bims-proteo Biomed News
on Proteostasis
Issue of 2021‒02‒14
forty-eight papers selected by
Eric Chevet
INSERM


  1. Mol Cell. 2021 Feb 03. pii: S1097-2765(20)30988-6. [Epub ahead of print]
    Heider M, Eichner R, Stroh J, Morath V, Kuisl A, Zecha J, Lawatscheck J, Baek K, Garz AK, Rudelius M, Deuschle FC, Keller U, Lemeer S, Verbeek M, Götze KS, Skerra A, Weber WA, Buchner J, Schulman BA, Kuster B, Fernández-Sáiz V, Bassermann F.
      The complex architecture of transmembrane proteins requires quality control (QC) of folding, membrane positioning, and trafficking as prerequisites for cellular homeostasis and intercellular communication. However, it has remained unclear whether transmembrane protein-specific QC hubs exist. Here we identify cereblon (CRBN), the target of immunomodulatory drugs (IMiDs), as a co-chaperone that specifically determines chaperone activity of HSP90 toward transmembrane proteins by means of counteracting AHA1. This function is abrogated by IMiDs, which disrupt the interaction of CRBN with HSP90. Among the multiple transmembrane protein clients of CRBN-AHA1-HSP90 revealed by cell surface proteomics, we identify the amino acid transporter LAT1/CD98hc as a determinant of IMiD activity in multiple myeloma (MM) and present an Anticalin-based CD98hc radiopharmaceutical for MM radio-theranostics. These data establish the CRBN-AHA1-HSP90 axis in the biogenesis of transmembrane proteins, link IMiD activity to tumor metabolism, and nominate CD98hc and LAT1 as attractive diagnostic and therapeutic targets in MM.
    Keywords:  CD98hc; CRBN; HSP90; IMiDs; LAT1; chaperones; multiple myeloma; protein quality control; radio-theranostics; ubiquitin
    DOI:  https://doi.org/10.1016/j.molcel.2020.12.046
  2. Matrix Biol. 2021 Feb 05. pii: S0945-053X(21)00014-7. [Epub ahead of print]
    Mende H, Müller S.
      The nucleolus functions as the cellular hub for the initiation and early steps of ribosome biogenesis. Ribosomes are key components of the translation machinery and, accordingly, their abundance needs to be adjusted to the cellular energy status. Further, to ensure translational fidelity, the integrity and quality of ribosomes needs to be monitored under conditions of cellular stress. Stressful insults, such as nutrient, genotoxic or proteotoxic stress, interfere with ribosome biogenesis and activate a cellular response referred to as nucleolar stress. This nucleolar stress response typically affects nucleolar integrity and is intricately linked to the activation of protein quality control pathways, including (i) the ubiquitin proteasome system (UPS) and (ii) the autophagy machinery, to restore cellular proteostasis. Here we will review some key features of the nucleolar stress response with a particular focus on the role of the UPS and autophagy in this process.
    Keywords:  Autophagy; nucleolar stress; p53; ribosome biogenesis; ubiquitin-proteasome system (UPS)
    DOI:  https://doi.org/10.1016/j.matbio.2021.02.001
  3. EMBO Rep. 2021 Feb 08. e50815
    Spits M, Heesterbeek IT, Voortman LM, Akkermans JJ, Wijdeven RH, Cabukusta B, Neefjes J.
      The endoplasmic reticulum (ER) is the largest organelle contacting virtually every other organelle for information exchange and control of processes such as transport, fusion, and fission. Here, we studied the role of the other organelles on ER network architecture in the cell periphery. We show that the co-migration of the ER with other organelles, called ER hitchhiking facilitated by late endosomes and lysosomes is a major mechanism controlling ER network architecture. When hitchhiking occurs, emerging ER structures may fuse with the existing ER tubules to alter the local ER architecture. This couples late endosomal/lysosomal positioning and mobility to ER network architecture. Conditions restricting late endosomal movement-including cell starvation-or the depletion of tether proteins that link the ER to late endosomes reduce ER dynamics and limit the complexity of the peripheral ER network architecture. This indicates that among many factors, the ER is controlled by late endosomal movement resulting in an alteration of the ER network architecture.
    Keywords:  endoplasmic reticulum; late endosomes; membrane contact sites; organelle hitchhiking; starvation
    DOI:  https://doi.org/10.15252/embr.202050815
  4. Biomedicines. 2021 Feb 05. pii: 156. [Epub ahead of print]9(2):
    Siwecka N, Rozpędek-Kamińska W, Wawrzynkiewicz A, Pytel D, Diehl JA, Majsterek I.
      Inositol-requiring enzyme type 1 (IRE1) is a serine/threonine kinase acting as one of three branches of the Unfolded Protein Response (UPR) signaling pathway, which is activated upon endoplasmic reticulum (ER) stress conditions. It is known to be capable of inducing both pro-survival and pro-apoptotic cellular responses, which are strictly related to numerous human pathologies. Among others, IRE1 activity has been confirmed to be increased in cancer, neurodegeneration, inflammatory and metabolic disorders, which are associated with an accumulation of misfolded proteins within ER lumen and the resulting ER stress conditions. Emerging evidence suggests that genetic or pharmacological modulation of IRE1 may have a significant impact on cell viability, and thus may be a promising step forward towards development of novel therapeutic strategies. In this review, we extensively describe the structural analysis of IRE1 molecule, the molecular dynamics associated with IRE1 activation, and interconnection between it and the other branches of the UPR with regard to its potential use as a therapeutic target. Detailed knowledge of the molecular characteristics of the IRE1 protein and its activation may allow the design of specific kinase or RNase modulators that may act as drug candidates.
    Keywords:  Regulated IRE1-Dependent Decay (RIDD); Unfolded Protein Response (UPR); apoptosis; c-Jun N-terminal kinase (JNK); drug development; endoplasmic reticulum (ER) stress; factor X-box binding protein 1 (XBP1); inositol-requiring enzyme type 1 (IRE1)
    DOI:  https://doi.org/10.3390/biomedicines9020156
  5. J Biol Chem. 2021 Feb 05. pii: S0021-9258(21)00155-1. [Epub ahead of print] 100383
    Lysyk L, Brassard R, Arutyunova E, Siebert V, Jiang Z, Takyi E, Morrison M, Young HS, Lemberg MK, O'Donoghue AJ, Lemieux MJ.
      The rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac/Diablo, which have crucial roles in mitochondrial quality control and apoptosis. However, the catalytic properties of PARL, including the effect of lipids on the protease, have never been characterized in vitro. To address this, we isolated human PARL expressed in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show PARL activity in detergent is enhanced by cardiolipin, a lipid enriched in the mitochondrial inner membrane. Significantly higher turnover rates were observed for PARL reconstituted in proteoliposomes, with Smac/Diablo being cleaved most rapidly at a rate of 1 min-1. In contrast, PGAM5 is cleaved with the highest efficiency (kcat/KM) compared to PINK1 and Smac/Diablo. In proteoliposomes, a truncated β-cleavage form of PARL, a physiological form known to affect mitochondrial fragmentation, is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5 and Smac/Diablo. Multiplex profiling of 228 peptides reveals that PARL prefers substrates with a bulky side chain such as Phe in P1, which is distinct from the preference for small side chain residues typically found with bacterial rhomboid proteases. This study using recombinant PARL provides fundamental insights into its catalytic activity and substrate preferences that enhance our understanding of its role in mitochondrial function and has implications for specific inhibitor design.
    Keywords:  GlpG; PGAM5; PINK1; Smac/Diablo; intramembrane proteolysis; membrane protease; mitochondria; rhomboid protease
    DOI:  https://doi.org/10.1016/j.jbc.2021.100383
  6. FEBS J. 2021 Feb 11.
    Hattori T, Hanafusa K, Wada I, Hosokawa N.
      Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). In mammalian cells, the HRD1-SEL1L membrane ubiquitin ligase complex plays a central role in this process. However, SEL1L is inherently unstable, and excess SEL1L is also degraded by ERAD. Accordingly, when proteasome activity is inhibited, multiple degradation intermediates of SEL1L appear in the cytosol. In this study, we searched for factors that inhibit SEL1L degradation, and identified OS-9 and XTP3-B, two ER lectins that regulate glycoprotein ERAD. SEL1L degradation was characterized by a ladder of degradation products, and the C-terminal Pro-rich region of SEL1L was responsible for generation of this pattern. In the cytosol, these degradation intermediates stimulated aggregation of polyglutamine-expanded Huntingtin protein (Htt-polyQ-GFP) by interacting with aggregation-prone proteins, including Htt-polyQ-GFP. Collectively, our findings indicate that peptide fragments of ER proteins generated during ERAD may affect protein aggregation in the cytosol, revealing the interconnection of protein homeostasis across subcellular compartments.
    Keywords:  ER-associated degradation (ERAD); HRD1-SEL1L ubiquitin ligase complex; OS-9; XTP3-B; polyQ aggregation
    DOI:  https://doi.org/10.1111/febs.15761
  7. Nat Cell Biol. 2021 Feb 11.
    Wei Y, Lee NN, Pan L, Dhakshnamoorthy J, Sun LL, Zofall M, Wheeler D, Grewal SIS.
      Cell proliferation and differentiation require signalling pathways that enforce appropriate and timely gene expression. We find that Tor2, the catalytic subunit of the TORC1 complex in fission yeast, targets a conserved nuclear RNA elimination network, particularly the serine and proline-rich protein Pir1, to control gene expression through RNA decay and facultative heterochromatin assembly. Phosphorylation by Tor2 protects Pir1 from degradation by the ubiquitin-proteasome system involving the polyubiquitin Ubi4 stress-response protein and the Cul4-Ddb1 E3 ligase. This pathway suppresses widespread and untimely gene expression and is critical for sustaining cell proliferation. Moreover, we find that the dynamic nature of Tor2-mediated control of RNA elimination machinery defines gene expression patterns that coordinate fundamental chromosomal events during gametogenesis, such as meiotic double-strand-break formation and chromosome segregation. These findings have important implications for understanding how the TOR signalling pathway reprogrammes gene expression patterns and contributes to diseases such as cancer.
    DOI:  https://doi.org/10.1038/s41556-021-00631-y
  8. Autophagy. 2021 Feb 08. 1-3
    Vats S, Galli T.
      Together with the proteasome, macroautophagy is a main pathway for the degradation of intracellular elements. Endoplasmic reticulum (ER)-autophagy i.e. reticulophagy/ER-phagy leads to the encapsulation of pieces of the ER in forming autophagosomes. This is generally followed by fusion with lysosomes and degradation of these ER components by lysosomal hydrolases. Recent work by our group shows that ER elements could also be incorporated into late endosomes and later be released by a secretory mechanism which we will herein refer to as secretory reticulophagy/ER-phagy (SERP). In the absence of macroautophagy, such as by knocking out Atg5, SERP is more efficient, leading to an increased secretion of MAP1LC3B-II and LC3-interacting region (LIR)-containing proteins of the ER, reticulons and atlastins. In this scenario, neurites grow longer and neuronal polarity is altered. In the absence of SERP, such as by knocking out Vamp7, secretion of MAP1LC3B-II, ER-LIR containing proteins and neurite growth are severely inhibited. We argue that SERP might be a main secretory mechanism bypassing the Golgi apparatus, and that it is particularly active and important in neurite growth.
    Keywords:  ATG5; ER-phagy; VAMP7; atlastins; autophagy; extracellular vesicles; late-endosome; reticulons; secretion
    DOI:  https://doi.org/10.1080/15548627.2021.1883886
  9. Biomolecules. 2021 Feb 10. pii: 255. [Epub ahead of print]11(2):
    Witting KF, Mulder MPC.
      Post-translational modification with Ubiquitin-like proteins represents a complex signaling language regulating virtually every cellular process. Among these post-translational modifiers is Ubiquitin-fold modifier (UFM1), which is covalently attached to its substrates through the orchestrated action of a dedicated enzymatic cascade. Originally identified to be involved embryonic development, its biological function remains enigmatic. Recent research reveals that UFM1 regulates a variety of cellular events ranging from DNA repair to autophagy and ER stress response implicating its involvement in a variety of diseases. Given the contribution of UFM1 to numerous pathologies, the enzymes of the UFM1 cascade represent attractive targets for pharmacological inhibition. Here we discuss the current understanding of this cryptic post-translational modification especially its contribution to disease as well as expand on the unmet needs of developing chemical and biochemical tools to dissect its role.
    Keywords:  UFM1; Ubiquitin-like modifiers; activity-based probes; substrates
    DOI:  https://doi.org/10.3390/biom11020255
  10. Sci Adv. 2021 Jan;pii: eabc9781. [Epub ahead of print]7(5):
    Srivastava S, Sahu U, Zhou Y, Hogan AK, Sathyan KM, Bodner J, Huang J, Wong KA, Khalatyan N, Savas JN, Ntziachristos P, Ben-Sahra I, Foltz DR.
      Ubiquitin protein ligase E3 component N-recognin 7 (UBR7) is the most divergent member of UBR box-containing E3 ubiquitin ligases/recognins that mediate the proteasomal degradation of its substrates through the N-end rule. Here, we used a proteomic approach and found phosphoribosyl pyrophosphate synthetases (PRPSs), the essential enzymes for nucleotide biosynthesis, as strong interacting partners of UBR7. UBR7 stabilizes PRPS catalytic subunits by mediating the polyubiquitination-directed degradation of PRPS-associated protein (PRPSAP), the negative regulator of PRPS. Loss of UBR7 leads to nucleotide biosynthesis defects. We define UBR7 as a transcriptional target of NOTCH1 and show that UBR7 is overexpressed in NOTCH1-driven T cell acute lymphoblastic leukemia (T-ALL). Impaired nucleotide biosynthesis caused by UBR7 depletion was concomitant with the attenuated cell proliferation and oncogenic potential of T-ALL. Collectively, these results establish UBR7 as a critical regulator of nucleotide metabolism through the regulation of the PRPS enzyme complex and uncover a metabolic vulnerability in NOTCH1-driven T-ALL.
    DOI:  https://doi.org/10.1126/sciadv.abc9781
  11. EMBO Rep. 2021 Feb 10. e49097
    Shiiba I, Takeda K, Nagashima S, Ito N, Tokuyama T, Yamashita SI, Kanki T, Komatsu T, Urano Y, Fujikawa Y, Inatome R, Yanagi S.
      Parkin promotes cell survival by removing damaged mitochondria via mitophagy. However, although some studies have suggested that Parkin induces cell death, the regulatory mechanism underlying the dual role of Parkin remains unknown. Herein, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) regulates Parkin-mediated cell death through the FKBP38-dependent dynamic translocation from the mitochondria to the ER during mitophagy. Mechanistically, MITOL mediates ubiquitination of Parkin at lysine 220 residue, which promotes its proteasomal degradation, and thereby fine-tunes mitophagy by controlling the quantity of Parkin. Deletion of MITOL leads to accumulation of the phosphorylated active form of Parkin in the ER, resulting in FKBP38 degradation and enhanced cell death. Thus, we have shown that MITOL blocks Parkin-induced cell death, at least partially, by protecting FKBP38 from Parkin. Our findings unveil the regulation of the dual function of Parkin and provide a novel perspective on the pathogenesis of PD.
    Keywords:  E3 ubiquitin ligase; MITOL/MARCH5; Parkin; mitochondria; mitophagy
    DOI:  https://doi.org/10.15252/embr.201949097
  12. Cell Death Discov. 2020 Apr 17. 6(1): 22
    Bellini L, Strub T, Habel N, Pandiani C, Marchetti S, Martel A, Baillif S, Bailly-Maitre B, Gual P, Ballotti R, Bertolotto C.
      To address unmet clinical need for uveal melanomas, we assessed the effects of BH3-mimetic molecules, the ABT family, known to exert pro-apoptotic activities in cancer cells. Our results uncovered that ABT-263 (Navitoclax), a potent and orally bioavailable BCL-2 family inhibitor, induced antiproliferative effects in metastatic human uveal melanoma cells through cell cycle arrest at the G0/G1 phase, loss of mitochondrial membrane potential, and subsequently apoptotic cell death monitored by caspase activation and poly-ADP ribose polymerase cleavage. ABT-263-mediated reduction in tumor growth was also observed in vivo. We observed in some cells that ABT-263 treatment mounted a pro-survival response through activation of the ER stress signaling pathway. Blocking the PERK signaling pathway increased the pro-apoptotic ABT-263 effect. We thus uncovered a resistance mechanism in uveal melanoma cells mediated by activation of endoplasmic reticulum stress pathway. Therefore, our study identifies ABT-263 as a valid therapeutic option for patients suffering from uveal melanoma.
    DOI:  https://doi.org/10.1038/s41420-020-0259-2
  13. Int J Mol Sci. 2021 Feb 04. pii: 1564. [Epub ahead of print]22(4):
    Ichhaporia VP, Hendershot LM.
      Cell surface and secreted proteins provide essential functions for multicellular life. They enter the endoplasmic reticulum (ER) lumen co-translationally, where they mature and fold into their complex three-dimensional structures. The ER is populated with a host of molecular chaperones, associated co-factors, and enzymes that assist and stabilize folded states. Together, they ensure that nascent proteins mature properly or, if this process fails, target them for degradation. BiP, the ER HSP70 chaperone, interacts with unfolded client proteins in a nucleotide-dependent manner, which is tightly regulated by eight DnaJ-type proteins and two nucleotide exchange factors (NEFs), SIL1 and GRP170. Loss of SIL1's function is the leading cause of Marinesco-Sjögren syndrome (MSS), an autosomal recessive, multisystem disorder. The development of animal models has provided insights into SIL1's functions and MSS-associated pathologies. This review provides an in-depth update on the current understanding of the molecular mechanisms underlying SIL1's NEF activity and its role in maintaining ER homeostasis and normal physiology. A precise understanding of the underlying molecular mechanisms associated with the loss of SIL1 may allow for the development of new pharmacological approaches to treat MSS.
    Keywords:  BiP/GRP78/HSPA5; HSP70 chaperones; Marinesco-Sjögren syndrome; SIL1; chemical chaperones; endoplasmic reticulum; gene therapy; glioma; metabolism; neurodegeneration; skeletal muscles; unfolded protein response
    DOI:  https://doi.org/10.3390/ijms22041564
  14. Mol Cell Proteomics. 2020 Dec 07. pii: S1535-9476(20)35130-6. [Epub ahead of print]20 100016
    Ross AB, Langer JD, Jovanovic M.
      In all cells, proteins are continuously synthesized and degraded to maintain protein homeostasis and modify gene expression levels in response to stimuli. Collectively, the processes of protein synthesis and degradation are referred to as protein turnover. At a steady state, protein turnover is constant to maintain protein homeostasis, but in dynamic responses, proteins change their rates of synthesis and degradation to adjust their proteomes to internal or external stimuli. Thus, probing the kinetics and dynamics of protein turnover lends insight into how cells regulate essential processes such as growth, differentiation, and stress response. Here, we outline historical and current approaches to measuring the kinetics of protein turnover on a proteome-wide scale in both steady-state and dynamic systems, with an emphasis on metabolic tracing using stable isotope-labeled amino acids. We highlight important considerations for designing proteome turnover experiments, key biological findings regarding the conserved principles of proteome turnover regulation, and future perspectives for both technological and biological investigation.
    Keywords:  Dynamic SILAC; Mass spectrometry; Protein degradation; Protein synthesis; Proteome turnover; Proteomics; pSILAC
    DOI:  https://doi.org/10.1074/mcp.R120.002190
  15. Mol Biol Cell. 2021 Feb 10. mbcE20110720
    Flagg MP, Wangeline MA, Holland SR, Duttke SH, Benner C, Neal S, Hampton RY.
      Prior to their delivery to and degradation by the 26S proteasome, misfolded transmembrane proteins of the ER and inner-nuclear membrane must be extracted from lipid bilayers. This extraction process, known as retrotranslocation, requires both quality-control E3 ubiquitin ligases and dislocation factors that diminish the energetic cost of dislodging the transmembrane segments of a protein. Recently, we showed that retrotranslocation of all ER transmembrane proteins requires the Dfm1 rhomboid pseudoprotease. However, we did not investigate whether Dfm1 also mediated retrotranslocation of transmembrane substrates in the inner-nuclear membrane (INM), which is contiguous with the ER but functionally separated from it by nucleoporins. Here, we show that canonical retrotranslocation occurs during INM-associated degradation (INMAD) but proceeds independently of Dfm1. Despite this independence, ERAD-M and INMAD cooperate to mitigate proteotoxicity. We show a novel misfolded-transmembrane-protein toxicity that elicits genetic suppression, demonstrating the cell's ability to tolerate a toxic burden of misfolded transmembrane proteins without functional INMAD or ERAD-M. This strikingly contrasted the suppression of the dfm1Δ null, which leads to the resumption of ERAD-M through HRD-complex remodeling. Thus, we conclude that INM retrotranslocation proceeds through a novel, private channel, which can be studied by virtue of its role in alleviating membrane-associated proteotoxicity.
    DOI:  https://doi.org/10.1091/mbc.E20-11-0720
  16. Trends Cell Biol. 2021 Feb 08. pii: S0962-8924(21)00009-X. [Epub ahead of print]
    Lu G, Wang L, Zhou J, Liu W, Shen HM.
      Autophagy and the ubiquitin-proteasome system (UPS) are two major pathways for protein degradation. The cullin-RING E3 ligases (CRLs) are the largest E3 ligase family and have key biological functions in maintaining protein homeostasis. We provide an updated review of the interactions between CRLs and autophagy, focusing on the regulatory effects of CRLs on the core autophagy machinery that consists of several autophagy-related protein (ATG) complexes and their key upstream signaling pathways. The involvement of such functional interactions in health and disease is also discussed. Understanding the role of CRLs in autophagy is helpful for the development of therapeutic strategies for diseases in which CRLs and autophagy are dysregulated, such as cancer and neurodegenerative conditions.
    Keywords:  MTORC1; autophagy; cancer; cullin-RING E3 ligases; neddylation; ubiquitination
    DOI:  https://doi.org/10.1016/j.tcb.2021.01.005
  17. J Med Chem. 2021 Feb 11. 64(3): 1626-1648
    Hanafi M, Chen X, Neamati N.
      Napabucasin, undergoing multiple clinical trials, was reported to inhibit the signal transducer and transcription factor 3 (STAT3). To better elucidate its mechanism of action, we designed a napabucasin-based proteolysis targeting chimera (PROTAC), XD2-149 that resulted in inhibition of STAT3 signaling in pancreatic cancer cell lines without inducing proteasome-dependent degradation of STAT3. Proteomics analysis of XD2-149 revealed the downregulation of the E3 ubiquitin-protein ligase ZFP91. XD2-149 degrades ZFP91 with DC50 values in the nanomolar range. The cytotoxicity of XD2-149 was significantly, but not fully, reduced with ZFP91 knockdown providing evidence for its multi-targeted mechanism of action. The NQO1 inhibitor, dicoumarol, rescued the cytotoxicity of XD2-149 but not ZFP91 degradation, suggesting that the NQO1-induced cell death is independent of ZFP91. ZFP91 plays a role in tumorigenesis and is involved in multiple oncogenic pathways including NF-κB and HIF-1α.
    DOI:  https://doi.org/10.1021/acs.jmedchem.0c01897
  18. J Cell Physiol. 2021 Feb 12.
    Huang KC, Chiang SF, Yang PC, Ke TW, Chen TW, Lin CY, Chang HY, Chen WT, Chao KC.
      AAA domain containing 3A (ATAD3A) is a nucleus-encoded mitochondrial protein with vital function in communication between endoplasmic reticulum (ER) and mitochondria which is participated in cancer metastasis. Here we show that elevated ATAD3A expression is clinically associated with poor 5-year disease-free survival in patients with colorectal cancer (CRC), especially high-risk CRC patients who received adjuvant chemotherapy. Our results indicated ATAD3A is significantly upregulated to reduce chemotherapy-induced cancer cell death. We found that knockdown of ATAD3A leads to dysregulation in protein processing for inducing ER stress by RNA sequencing (RNA-seq). In response to chemotherapy-induced ER stress, ATAD3A interacts with elevated GRP78 protein to assist protein folding and alleviate ER stress for cancer cell survival. This reduction of ER stress leads to reduce the surface exposure of calreticulin, which is the initiator of immunogenic cell death and antitumor immunity. However, silencing of ATAD3A enhances cell death, triggers the feasibility of chemotherapy-induced ER stress for antitumor immunity, increases infiltration of T lymphocytes and delays tumor regrowth in vitro and in vivo. Clinically, CRC patients with less ATAD3A have high density of CD45+ intratumoral infiltrating lymphocytes (TILs) and memory CD45RO+ TILs. Taken together, our results suggest that pharmacologic targeting to ATAD3A might be a potential therapeutic strategy to enhance antitumor immunity for CRC patients who received adjuvant chemotherapy.
    Keywords:  ATPase family AAA domain containing 3A (ATAD3A); ER stress; colon carcinoma; glucose-regulated protein 78 kDa (GRP78); immunogenic cell death
    DOI:  https://doi.org/10.1002/jcp.30323
  19. Cell Rep. 2021 Feb 09. pii: S2211-1247(21)00039-5. [Epub ahead of print]34(6): 108726
    Ma S, McGuire MH, Mangala LS, Lee S, Stur E, Hu W, Bayraktar E, Villar-Prados A, Ivan C, Wu SY, Yokoi A, Dasari SK, Jennings NB, Liu J, Lopez-Berestein G, Ram P, Sood AK.
      Tumor and stromal interactions consist of reciprocal signaling through cytokines, growth factors, direct cell-cell interactions, and extracellular vesicles (EVs). Small EVs (≤200 nm) have been considered critical messengers of cellular communication during tumor development. Here, we demonstrate that gain-of-function (GOF) p53 protein can be packaged into small EVs and transferred to fibroblasts. GOF p53 protein is selectively bound by heat shock protein 90 (HSP90), a chaperone protein, and packaged into small EVs. Inhibition of HSP90 activity blocks packaging of GOF, but not wild-type, p53 in small EVs. GOF p53-containing small EVs result in their conversion to cancer-associated fibroblasts. In vivo studies reveal that GOF p53-containing small EVs can enhance tumor growth and promote fibroblast transformation into a cancer-associated phenotype. These findings provide a better understanding of the complex interactions between cancer and stromal cells and may have therapeutic implications.
    Keywords:  CAFs; HSP90; Nrf2; p53; small EVs
    DOI:  https://doi.org/10.1016/j.celrep.2021.108726
  20. Autophagy. 2021 Feb 10.
    Bao Y, Qian C, Liu MY, Jiang F, Jiang X, Liu H, Zhang Z, Sun F, Fu N, Hou Z, Ke Y, Li Y, Qian ZM.
      RASAL2 (RAS protein activator like 2), a RASGTPase activating protein, can catalyze the hydrolysis of RAS-GTP into RAS-GDP to inactivate the RAS pathway in various types of cancer cells. However, the cellular function of RASAL2 remains elusive. Here we showed that RASAL2 can attenuate PRKAA/AMPKα phosphorylation by recruiting phosphatase PPM1B/pp2cβ, thus inhibiting the initiation of basal autophagy under normal conditions. In addition, we found that glucose starvation could induce dissociation of PPM1B from RASAL2 and then RASAL2 at S351 be phosphorylated by PRKAA, followed by the binding of phosphorylated-RASAL2 with to PIK3C3/VPS34-ATG14-BECN1/Beclin1 complex to increase PIK3C3 activity and autophagy. Furthermore, RASAL2 S351 phosphorylation facilitated breast tumor growth and correlated to poor clinical outcomes in breast cancer patients. Our study demonstrated that the phosphorylation status of RASAL2 S351 can function as a molecular switch to either suppress or promote AMPK-mediated autophagy. Inhibition of RASAL2 S351 phosphorylation might be a potential therapeutic strategy to overcome the resistance of AMPK-activation agents.
    Keywords:  Autophagy; PP1MB; PRKAA; RASAL2; breast cancer
    DOI:  https://doi.org/10.1080/15548627.2021.1886767
  21. Cell Mol Life Sci. 2021 Feb 13.
    Poole LP, Macleod KF.
      Cells use mitophagy to remove dysfunctional or excess mitochondria, frequently in response to imposed stresses, such as hypoxia and nutrient deprivation. Mitochondrial cargo receptors (MCR) induced by these stresses target mitochondria to autophagosomes through interaction with members of the LC3/GABARAP family. There are a growing number of these MCRs, including BNIP3, BNIP3L, FUNDC1, Bcl2-L-13, FKBP8, Prohibitin-2, and others, in addition to mitochondrial protein targets of PINK1/Parkin phospho-ubiquitination. There is also an emerging link between mitochondrial lipid signaling and mitophagy where ceramide, sphingosine-1-phosphate, and cardiolipin have all been shown to promote mitophagy. Here, we review the upstream signaling mechanisms that regulate mitophagy, including components of the mitochondrial fission machinery, AMPK, ATF4, FoxOs, Sirtuins, and mtDNA release, and address the significance of these pathways for stress responses in tumorigenesis and metastasis. In particular, we focus on how mitophagy modulators intersect with cell cycle control and survival pathways in cancer, including following ECM detachment and during cell migration and metastasis. Finally, we interrogate how mitophagy affects tissue atrophy during cancer cachexia and therapy responses in the clinic.
    Keywords:  AMPK; ATF4; Autophagy; BCL2-L-13; BNIP3/BNIP3L; Cachexia; DRP1; Electron transport chain; FUNDC1; Fission; FoxOs; LC3/GABARAP; Metabolism; Metastasis; Mitochondria; Mitohormesis; Mitophagy; NAD+; PARP; PINK1/Parkin; ROS; Respiration; Sirtuins; UPRmt
    DOI:  https://doi.org/10.1007/s00018-021-03774-1
  22. EMBO J. 2021 Feb 08. e106449
    Ranjan N, Pochopien AA, Chih-Chien Wu C, Beckert B, Blanchet S, Green R, V Rodnina M, Wilson DN.
      In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the ribosomal A and E sites, its exact mechanism of action is unclear. Here, we show that eEF3 acts at the mRNA-tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl-tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slowdown in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo-EM analysis of native eEF3-ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non-rotated ribosomal states, as well as by opening the L1 stalk to release the E-site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.
    Keywords:  ABC ATPase; E-site tRNA; L1 stalk; cryo-EM; eEF3
    DOI:  https://doi.org/10.15252/embj.2020106449
  23. J Biol Chem. 2021 Feb 07. pii: S0021-9258(21)00168-X. [Epub ahead of print] 100396
    Perry M, Biegert M, Kollala SS, Mallard H, Su G, Kodavati M, Kreiling N, Holbrook A, Ghosal G.
      DNA-protein crosslinks (DPCs) are toxic DNA lesions that interfere with DNA metabolic processes such as replication, transcription and recombination. USP11 deubiquitinase participates in DNA repair, but the role of USP11 in DPC repair is not known. SPRTN is a replication-coupled DNA-dependent metalloprotease that cleaves proteins crosslinked to DNA to promote DPC repair. SPRTN function is tightly regulated by a monoubiquitin switch that controls SPRTN auto-proteolysis and chromatin accessibility during DPC repair. Previously, VCPIP1 and USP7 deubiquitinases have been shown to regulate SPRTN. Here, we identify USP11 as a SPRTN deubiquitinase. USP11 interacts with SPRTN and cleaves monoubiquitinated SPRTN in cells and in vitro. USP11 depletion impairs SPRTN deubiquitination and promotes SPRTN auto-proteolysis in response to formaldehyde-induced DPCs. Loss of USP11 causes an accumulation of unrepaired DPCs and cellular hypersensitivity to treatment with DPC-inducing agents. Our findings show that USP11 regulates SPRTN auto-proteolysis and SPRTN-mediated DPC repair to maintain genome stability.
    Keywords:  DPC; DPC repair; SPRTN; TOP1-ccs; USP11; deubiquitination; monoubiquitinated SPRTN
    DOI:  https://doi.org/10.1016/j.jbc.2021.100396
  24. Mol Cell. 2021 Jan 28. pii: S1097-2765(21)00016-2. [Epub ahead of print]
    Rawat P, Boehning M, Hummel B, Aprile-Garcia F, Pandit AS, Eisenhardt N, Khavaran A, Niskanen E, Vos SM, Palvimo JJ, Pichler A, Cramer P, Sawarkar R.
      In response to stress, human cells coordinately downregulate transcription and translation of housekeeping genes. To downregulate transcription, the negative elongation factor (NELF) is recruited to gene promoters impairing RNA polymerase II elongation. Here we report that NELF rapidly forms nuclear condensates upon stress in human cells. Condensate formation requires NELF dephosphorylation and SUMOylation induced by stress. The intrinsically disordered region (IDR) in NELFA is necessary for nuclear NELF condensation and can be functionally replaced by the IDR of FUS or EWSR1 protein. We find that biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation. Importantly, NELF condensation is required for cellular viability under stressful conditions. We propose that stress-induced NELF condensates reported here are nuclear counterparts of cytosolic stress granules. These two stress-inducible condensates may drive the coordinated downregulation of transcription and translation, likely forming a critical node of the stress survival strategy.
    Keywords:  CDK9; RNA polymerase II; SUMO; heat shock; negative elongation factor (NELF); pausing; phase separation; proteostasis; transcriptional condensates; transcriptional stress response
    DOI:  https://doi.org/10.1016/j.molcel.2021.01.016
  25. Proc Natl Acad Sci U S A. 2021 Feb 16. pii: e2014457118. [Epub ahead of print]118(7):
    Theodoridis PR, Bokros M, Marijan D, Balukoff NC, Wang D, Kirk CC, Budine TD, Goldsmith HD, Wang M, Audas TE, Lee S.
      Biomolecular condensates concentrate molecules to facilitate basic biochemical processes, including transcription and DNA replication. While liquid-like condensates have been ascribed various functions, solid-like condensates are generally thought of as amorphous sites of protein storage. Here, we show that solid-like amyloid bodies coordinate local nuclear protein synthesis (LNPS) during stress. On stimulus, translationally active ribosomes accumulate along fiber-like assemblies that characterize amyloid bodies. Mass spectrometry analysis identified regulatory ribosomal proteins and translation factors that relocalize from the cytoplasm to amyloid bodies to sustain LNPS. These amyloidogenic compartments are enriched in newly transcribed messenger RNA by Heat Shock Factor 1 (HSF1). Depletion of stress-induced ribosomal intergenic spacer noncoding RNA (rIGSRNA) that constructs amyloid bodies prevents recruitment of the nuclear protein synthesis machinery, abolishes LNPS, and impairs the nuclear HSF1 response. We propose that amyloid bodies support local nuclear translation during stress and that solid-like condensates can facilitate complex biochemical reactions as their liquid counterparts can.
    Keywords:  HSR; Hsp70; acidosis; hypoxia; long noncoding RNA
    DOI:  https://doi.org/10.1073/pnas.2014457118
  26. Plant Physiol Biochem. 2021 Jan 24. pii: S0981-9428(21)00044-9. [Epub ahead of print]160 397-403
    Kaur N, Kaitheri Kandoth P.
      Environmental stresses activate endoplasmic reticulum (ER) stress response pathways, collectively known as the unfolded protein response (UPR). IRE1/bZIP60 pathway is the most conserved of all UPR pathways from yeast to plants. Transcription factor bZIP60 is activated by the cytoplasmic splicing of its mRNA by Inositol Requiring Enzyme1 (IRE1) protein. bZIP60 mRNA has a typical stem-loop structure that is required for its splicing by IRE1 ribonuclease. We identified the tomato bZIP60 (SlbZIP60) and secondary structure prediction showed that it has the conserved stem-loop structure. Further, we demonstrate that SlbZIP60 is spliced upon treatment with an ER stress-inducing agent, tunicamycin. Tunicamycin also upregulated the expression of SlbZIP60. Finally, we show that SlbZIP60 undergo physiologically activated splicing in certain tissues of the plant and respond to environmental stresses, heat, and virus infection. This study will help for a deeper understanding of ER stress pathways and how they contribute to the stress tolerance of tomato, one of the important vegetable crops, cultivated under varied environmental conditions.
    Keywords:  ER stress; Heat; IRE1; Tomato; Unfolded protein response (UPR); bZIP60
    DOI:  https://doi.org/10.1016/j.plaphy.2021.01.033
  27. Mol Cell. 2021 Feb 03. pii: S1097-2765(21)00043-5. [Epub ahead of print]
    Kaiho-Soma A, Akizuki Y, Igarashi K, Endo A, Shoda T, Kawase Y, Demizu Y, Naito M, Saeki Y, Tanaka K, Ohtake F.
      Targeted protein degradation is an emerging therapeutic paradigm. Small-molecule degraders such as proteolysis-targeting chimeras (PROTACs) induce the degradation of neo-substrates by hijacking E3 ubiquitin ligases. Although ubiquitylation of endogenous substrates has been extensively studied, the mechanism underlying forced degradation of neo-substrates is less well understood. We found that the ubiquitin ligase TRIP12 promotes PROTAC-induced and CRL2VHL-mediated degradation of BRD4 but is dispensable for the degradation of the endogenous CRL2VHL substrate HIF-1α. TRIP12 associates with BRD4 via CRL2VHL and specifically assembles K29-linked ubiquitin chains, facilitating the formation of K29/K48-branched ubiquitin chains and accelerating the assembly of K48 linkage by CRL2VHL. Consequently, TRIP12 promotes the PROTAC-induced apoptotic response. TRIP12 also supports the efficiency of other degraders that target CRABP2 or TRIM24 or recruit CRBN. These observations define TRIP12 and K29/K48-branched ubiquitin chains as accelerators of PROTAC-directed targeted protein degradation, revealing a cooperative mechanism of branched ubiquitin chain assembly unique to the degradation of neo-substrates.
    Keywords:  apoptosis; cancer; cullin-RING ligase; epigenetics; targeted protein degradation; ubROTAC; ubiquitin
    DOI:  https://doi.org/10.1016/j.molcel.2021.01.023
  28. Cells. 2021 Feb 05. pii: 335. [Epub ahead of print]10(2):
    Cristofani R, Piccolella M, Crippa V, Tedesco B, Montagnani Marelli M, Poletti A, Moretti RM.
      The cellular response to cancer-induced stress is one of the major aspects regulating cancer development and progression. The Heat Shock Protein B8 (HSPB8) is a small chaperone involved in chaperone-assisted selective autophagy (CASA). CASA promotes the selective degradation of proteins to counteract cell stress such as tumor-induced stress. HSPB8 is also involved in (i) the cell division machinery regulating chromosome segregation and cell cycle arrest in the G0/G1 phase and (ii) inflammation regulating dendritic cell maturation and cytokine production. HSPB8 expression and role are tumor-specific, showing a dual and opposite role. Interestingly, HSPB8 may be involved in the acquisition of chemoresistance to drugs. Despite the fact the mechanisms of HSPB8-mediated CASA activation in tumors need further studies, HSPB8 could represent an important factor in cancer induction and progression and it may be a potential target for anticancer treatment in specific types of cancer. In this review, we will discuss the molecular mechanism underlying HSPB8 roles in normal and cancer conditions. The basic mechanisms involved in anti- and pro-tumoral activities of HSPB8 are deeply discussed together with the pathways that modulate HSPB8 expression, in order to outline molecules with a beneficial effect for cancer cell growth, migration, and death.
    Keywords:  CASA; HSPB8; PQC; autophagy; cancer; chaperones
    DOI:  https://doi.org/10.3390/cells10020335
  29. Trends Biochem Sci. 2021 Feb 05. pii: S0968-0004(21)00006-2. [Epub ahead of print]
    Nieto-Torres JL, Leidal AM, Debnath J, Hansen M.
      The ATG8 family proteins are critical players in autophagy, a cytoprotective process that mediates degradation of cytosolic cargo. During autophagy, ATG8s conjugate to autophagosome membranes to facilitate cargo recruitment, autophagosome biogenesis, transport, and fusion with lysosomes, for cargo degradation. In addition to these canonical functions, recent reports demonstrate that ATG8s are also delivered to single-membrane organelles, which leads to highly divergent degradative or secretory fates, vesicle maturation, and cargo specification. The association of ATG8s with different vesicles involves complex regulatory mechanisms still to be fully elucidated. Whether individual ATG8 family members play unique canonical or non-canonical roles, also remains unclear. This review summarizes the many open molecular questions regarding ATG8s that are only beginning to be unraveled.
    Keywords:  GABARAP; LC3; LC3-associated phagocytosis; multivesicular bodies; non-canonical autophagy; unconventional secretion
    DOI:  https://doi.org/10.1016/j.tibs.2021.01.004
  30. Trends Biochem Sci. 2021 Feb 08. pii: S0968-0004(21)00019-0. [Epub ahead of print]
    Satapathy S, Wilson MR.
      Clusterin (CLU) was the first reported secreted mammalian chaperone and impacts on serious diseases associated with inappropriate extracellular protein aggregation. Many studies have described intracellular CLU in locations outside the secretory system and recent work has shown that CLU can be released into the cytosol during cell stress. In this article, we critically evaluate evidence relevant to the proposed origins of cellular CLU found outside the secretory system, and advance the hypothesis that the cytosolic release of CLU induced by stress serves to facilitate the trafficking of misfolded proteins to the proteasome and autophagy for degradation. We also propose future research directions that could help establish CLU as a unique chaperone performing critical and synergic roles in both intracellular and extracellular proteostasis.
    Keywords:  autophagy; chaperone; clusterin; misfolded proteins; neurodegenerative diseases; proteasome; proteostasis
    DOI:  https://doi.org/10.1016/j.tibs.2021.01.005
  31. Mol Cell Oncol. 2021 ;8(1): 1837582
    Zhang S, Sun Y.
      CRL and APC/C belong to the RING-finger-type E3 ligases, and both play important roles in cell cycle regulation. Recently, we found that SAG, a RING component of CRL, acts as an endogenous inhibitor of APC/C by competing with APC2 for E2s binding; while APC/CCDH1 targets SAG for ubiquitylation and degradation at G1 phase. The negative crosstalk between these two E3s ensures the orderly cell cycle progression.
    Keywords:  APC/C; SAG; UBE2C; UBE2S; cell cycle
    DOI:  https://doi.org/10.1080/23723556.2020.1837582
  32. Nucleic Acids Res. 2021 Feb 12. pii: gkab065. [Epub ahead of print]
    Hou G, Zhao X, Li L, Yang Q, Liu X, Huang C, Lu R, Chen R, Wang Y, Jiang B, Yu J.
      N 6-Methyladenosine (m6A) is the most abundant modification within diverse RNAs including mRNAs and lncRNAs and is regulated by a reversible process with important biological functions. Human YTH domain family 2 (YTHDF2) selectively recognized m6A-RNAs to regulate degradation. However, the possible regulation of YTHDF2 by protein post-translational modification remains unknown. Here, we show that YTHDF2 is SUMOylated in vivo and in vitro at the major site of K571, which can be induced by hypoxia while reduced by oxidative stress and SUMOylation inhibitors. SUMOylation of YTHDF2 has little impact on its ubiquitination and localization, but significantly increases its binding affinity of m6A-modified mRNAs and subsequently results in deregulated gene expressions which accounts for cancer progression. Moreover, Disease-free survival analysis of patients with lung adenocarcinoma derived from TCGA dataset reveals that higher expression of YTHDF2 together with higher expression of SUMO1 predicts poor prognosis. Our works uncover a new regulatory mechanism for YTHDF2 recognition of m6A-RNAs and highlight the importance of YTHDF2 SUMOylation in post-transcriptional gene expression regulation and cancer progression.
    DOI:  https://doi.org/10.1093/nar/gkab065
  33. Cell Death Differ. 2021 Feb 08.
    Jie X, Fong WP, Zhou R, Zhao Y, Zhao Y, Meng R, Zhang S, Dong X, Zhang T, Yang K, Wu G, Xu S.
      Radioresistance is regarded as the main barrier to effective radiotherapy in lung cancer. However, the underlying mechanisms of radioresistance remain elusive. Here, we show that lysine-specific demethylase 4C (KDM4C) is overexpressed and correlated with poor prognosis in lung cancer patients. We provide evidence that genetical or pharmacological inhibition of KDM4C impairs tumorigenesis and radioresistance in lung cancer in vitro and in vivo. Moreover, we uncover that KDM4C upregulates TGF-β2 expression by directly reducing H3K9me3 level at the TGF-β2 promoter and then activates Smad/ATM/Chk2 signaling to confer radioresistance in lung cancer. Using tandem affinity purification technology, we further identify deubiquitinase USP9X as a critical binding partner that deubiquitinates and stabilizes KDM4C. More importantly, depletion of USP9X impairs TGF-β2/Smad signaling and radioresistance by destabilizing KDM4C in lung cancer cells. Thus, our findings demonstrate that USP9X-mediated KDM4C deubiquitination activates TGF-β2/Smad signaling to promote radioresistance, suggesting that targeting KDM4C may be a promising radiosensitization strategy in the treatment of lung cancer.
    DOI:  https://doi.org/10.1038/s41418-021-00740-z
  34. J Biol Chem. 2021 Feb 09. pii: S0021-9258(21)00177-0. [Epub ahead of print] 100405
    Jia R, Bonifacino JS.
      Components of the autophagy machinery are subject to regulation by various post-translational modifications. Previous studies have shown that monoubiquitination of LC3B catalyzed by the ubiquitin-activating enzyme UBA6 and ubiquitin-conjugating enzyme/ubiquitin ligase BIRC6 targets LC3B for proteasomal degradation, thus reducing LC3B levels and autophagic activity under conditions of stress. However, mechanisms capable of counteracting this process are not known. Herein, we report that LC3B ubiquitination is reversed by the action of the deubiquitinating enzyme USP10. We identified USP10 in a CRISPR-Cas9 knockout screen for ubiquitination-related genes that regulate LC3B levels. Biochemical analyses showed that silencing of USP10 reduces the levels of both the LC3B-I and LC3B-II forms of LC3B through increased ubiquitination and proteasomal degradation. In turn, the reduced LC3B levels result in slower degradation of the autophagy receptors SQSTM1 and NBR1, and an increased accumulation of puromycin-induced aggresome-like structures. Taken together, these findings indicate that the levels of LC3B and autophagic activity are controlled through cycles of LC3B ubiquitination and deubiquitination.
    Keywords:  CRISPR/Cas; LC3; USP10; autophagy; deubiquitination; protein aggregation; ubiquitin
    DOI:  https://doi.org/10.1016/j.jbc.2021.100405
  35. EMBO J. 2021 Feb 12. e106094
    Pluska L, Jarosch E, Zauber H, Kniss A, Waltho A, Bagola K, von Delbrück M, Löhr F, Schulman BA, Selbach M, Dötsch V, Sommer T.
      The assembly of a specific polymeric ubiquitin chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The homologous ubiquitin-conjugating (E2) enzymes Ubc1 (budding yeast) and Ube2K (mammals) exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a ubiquitin-binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with ubiquitin chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments, and NMR studies, we propose that the UBA domain aligns Ubc1 with K63-linked polyubiquitin and facilitates the selective assembly of K48/K63-branched ubiquitin conjugates. Genetic and proteomics experiments link the activity of the UBA domain, and hence the formation of this unusual ubiquitin chain topology, to the maintenance of cellular proteostasis.
    Keywords:  K48-linked; K63-linked; cell stress; polyubiquitin; ubiquitin-conjugating enzymes
    DOI:  https://doi.org/10.15252/embj.2020106094
  36. Cancer Lett. 2021 Feb 05. pii: S0304-3835(21)00014-8. [Epub ahead of print]
    Kang SU, Kim DH, Lee YS, Huang M, Byeon HK, Lee SH, Baek SJ, Kim CH.
      Despite technological advances in cancer treatment, the survival rate of patients with head and neck cancer (HNC) has not improved significantly. Many studies have shown that endoplasmic reticulum (ER) stress-related signals are associated with mitochondrial damage and that these signals determine whether cells maintain homeostasis or activate cell death programs. The unfolded protein response (UPR) is regulated by ER membrane proteins such as double-stranded RNA-activated protein kinase R(PKR)-like ER kinase (PERK), which directly activate transcription of chaperones or genes that function in redox homeostasis, protein secretion, or cell death programs. In this study, we focused on the role of mitophagy and ER stress-mediated cell death induced by DIM-C-pPhtBu in HNC cancer. We found that DIM-C-pPhtBu, a compound that activates ER stress in many cancers, induced lysosomal dysfunction, excessive mitophagy, and cell death in HNC cells. Moreover, DIM-C-pPhtBu strongly inhibited HNC progression in a xenograft model by altering mitophagy related protein expression. Taken together, the results demonstrate that DIM-C-pPhtBu induces excessive mitophagy and eventually UPR-mediated cell death in HNC cells, suggesting that new anti-cancer drugs could be developed based on the connection between mitophagy and cancer cell death.
    Keywords:  3,3′-diindolylmethane (DIM); Apoptosis-linked gene 2-interacting protein X (AIP1/Alix); Differentially expressed genes (DEGs); Double-stranded RNA-Activated protein kinase-like ER kinase (PERK); Endoplasmic reticulum stress; HNC (Head and neck cancer)
    DOI:  https://doi.org/10.1016/j.canlet.2021.01.005
  37. Int J Mol Sci. 2021 Jan 25. pii: 1168. [Epub ahead of print]22(3):
    Wu Y, Zhang W.
      Pluripotent embryonic stem cells (ESCs) are derived from early embryos and can differentiate into any type of cells in living organisms. Induced pluripotent stem cells (iPSCs) resemble ESCs, both of which serve as excellent sources to study early embryonic development and realize cell replacement therapies for age-related degenerative diseases and other cell dysfunction-related illnesses. To achieve these valuable applications, comprehensively understanding of the mechanisms underlying pluripotency maintenance and acquisition is critical. Ubiquitination modifies proteins with Ubiquitin (Ub) at the post-translational level to monitor protein stability and activity. It is extensively involved in pluripotency-specific regulatory networks in ESCs and iPSCs. Ubiquitination is achieved by sequential actions of the Ub-activating enzyme E1, Ub-conjugating enzyme E2, and Ub ligase E3. Compared with E1s and E2s, E3s are most abundant, responsible for substrate selectivity and functional diversity. In this review, we focus on E3 ligases to discuss recent progresses in understanding how they regulate pluripotency and somatic cell reprogramming through ubiquitinating core ESC regulators.
    Keywords:  embryonic stem cells; induced pluripotent stem cells; pluripotency; ubiquitination
    DOI:  https://doi.org/10.3390/ijms22031168
  38. Virus Res. 2021 Feb 09. pii: S0168-1702(21)00045-9. [Epub ahead of print] 198338
    Rother M, Naumann M.
      Zika is a major teratogenic virus that can be transmitted from pregnant women to the fetus via the transplacental route. At present, no specific vaccines or treatments are available. Large-scale functional genomics approaches for the analysis of host cell function in infection greatly improve the understanding of molecular infection processes and advance the discovery of antiviral targets. We conducted a pooled CRISPR/Cas9 screen to explore trophoblast function upon Zika infection. The identified Zika virus host factors enrich in the ER membrane complex and the signal peptide processing pathway. Finally, we demonstrate that signal peptidase complex subunit 1 (SPCS1) is crucial for virus replication in trophoblasts.
    Keywords:  CRISPR screen; ER membrane complex; Guillain-Barré syndrome; SPCS1; ZIKV; microcephaly
    DOI:  https://doi.org/10.1016/j.virusres.2021.198338
  39. Mol Cell. 2021 Feb 01. pii: S1097-2765(21)00040-X. [Epub ahead of print]
    Tokheim C, Wang X, Timms RT, Zhang B, Mena EL, Wang B, Chen C, Ge J, Chu J, Zhang W, Elledge SJ, Brown M, Liu XS.
      The ubiquitin-proteasome system (UPS) is the primary route for selective protein degradation in human cells. The UPS is an attractive target for novel cancer therapies, but the precise UPS genes and substrates important for cancer growth are incompletely understood. Leveraging multi-omics data across more than 9,000 human tumors and 33 cancer types, we found that over 19% of all cancer driver genes affect UPS function. We implicate transcription factors as important substrates and show that c-Myc stability is modulated by CUL3. Moreover, we developed a deep learning model (deepDegron) to identify mutations that result in degron loss and experimentally validated the prediction that gain-of-function truncating mutations in GATA3 and PPM1D result in increased protein stability. Last, we identified UPS driver genes associated with prognosis and the tumor microenvironment. This study demonstrates the important role of UPS dysregulation in human cancer and underscores the potential therapeutic utility of targeting the UPS.
    DOI:  https://doi.org/10.1016/j.molcel.2021.01.020
  40. EMBO J. 2021 Feb 12. e106292
    Tobiasson V, Gahura O, Aibara S, Baradaran R, Zíková A, Amunts A.
      Mitoribosomes consist of ribosomal RNA and protein components, coordinated assembly of which is critical for function. We used mitoribosomes from Trypanosoma brucei with reduced RNA and increased protein mass to provide insights into the biogenesis of the mitoribosomal large subunit. Structural characterization of a stable assembly intermediate revealed 22 assembly factors, some of which have orthologues/counterparts/homologues in mammalian genomes. These assembly factors form a protein network that spans a distance of 180 Å, shielding the ribosomal RNA surface. The central protuberance and L7/L12 stalk are not assembled entirely and require removal of assembly factors and remodeling of the mitoribosomal proteins to become functional. The conserved proteins GTPBP7 and mt-EngA are bound together at the subunit interface in proximity to the peptidyl transferase center. A mitochondrial acyl-carrier protein plays a role in docking the L1 stalk, which needs to be repositioned during maturation. Additional enzymatically deactivated factors scaffold the assembly while the exit tunnel is blocked. Together, this extensive network of accessory factors stabilizes the immature sites and connects the functionally important regions of the mitoribosomal large subunit.
    Keywords:  assembly; mitochondria; mitoribosome; translation; trypanosoma
    DOI:  https://doi.org/10.15252/embj.2020106292
  41. J Clin Invest. 2021 Feb 11. pii: 144888. [Epub ahead of print]
    Yan S, Kumari M, Xiao H, Jacobs C, Kochumon S, Jedrychowski M, Chouchani E, Ahmad R, Rosen ED.
      Adipose thermogenesis is repressed in obesity, reducing the homeostatic capacity to compensate for chronic overnutrition. Inflammation inhibits adipose thermogenesis, but little is known about how this occurs. Here we show that the innate immune transcription factor IRF3 is a strong repressor of thermogenic gene expression and oxygen consumption in adipocytes. IRF3 achieves this by driving expression of the ubiquitin-like modifier ISG15, which becomes covalently attached to glycolytic enzymes, thus reducing their function and decreasing lactate production. Lactate repletion is able to restore thermogenic gene expression, even when the IRF3-ISG15 axis is activated. Mice lacking ISG15 phenocopy mice lacking IRF3 in adipocytes, as both have elevated energy expenditure and are resistant to diet-induced obesity. These studies provide a deep mechanistic understanding of how the chronic inflammatory milieu of adipose tissue in obesity prevents thermogenic compensation for overnutrition.
    Keywords:  Adipose tissue; Innate immunity; Metabolism
    DOI:  https://doi.org/10.1172/JCI144888
  42. Mol Cell Proteomics. 2021 Feb 06. pii: S1535-9476(21)00026-8. [Epub ahead of print] 100053
    Guo Z, Pan F, Peng L, Tian S, Jiao J, Liao L, Lu C, Zhai G, Wu Z, Dong H, Xu X, Wu J, Chen P, Bai X, Lin D, Xu L, Li E, Zhang K.
      Esophageal squamous cell cancer (ESCC) is an aggressive malignancy with poor therapeutic outcomes. However, the alterations in proteins and post-translational modifications (PTMs) leading to the pathogenesis of ESCC remains unclear. Here, we provide the comprehensive characterization of the proteome, phosphorylome, lysine acetylome and succinylome for ESCC and matched control cells using quantitative proteomic approach. We identify abnormal protein and post-translational modification (PTM) pathways, including significantly downregulated lysine succinylation sites in cancer cells. Focusing on hyposuccinylation, we reveal that this altered PTM was enriched on enzymes of metabolic pathways inextricably linked with cancer metabolism. Importantly, ESCC malignant behaviors such as cell migration are inhibited once the level of succinylation was restored in vitro or in vivo. This effect was further verified by mutations to disrupt succinylation sites in candidate proteins. Meanwhile, we found that succinylation has a negative regulatory effect on histone methylation to promote cancer migration. Finally, hyposuccinylation is confirmed in primary ESCC specimens. Our findings together demonstrate that lysine succinylation may alter ESCC metabolism and migration, providing new insights into the functional significance of PTM in cancer biology.
    Keywords:  Esophageal squamous; Migration; Post-translational modification; Succinylation
    DOI:  https://doi.org/10.1074/mcp.RA120.002150
  43. Cell Signal. 2021 Feb 03. pii: S0898-6568(21)00034-6. [Epub ahead of print]81 109946
    Zhang X, Chen XL.
      The ubiquitin related modifier Urm1 protein was firstly identified in the yeast Saccharomyces cerevisiae, and was later found to play important roles in different eukaryotes. By the assistance of an E1-like activation enzyme Uba4, Urm1 can function as a modifier to target proteins, called urmylation. The thioredoxin peroxidase Ahp1 was the only identified Urm1 target in the early time. Recently, many other Urm1 targets were identified, which is important for us to fully understand functions of urmylation. Urm1 can also function as a sulfur carrier to play a key role in tRNAs thiolation. Mechanisms of the Urm1 in protein and RNA modifications were finely revealed in the past few years. Biological and physiological functions of Urm1 were also found in different organisms. In this review, we will summarize these emerging progresses.
    Keywords:  Stress response; Ubiquitin related modifier; Urm1; Urmylation; tRNAs thiolation
    DOI:  https://doi.org/10.1016/j.cellsig.2021.109946
  44. EMBO J. 2021 Feb 08. e105853
    Kaneko Y, Shimoda K, Ayala R, Goto Y, Panico S, Zhang X, Kondo H.
      p97ATPase-mediated membrane fusion is required for the biogenesis of the Golgi complex. p97 and its cofactor p47 function in soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) priming, but the tethering complex for p97/p47-mediated membrane fusion remains unknown. In this study, we identified formiminotransferase cyclodeaminase (FTCD) as a novel p47-binding protein. FTCD mainly localizes to the Golgi complex and binds to either p47 or p97 via its association with their polyglutamate motifs. FTCD functions in p97/p47-mediated Golgi reassembly at mitosis in vivo and in vitro via its binding to p47 and to p97. We also showed that FTCD, p47, and p97 form a big FTCD-p97/p47-FTCD tethering complex. In vivo tethering assay revealed that FTCD that was designed to localize to mitochondria caused mitochondria aggregation at mitosis by forming a complex with endogenous p97 and p47, which support a role for FTCD in tethering biological membranes in cooperation with the p97/p47 complex. Therefore, FTCD is thought to act as a tethering factor by forming the FTCD-p97/p47-FTCD complex in p97/p47-mediated Golgi membrane fusion.
    Keywords:  Golgi apparatus; membrane fusion; mitosis; p97ATPase; tethering
    DOI:  https://doi.org/10.15252/embj.2020105853
  45. Nucleic Acids Res. 2021 Feb 09. pii: gkab036. [Epub ahead of print]
    Karasik A, Jones GD, DePass AV, Guydosh NR.
      Ribonuclease L (RNase L) is activated as part of the innate immune response and plays an important role in the clearance of viral infections. When activated, it endonucleolytically cleaves both viral and host RNAs, leading to a global reduction in protein synthesis. However, it remains unknown how widespread RNA decay, and consequent changes in the translatome, promote the elimination of viruses. To study how this altered transcriptome is translated, we assayed the global distribution of ribosomes in RNase L activated human cells with ribosome profiling. We found that RNase L activation leads to a substantial increase in the fraction of translating ribosomes in ORFs internal to coding sequences (iORFs) and ORFs within 5' and 3' UTRs (uORFs and dORFs). Translation of these alternative ORFs was dependent on RNase L's cleavage activity, suggesting that mRNA decay fragments are translated to produce short peptides that may be important for antiviral activity.
    DOI:  https://doi.org/10.1093/nar/gkab036
  46. Nat Commun. 2021 02 11. 12(1): 946
    Ryder BD, Matlahov I, Bali S, Vaquer-Alicea J, van der Wel PCA, Joachimiak LA.
      The Hsp40/Hsp70 chaperone families combine versatile folding capacity with high substrate specificity, which is mainly facilitated by Hsp40s. The structure and function of many Hsp40s remain poorly understood, particularly oligomeric Hsp40s that suppress protein aggregation. Here, we used a combination of biochemical and structural approaches to shed light on the domain interactions of the Hsp40 DnaJB8, and how they may influence recruitment of partner Hsp70s. We identify an interaction between the J-Domain (JD) and C-terminal domain (CTD) of DnaJB8 that sequesters the JD surface, preventing Hsp70 interaction. We propose a model for DnaJB8-Hsp70 recruitment, whereby the JD-CTD interaction of DnaJB8 acts as a reversible switch that can control the binding of Hsp70. These findings suggest that the evolutionarily conserved CTD of DnaJB8 is a regulatory element of chaperone activity in the proteostasis network.
    DOI:  https://doi.org/10.1038/s41467-021-21147-x
  47. Neuroscience. 2021 Feb 09. pii: S0306-4522(21)00071-3. [Epub ahead of print]
    Liu X, Moussa C.
      Ubiquitin specific protease (USP)-13 is a de-ubiquitinase member of the cysteine-dependent protease superfamily that cleaves ubiquitin off protein substrates to reverse ubiquitin-mediated protein degradation. Several findings implicate USPs in neurodegeneration. Ubiquitin targets proteins to major degradation pathways, including the proteasome and the lysosome. In melanoma cells, USP13 regulates the degradation of several proteins primarily via ubiquitination and de-ubiquitination. However, the significance of USP13 in regulating protein clearance in neurodegeneration is largely unknown. This mini-review summarizes the most recent evidence pertaining to the role of USP13 in protein clearance via autophagy and the proteasome in neurodegenerative diseases.
    Keywords:  USP13; autophagy; neurodegenerative diseases; ubiquitin
    DOI:  https://doi.org/10.1016/j.neuroscience.2021.02.004
  48. Nat Struct Mol Biol. 2021 Feb 08.
    Wang Y, Niu Y, Zhang Z, Gable K, Gupta SD, Somashekarappa N, Han G, Zhao H, Myasnikov AG, Kalathur RC, Dunn TM, Lee CH.
      Sphingolipids are essential lipids in eukaryotic membranes. In humans, the first and rate-limiting step of sphingolipid synthesis is catalyzed by the serine palmitoyltransferase holocomplex, which consists of catalytic components (SPTLC1 and SPTLC2) and regulatory components (ssSPTa and ORMDL3). However, the assembly, substrate processing and regulation of the complex are unclear. Here, we present 8 cryo-electron microscopy structures of the human serine palmitoyltransferase holocomplex in various functional states at resolutions of 2.6-3.4 Å. The structures reveal not only how catalytic components recognize the substrate, but also how regulatory components modulate the substrate-binding tunnel to control enzyme activity: ssSPTa engages SPTLC2 and shapes the tunnel to determine substrate specificity. ORMDL3 blocks the tunnel and competes with substrate binding through its amino terminus. These findings provide mechanistic insights into sphingolipid biogenesis governed by the serine palmitoyltransferase complex.
    DOI:  https://doi.org/10.1038/s41594-020-00551-9