylation of an endocytic tyrosine motif (4Y motif) in ZNRF3 by protein tyrosine phosphatase receptor-type kappa (PTPRK) promotes ZNRF3 internalization and Wnt receptor degradation (Chang et al. 2020). However, a responsible protein tyrosine kinase(s) (PTK) phosphorylating the 4Y motif remained elusive. Here we identify the proto-oncogene MET (mesenchymal-epithelial transition factor) as a 4Y kinase. MET binds to ZNRF3 and induces 4Y phosphorylation, stimulated by the MET ligand HGF (hepatocyte growth factor, scatter factor). HGF-MET signalling reduces ZNRF3-dependent Wnt receptor degradation thereby enhancing Wnt/b-catenin signalling. Conversely, depletion or pharmacological inhibition of MET promotes internalization of ZNRF3 and Wnt receptor degradation. We conclude that HGF-MET signalling phosphorylates- and PTPRK dephosphorylates ZNRF3 to regulate ZNRF3 internalization, functioning as a rheostat for Wnt signalling that may offer novel opportunities for therapeutic intervention.
    Keywords:  cell biology; human
  10. Cell Stress Chaperones. 2021 Sep 29.
      Heat shock protein-90 (Hsp90) is an essential molecular chaperone in eukaryotes that plays a vital role in protecting and maintaining the functional integrity of deregulated signaling proteins in tumors. We have previously reported that the stability and activity of the mitotic checkpoint kinase Mps1 depend on Hsp90. In turn, Mps1-mediated phosphorylation Hsp90 regulates its chaperone function and is essential for the mitotic arrest. Cdc14-assisted dephosphorylation of Hsp90 is vital for the mitotic exit. Post-translational regulation of Hsp90 function is also known as the Hsp90 "Chaperone Code." Here, we demonstrate that only the active Mps1 is ubiquitinated on K86, K827, and K848 by the tumor suppressor von Hippel-Lindau (VHL) containing E3 enzyme, in a prolyl hydroxylation-independent manner and degraded in the proteasome. Furthermore, we show that this process regulates cell exit from the mitotic checkpoint. Collectively, our data demonstrates an interplay between the Hsp90 chaperone and VHL degradation machinery in regulating mitosis.
    Keywords:  Chaperone code; Clear cell renal cell carcinoma; Heat shock protein 90; Kinase; Mitotic checkpoint; Molecular chaperones; Mps1; Phosphorylation; Tumor suppressor; Ubiquitination; VHL; Von Hippel-Lindau
  11. Life Sci Alliance. 2021 Dec;pii: e202101182. [Epub ahead of print]4(12):
      The mitochondrial unfolded protein response (mitoUPR) is an evolutionarily conserved pathway that responds to mitochondria insults through transcriptional changes, mediated by the transcription factor ATFS-1/ATF-5, which acts to restore mitochondrial homeostasis. In this work, we characterized the role of ATFS-1 in responding to organismal stress. We found that activation of ATFS-1 is sufficient to cause up-regulation of genes involved in multiple stress response pathways including the DAF-16-mediated stress response pathway, the cytosolic unfolded protein response, the endoplasmic reticulum unfolded protein response, the SKN-1-mediated oxidative stress response pathway, the HIF-1-mediated hypoxia response pathway, the p38-mediated innate immune response pathway, and antioxidant genes. Constitutive activation of ATFS-1 increases resistance to multiple acute exogenous stressors, whereas disruption of atfs-1 decreases stress resistance. Although ATFS-1-dependent genes are up-regulated in multiple long-lived mutants, constitutive activation of ATFS-1 decreases lifespan in wild-type animals. Overall, our work demonstrates that ATFS-1 serves a vital role in organismal survival of acute stressors through its ability to activate multiple stress response pathways but that chronic ATFS-1 activation is detrimental for longevity.
  12. Hepatology. 2021 Sep 28.
      BACKGROUND & AIMS: Hepatitis B virus (HBV) infection has been reported to trigger endoplasmic reticulum (ER) stress and initiate autophagy. However, how ER stress and autophagy influence HBV production remains elusive. Here, we studied the effect of tunicamycin (TM), an N-glycosylation inhibitor and ER stress inducer, on HBV replication and secretion, and examined the underlying mechanisms.APPROACH & RESULTS: PDI (an ER marker), LC3 (an autophagosome marker) and p62 (a typical cargo for autophagic degradation) expression were tested in the liver tissues of patients with chronic HBV infection and hepatoma cell lines. The role of TM treatment in HBV production and trafficking was examined in hepatoma cell lines. TM treatment that mimics HBV infection triggered ER stress and increased autophagosome formation, resulting in enhanced HBV replication and the secretion of subviral particles (SVPs) and naked capsids. Additionally, TM reduced the number of early endosomes and HBV surface antigen (HBsAg) localization in this compartment, causing HBsAg/SVPs accumulate in the ER. Thus, TM-induced autophagosome formation serves as an alternative pathway for HBsAg/SVPs trafficking. Importantly, TM inhibited autophagosome-lysosome fusion, accompanied by enhanced autophagosome-late endosome/multivesicular body (MVB) fusion to release HBsAg/SVPs through or along with exosome release. Notably, TM treatment inhibited the HBsAg glycosylation, resulting in impairment of the HBV virions envelopment and secretion, but it was not critical for HBsAg/SVPs trafficking in our cell systems.
    CONCLUSIONS: TM-induced ER stress and autophagic flux promoted HBV replication and the release of SVPs and naked capsids through the autophagosome-late endosome/MVB axis.
    Keywords:  Hepatitis B virus; autophagosome; multivesicular bodies/MVBs; vesicular trafficking
  13. Nat Commun. 2021 Sep 27. 12(1): 5666
      In eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70 Ssb, atypical Hsp70 Ssz1 and J-domain protein cochaperone Zuo1. Zuo1 binds the ribosome at the tunnel exit. Zuo1 also binds Ssz1, tethering it to the ribosome, while its J-domain stimulates Ssb's ATPase activity to drive efficient nascent chain interaction. But the function of Ssz1 and how Ssb engages at the ribosome are not well understood. Employing in vivo site-specific crosslinking, we found that Ssb(ATP) heterodimerizes with Ssz1. Ssb, in a manner consistent with the ADP conformation, also crosslinks to ribosomal proteins across the tunnel exit from Zuo1. These two modes of Hsp70 Ssb interaction at the ribosome suggest a functionally efficient interaction pathway: first, Ssb(ATP) with Ssz1, allowing optimal J-domain and nascent chain engagement; then, after ATP hydrolysis, Ssb(ADP) directly with the ribosome.
  14. Cell. 2021 Sep 20. pii: S0092-8674(21)01053-9. [Epub ahead of print]
      Despite remarkable clinical efficacy of immune checkpoint blockade (ICB) in cancer treatment, ICB benefits for triple-negative breast cancer (TNBC) remain limited. Through pooled in vivo CRISPR knockout (KO) screens in syngeneic TNBC mouse models, we found that deletion of the E3 ubiquitin ligase Cop1 in cancer cells decreases secretion of macrophage-associated chemokines, reduces tumor macrophage infiltration, enhances anti-tumor immunity, and strengthens ICB response. Transcriptomics, epigenomics, and proteomics analyses revealed that Cop1 functions through proteasomal degradation of the C/ebpδ protein. The Cop1 substrate Trib2 functions as a scaffold linking Cop1 and C/ebpδ, which leads to polyubiquitination of C/ebpδ. In addition, deletion of the E3 ubiquitin ligase Cop1 in cancer cells stabilizes C/ebpδ to suppress expression of macrophage chemoattractant genes. Our integrated approach implicates Cop1 as a target for improving cancer immunotherapy efficacy in TNBC by regulating chemokine secretion and macrophage infiltration in the tumor microenvironment.
    Keywords:  C/ebpδ; CRISPR screening; Cop1; E3 ubiquitin ligase; immunotherapy; triple-negative breast cancer
  15. Mol Cell Biol. 2021 Sep 27. MCB0013521
      RNA polymerase II-associated factor 1 (PAF1)/pancreatic differentiation 2 (PD2) is a core subunit of the human PAF1 complex (PAF1C) that regulates the RNA polymerase II function during transcriptional elongation. PAF1/PD2 has also been linked to the oncogenesis of pancreatic ductal adenocarcinoma (PDAC). Here, we report that PAF1/PD2 undergoes post-translational modification (PTM) through SUMOylation, enhancing the radiation resistance of PDAC cells. We identified that PAF1/PD2 is preferentially modified by small ubiquitin-related modifier 1 (SUMO 1), and mutating the residues (K)-150 and 154 by site-directed mutagenesis reduces the SUMOylation. Interestingly, PAF1/PD2 was found to directly interact with the promyelocytic leukemia (PML) protein in response to radiation and inhibiton of PAF1/PD2 SUMOylation at K-150/154 affects its interaction with PML. Our results demonstrate that SUMOylation of PAF1/PD2 increased in the radiated pancreatic cancer cells. Further, inhibition of SUMOylation or PML reduces the cell growth and proliferation of PDAC cells after radiation treatment. These results suggest that SUMOylation of PAF1/PD2 interacts with PTM for PDAC cell survival. Furthermore, abolishing the SUMOylation in PDAC cells enhances the effectiveness of radiotherapy. Overall, our results demonstrate a novel PTM and PAF1/PD2 interaction through SUMOylation and inhibiting the SUMOylation of PAF1/PD2 enhance the therapeutic efficacy for PDAC.
  16. Mol Biol Cell. 2021 Sep 29. mbcE21040219
      The family of Bro1 proteins coordinates the activity of the Endosomal Sorting Complexes Required for Transport (ESCRTs) to mediate a number of membrane remodeling events. These events culminate in membrane scission catalyzed by ESCRT-III, whose polymerization and disassembly is controlled by the AAA-ATPase, Vps4. Bro1-family members Alix and HD-PTP as well as yeast Bro1 have a central 'V' domains that non-covalently bind Ub and connect ubiquitinated proteins to ESCRT-driven functions such as the incorporation of ubiquitinated membrane proteins into intralumenal vesicles of multivesicular bodies. Recently, it was discovered that the V domain of yeast Bro1 binds the MIT domain of Vps4 to stimulate its ATPase activity. Here we determine the structural basis for how the V domain of human HD-PTP binds ubiquitin. The HD-PTP V domain also binds the MIT domain of Vps4 and ubiquitin-binding to the HD-PTP V domain enhances its ability to stimulate Vps4 ATPase activity. Additionally, we found V domains of both HD-PTP and Bro1 bind CHMP5 and Vps60, respectively, providing another potential molecular mechanism to alter Vps4 activity. These data support a model whereby contacts between ubiquitin, ESCRT-III, and Vps4 by V domains of the Bro1 family may coordinate late events in ESCRT-driven membrane remodeling events.
  17. Cell Rep. 2021 Sep 28. pii: S2211-1247(21)01216-X. [Epub ahead of print]36(13): 109762
      The evolutionarily conserved ULK1 kinase complex acts as gatekeeper of canonical autophagy and regulates induction of autophagosome biogenesis. To better understand control of ULK1 and analyze whether ULK1 has broader functions that are also linked to the later steps of autophagy, we perform comprehensive phosphoproteomic analyses. Combining in vivo with in vitro data, we identify numerous direct ULK1 target sites within autophagy-relevant proteins that are critical for autophagosome maturation and turnover. In addition, we highlight an intimate crosstalk between ULK1 and several phosphatase complexes. ULK1 is not only a PP2A target but also directly phosphorylates the regulatory PP2A subunit striatin, activating PP2A and serving as positive feedback to promote autophagy-dependent protein turnover. Thus, ULK1 and phosphatase activities are tightly coordinated to robustly regulate protein degradation by autophagy.
    Keywords:  PP2A; STRIPAK; STRN; autophagy; in vitro kinase assay; kinase; mass spectrometry; phosphatase; phosphoproteomics; signaling
  18. J Immunol. 2021 Sep 27. pii: ji2100258. [Epub ahead of print]
      Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) regulate the vesicle transport machinery in phagocytic cells. Within the secretory pathway, Sec22b is an endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-resident SNARE that controls phagosome maturation and function in macrophages and dendritic cells. The secretory pathway controls the release of cytokines and may also impact the secretion of NO, which is synthesized by the Golgi-active inducible NO synthase (iNOS). Whether ERGIC SNARE Sec22b controls NO and cytokine secretion is unknown. Using murine bone marrow-derived dendritic cells, we demonstrated that inducible NO synthase colocalizes with ERGIC/Golgi markers, notably Sec22b and its partner syntaxin 5, in the cytoplasm and at the phagosome. Pharmacological blockade of the secretory pathway hindered NO and cytokine release, and inhibited NF-κB translocation to the nucleus. Importantly, RNA interference-mediated silencing of Sec22b revealed that NO and cytokine production were abrogated at the protein and mRNA levels. This correlated with reduced nuclear translocation of NF-κB. We also found that Sec22b co-occurs with NF-κB in both the cytoplasm and nucleus, pointing to a role for this SNARE in the shuttling of NF-κB. Collectively, our data unveiled a novel function for the ERGIC/Golgi, and its resident SNARE Sec22b, in the production and release of inflammatory mediators.
  19. Cell Calcium. 2021 Sep 20. pii: S0143-4160(21)00131-7. [Epub ahead of print]100 102477
      The aberrant release of endoplasmic reticulum (ER) calcium leads to the disruption of intracellular calcium homeostasis, which is associated with the occurrence of ER stress and closely related to the pathogenesis of liver damage. Mannan-binding lectin (MBL) is a soluble calcium-dependent protein synthesized primarily in hepatocytes and is a pattern recognition molecule in the innate immune system. MBL deficiency is highly prevalent in the population and has been reported to be associated with susceptibility to several liver diseases. We here showed that genetic MBL ablation strongly sensitized mice to ER stress-induced liver injury. Mechanistic studies established that MBL directly interacted with ER-resident chaperone immunoglobulin heavy chain binding protein (BiP), and MBL deficiency accelerated the separation of PKR-like ER kinase (PERK) from BiP during hepatic ER stress. Moreover, MBL deficiency led to enhanced activation of the PERK-C/EBP-homologous protein (CHOP) pathway and initiates an inositol 1,4,5-trisphosphate receptor (IP3R)-mediated calcium release from the ER, thereby aggravating the hepatic ER stress response. Our results demonstrate an unexpected function of MBL in ER calcium homeostasis and ER stress response, thus providing new insight into the liver injury related to ER stress in patients with MBL deficiency.
    Keywords:  Calcium; Endoplasmic reticulum stress; Inositol 1,4,5-trisphosphate receptor; Liver injury; Mannan-binding lectin
  20. Autophagy. 2021 Sep 29. 1-24
      Owing to the dominant functions of mitochondria in multiple cellular metabolisms and distinct types of regulated cell death, maintaining a functional mitochondrial network is fundamental for the cellular homeostasis and body fitness in response to physiological adaptations and stressed conditions. The process of mitophagy, in which the dysfunctional or superfluous mitochondria are selectively engulfed by autophagosome and subsequently degraded in lysosome, has been well formulated as one of the major mechanisms for mitochondrial quality control. To date, the PINK1-PRKN-dependent and receptors (including proteins and lipids)-dependent pathways have been characterized to determine the mitophagy in mammalian cells. The mitophagy is highly responsive to the dynamics of endogenous metabolites, including iron-, calcium-, glycolysis-TCA-, NAD+-, amino acids-, fatty acids-, and cAMP-associated metabolites. Herein, we summarize the recent advances toward the molecular details of mitophagy regulation in mammalian cells. We also highlight the key regulations of mammalian mitophagy by endogenous metabolites, shed new light on the bidirectional interplay between mitophagy and cellular metabolisms, with attempting to provide a perspective insight into the nutritional intervention of metabolic disorders with mitophagy deficit.Abbreviations: acetyl-CoA: acetyl-coenzyme A; ACO1: aconitase 1; ADCYs: adenylate cyclases; AMPK: AMP-activated protein kinase; ATM: ATM serine/threonine kinase; BCL2L1: BCL2 like 1; BCL2L13: BCL2 like 13; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; Ca2+: calcium ion; CALCOCO2: calcium binding and coiled-coil domain 2; CANX: calnexin; CO: carbon monoxide; CYCS: cytochrome c, somatic; DFP: deferiprone; DNM1L: dynamin 1 like; ER: endoplasmic reticulum; FKBP8: FKBP prolyl isomerase 8; FOXO3: forkhead box O3; FTMT: ferritin mitochondrial; FUNDC1: FUN14 domain containing 1; GABA: γ-aminobutyric acid; GSH: glutathione; HIF1A: hypoxia inducible factor 1 subunit alpha; IMMT: inner membrane mitochondrial protein; IRP1: iron regulatory protein 1; ISC: iron-sulfur cluster; ITPR2: inositol 1,4,5-trisphosphate type 2 receptor; KMO: kynurenine 3-monooxygenase; LIR: LC3 interacting region; MAM: mitochondria-associated membrane; MAP1LC3: microtubule associated protein 1 light chain 3; MFNs: mitofusins; mitophagy: mitochondrial autophagy; mPTP: mitochondrial permeability transition pore; MTOR: mechanistic target of rapamycin kinase; NAD+: nicotinamide adenine dinucleotide; NAM: nicotinamide; NMN: nicotinamide mononucleotide; NO: nitric oxide; NPA: Niemann-Pick type A; NR: nicotinamide riboside; NR4A1: nuclear receptor subfamily 4 group A member 1; NRF1: nuclear respiratory factor 1; OPA1: OPA1 mitochondrial dynamin like GTPase; OPTN: optineurin; PARL: presenilin associated rhomboid like; PARPs: poly(ADP-ribose) polymerases; PC: phosphatidylcholine; PHB2: prohibitin 2; PINK1: PTEN induced kinase 1; PPARG: peroxisome proliferator activated receptor gamma; PPARGC1A: PPARG coactivator 1 alpha; PRKA: protein kinase AMP-activated; PRKDC: protein kinase, DNA-activated, catalytic subunit; PRKN: parkin RBR E3 ubiquitin protein ligase; RHOT: ras homolog family member T; ROS: reactive oxygen species; SIRTs: sirtuins; STK11: serine/threonine kinase 11; TCA: tricarboxylic acid; TP53: tumor protein p53; ULK1: unc-51 like autophagy activating kinase 1; VDAC1: voltage dependent anion channel 1.
    Keywords:  Cell metabolism; metabolite; mitochondria; mitophagy; mitophagy receptor
  21. J Am Chem Soc. 2021 Oct 01.
      The targeted degradation of membrane proteins would afford an attractive and general strategy for treating various diseases that remain difficult with the current proteolysis-targeting chimera (PROTAC) methodology. We herein report a covalent nanobody-based PROTAC strategy, termed GlueTAC, for targeted membrane protein degradation with high specificity and efficiency. We first established a mass-spectrometry-based screening platform for the rapid development of a covalent nanobody (GlueBody) that allowed proximity-enabled cross-linking with surface antigens on cancer cells. By conjugation with a cell-penetrating peptide and a lysosomal-sorting sequence, the resulting GlueTAC chimera triggered the internalization and degradation of programmed death-ligand 1 (PD-L1), which provides a new avenue to target and degrade cell-surface proteins.
  22. Mol Cell. 2021 Sep 21. pii: S1097-2765(21)00736-X. [Epub ahead of print]
      mRNA translation is a highly conserved and tightly controlled mechanism for protein synthesis. Despite protein quality control mechanisms, amino acid shortage in melanoma induces aberrant proteins by ribosomal frameshifting. The extent and the underlying mechanisms related to this phenomenon are yet unknown. Here, we show that tryptophan depletion-induced ribosomal frameshifting is a widespread phenomenon in cancer. We termed this event sloppiness and strikingly observed its association with MAPK pathway hyperactivation. Sloppiness is stimulated by RAS activation in primary cells, suppressed by pharmacological inhibition of the oncogenic MAPK pathway in sloppy cells, and restored in cells with acquired resistance to MAPK pathway inhibition. Interestingly, sloppiness causes aberrant peptide presentation at the cell surface, allowing recognition and specific killing of drug-resistant cancer cells by T lymphocytes. Thus, while oncogenes empower cancer progression and aggressiveness, they also expose a vulnerability by provoking the production of aberrant peptides through sloppiness.
    Keywords:  MAPK pathway; T cell killing; T cell recognition; aberrant peptides; acquired drug resistance; antigen presentation; cancer; protein synthesis; ribosomal frameshifting
  23. Curr Biol. 2021 Sep 27. pii: S0960-9822(21)01143-X. [Epub ahead of print]31(18): R1084-R1087
      Glycosylphosphatidylinositol-anchored proteins are a class of lipid-anchored membrane proteins found at the surface of all eukaryotic cells. New work provides genome-wide insights into mechanisms that mediate quality control of the folding of this important protein family.
  24. iScience. 2021 Sep 24. 24(9): 103086
      Using antibody arrays, we found that the RNA helicase DDX3 modulates the expression of secreted signaling factors in oral squamous cell carcinoma (OSCC) cells. Ribo-seq analysis confirmed amphiregulin (AREG) as a translational target of DDX3. AREG exerts important biological functions in cancer, including promoting cell migration and paracrine effects of OSCC cells and reprogramming the tumor microenvironment (TME) of OSCC in mice. DDX3-mediated translational control of AREG involves its 3'-untranslated region. Proteomics identified the signal recognition particle (SRP) as an unprecedented interacting partner of DDX3. DDX3 and SRP54 were located near the endoplasmic reticulum, regulated the expression of a common set of secreted factors, and were essential for targeting AREG mRNA to membrane-bound polyribosomes. Finally, OSCC-associated mutant DDX3 increased the expression of AREG, emphasizing the role of DDX3 in tumor progression via SRP-dependent, endoplasmic reticulum-associated translation. Therefore, pharmacological targeting of DDX3 may inhibit the tumor-promoting functions of the TME.
    Keywords:  Cancer; Cell biology; Molecular biology
  25. Blood Cancer Discov. 2021 Sep;2(5): 468-483
      Proteasome inhibitor bortezomib induces apoptosis in multiple myeloma (MM) cells, and has transformed patient outcome. Using in vitro as well as in vivo immunodeficient and immunocompetent murine MM models, we here show that bortezomib also triggers immunogenic cell death (ICD) characterized by exposure of calreticulin on dying MM cells, phagocytosis of tumor cells by dendritic cells, and induction of MM specific immunity. We identify a bortezomib-triggered specific ICD-gene signature associated with better outcome in two independent MM patient cohorts. Importantly, bortezomib stimulates MM cells immunogenicity via activation of cGAS/STING pathway and production of type-I interferons; and STING agonists significantly potentiate bortezomib-induced ICD. Our studies therefore delineate mechanisms whereby bortezomib exerts immunotherapeutic activity, and provide the framework for clinical trials of STING agonists with bortezomib to induce potent tumor-specific immunity and improve patient outcome in MM.
    Keywords:  STING; bortezomib; immunogenic cell death (ICD); immunotherapy; multiple myeloma
  26. Trends Cancer. 2021 Sep 24. pii: S2405-8033(21)00176-X. [Epub ahead of print]
      Increasing evidence indicates that a mitochondria-specific stress response referred to as the 'mitochondrial unfolded protein response' (UPRmt) is activated to maintain mitochondrial integrity and support tumor growth. In this forum article, we discuss the recent advances and current challenges in therapeutically targeting UPRmt in cancer.
    Keywords:  cancer; mitochondrial chaperonins; mitochondrial proteases; mitochondrial proteostasis; mitochondrial unfolded protein response
  27. Biochemistry. 2021 Sep 27.
      The advent of multi-specific targeted protein degradation (TPD) therapies has made it possible to drug targets that have long been considered to be inaccessible. For this reason, the foremost TPD modalities - molecular glues and proteolysis targeting chimeras (PROTACs) -have been widely adopted and developed in therapeutic programs across the pharmaceutical and biotechnology industries. While there are many clear advantages to these two approaches, there are also blind spots. Specifically, PROTACs and molecular glues are inherently mechanistically analogous in that targets of both are degraded via the 26s proteasome; however, not all disease-relevant targets are suitable for ubiquitin proteasome system (UPS)-mediated degradation. The alternative mammalian protein degradation pathway, the autophagy-lysosome system (or ALS), is capable of degrading targets that elude the UPS such as long-lived proteins, insoluble protein aggregates, and even abnormal organelles. Emerging TPD strategies- such as ATTEC, AUTAC, and LYTAC- take advantage of the substrate diversity of the ALS to greatly expand the clinical utility of TPD. In this Perspective, we will discuss the array of current TPD modalities, with a focus on critical evaluation of these novel ALS-mediated degradation techniques.
  28. Cell Death Differ. 2021 Sep 28.
      Renal fibrosis and inflammation are critical for the initiation and progression of hypertensive renal disease (HRD). However, the signaling mechanisms underlying their induction are poorly understood, and the role of tripartite motif-containing protein 31 (TRIM31), an E3 ubiquitin ligase, in HRD remains unclear. This study aimed to elucidate the role of TRIM31 in the pathogenesis of HRD, discover targets of TRIM31, and explore the underlying mechanisms. Pathological specimens of human HRD kidney were collected and an angiotensin II (AngII)-induced HRD mouse model was developed. We found that TRIM31 was markedly reduced in both human and mouse HRD renal tissues. A TRIM31-/- mice was thus constructed and showed significantly aggravated hypertension-induced renal dysfunction, fibrosis, and inflammation, following chronic AngII infusion compared with TRIM31+/+ mice. In contrast, overexpression of TRIM31 by injecting adeno-associated virus (AAV) 9 into C57BL/6J mice markedly ameliorated renal dysfunction, fibrotic and inflammatory response in AngII-induced HRD relative to AAV-control mice. Mechanistically, TRIM31 interacted with and catalyzed the K48-linked polyubiquitination of lysine 72 on Mitogen-activated protein kinase kinase kinase 7 (MAP3K7), followed by the proteasomal degradation of MAP3K7, which further negatively regulated TGF-β1-mediated Smad and MAPK/NF-κB signaling pathways. In conclusion, this study has demonstrated for the first time that TRIM31 serves as an important regulator in AngII-induced HRD by promoting MAP3K7 K48-linked polyubiquitination and inhibiting the TGF-β1 signaling pathway.
  29. Angew Chem Int Ed Engl. 2021 Sep 29.
      The autophagic ubiquitin-like protein LC3 functions through interactions with LC3-interaction regions (LIRs) of other autophagy proteins including autophagy receptors, which stands out as a promising protein-protein interaction (PPI) target for the intervention of autophagy. Post-translational modifications like acetylation of Lys49 on the LIR-interacting surface could disrupt the interaction, offering an opportunity to design covalent small molecules interfering the interface. Through screening covalent compounds, we discover a small molecule modulator of LC3A/B that covalently modifies LC3A/B protein at Lys49. Activity-based protein profiling (ABPP) based evaluations reveal that a derivative molecule DC-LC3in-D5 exhibits a potent covalent reactivity and selectivity to LC3A/B in HeLa cells. DC-LC3in-D5 compromises LC3B lipidation in vitro and in HeLa cells, leading to deficiency in the formation of autophagic structures and autophagic substrate degradation. DC-LC3in-D5 could serve as a powerful tool for autophagy research as well as for therapeutic interventions.
    Keywords:  LC3, covalent modification, post-translational modification, autophagy
  30. Biochem J. 2021 Sep 30. 478(18): 3467-3483
      Mutations in breast cancer type 1 susceptibility protein (BRCA1) and its heterodimeric binding partner BARD1 confer a high risk for the development of breast and ovarian cancers. The sole enzymatic function of the BRCA1/BARD1 complex is as a RING-type E3 ubiquitin (Ub) ligase, leading to the deposition of Ub signals onto a variety of substrate proteins. Distinct types of Ub signals deposited by BRCA1/BARD1 (i.e. degradative vs. non-degradative; mono-Ub vs. poly-Ub chains) on substrate proteins mediate aspects of its function in DNA double-stranded break repair, cell-cycle regulation, and transcriptional regulation. While cancer-predisposing mutations in both subunits lead to the inactivation of BRCA1/BARD1 ligase activity, controversy remains as to whether its Ub ligase activity directly inhibits tumorigenesis. Investigation of BRCA1/BARD1 substrates using rigorous, well-validated mutants and experimental systems will ultimately clarify the role of its ligase activity in cancer and possibly establish prognostic and diagnostic metrics for patients with mutations. In this review, we discuss the Ub ligase function of BRCA1/BARD1, highlighting experimental approaches, mechanistic considerations, and reagents that are useful in the study of substrate ubiquitylation. We also discuss the current understanding of two well-established BRCA1/BARD1 substrates (nucleosomal H2A and estrogen receptor α) and several recently discovered substrates (p50, NF2, Oct1, and LARP7). Lessons from the current body of work should provide a road map to researchers examining novel substrates and biological functions attributed to BRCA1/BARD1 Ub ligase activity.
    Keywords:  BARD1; BRCA1; ubiquitin ligases; ubiquitin signaling; ubiquitins
  31. Nat Commun. 2021 Sep 29. 12(1): 5708
      Ufmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1's active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1's conjugation machinery and may serve as a basis for the development of ufmylation inhibitors.
  32. Mol Cell Proteomics. 2021 Sep 27. pii: S1535-9476(21)00126-2. [Epub ahead of print] 100154
      Robust methods for deep-scale enrichment and site-specific identification of ubiquitylation sites are necessary for characterizing the myriad roles of protein ubiquitylation. To this end we previously developed UbiFast, a sensitive method for highly multiplexed ubiquitylation profiling where K-ɛ-GG peptides are enriched with anti-K-ε-GG antibody and labeled on-antibody with isobaric labeling reagents for sample multiplexing. Here, we present robotic automation of the UbiFast method using a magnetic bead-conjugated K-ε-GG antibody (mK-ε-GG) and a magnetic particle processor. We report the identification of ∼20,000 ubiquitylation sites from a TMT10-plex with 500 μg input per sample processed in ∼2 hours. Automation of the UbiFast method greatly increased the number of identified and quantified ubiquitylation sites, improved reproducibility and significantly reduced processing time. The automated method also significantly reduced variability across process replicates compared to the manual method. The workflow enables processing of up to 96 samples in a single day making it suitable to study ubiquitylation in large sample sets. Here we demonstrate the applicability of the method to profile small amounts of tissue using breast cancer patient-derived xenograft (PDX) tissue samples.
  33. STAR Protoc. 2021 Sep 17. 2(3): 100809
      Senescent cells constantly experience stressful conditions and restrain their protein translation to cope with it. Here, we present a detailed protocol to measure the rate of global protein synthesis using L-azidohomoalanine (L-AHA)-based click chemistry in human senescent fibroblasts. We optimized several aspects of the procedure, including senescence induction, a flow cytometry analysis of senescent cells, and the duration of L-AHA incorporation. This protocol uses senescent human fibroblasts but can be applied to other types of cells or circumstances. For complete details on the use and execution of this protocol, please refer to Lee et al. (2021).
    Keywords:  Cell Biology; Cell-based Assays; Flow Cytometry/Mass Cytometry; Molecular Biology; Molecular/Chemical Probes; Protein Biochemistry
  34. Trends Biochem Sci. 2021 Sep 25. pii: S0968-0004(21)00187-0. [Epub ahead of print]
      Lipid droplets (LDs) are the main organelles for lipid storage, and their surfaces contain unique proteins with diverse functions, including those that facilitate the deposition and mobilization of LD lipids. Among organelles, LDs have an unusual structure with an organic, hydrophobic oil phase covered by a phospholipid monolayer. The unique properties of LD monolayer surfaces require proteins to localize to LDs by distinct mechanisms. Here we review the two pathways known to mediate direct LD protein localization: the CYTOLD pathway mediates protein targeting from the cytosol toLDs, and the ERTOLD pathway functions in protein targeting from the endoplasmic reticulum toLDs. We describe the emerging principles for each targeting pathway in animal cells and highlight open questions in the field.
    Keywords:  amphipathic helix; bilayer membranes; hairpin motif; lipid droplets; phospholipid monolayer; protein targeting