bims-proteo Biomed News
on Proteostasis
Issue of 2023‒05‒07
thirty-two papers selected by
Eric Chevet
INSERM
  1. Ufmylation bridges autophagy and ER homeostasis in plants.
  2. Structural remodeling of the endoplasmic reticulum in response to extracellular signals requires αTAT1-induced microtubule acetylation.
  3. Recognition of an Ala-rich C-degron by the E3 ligase Pirh2.
  4. Substrate-specific effects of natural genetic variation on proteasome activity.
  5. Oligomeric scaffolding for curvature generation by ER tubule-forming proteins.
  6. Ubiquitin proteomics identifies RNA polymerase I as a target of the Smc5/6 complex.
  7. SEL1L-HRD1 endoplasmic reticulum-associated degradation controls STING-mediated innate immunity by limiting the size of the activable STING pool.
  8. Redox-regulated chaperones in cell stress responses.
  9. Ubx5-Cdc48 assists the protease Wss1 at DNA-protein crosslink sites in yeast.
  10. Clp-targeting BacPROTACs impair mycobacterial proteostasis and survival.
  11. The Brucella abortus Type IV Effector BspA Inhibits MARCH6-Dependent ERAD To Promote Intracellular Growth.
  12. Ubiquitination of non-protein substrates.
  13. Proteomic approaches to study ubiquitinomics.
  14. Actionable cancer vulnerability due to translational arrest, p53 aggregation and ribosome biogenesis stress evoked by the disulfiram metabolite CuET.
  15. The multiple ubiquitination mechanisms in CFTR peripheral quality control.
  16. Dynamics and composition of small heat shock protein condensates and aggregates.
  17. Atransgenic mouse line for assaying tissue-specific changes in endoplasmic reticulum proteostasis.
  18. Termination of STING responses is mediated via ESCRT-dependent degradation.
  19. Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants.
  20. Nuclear bodies protect phase separated proteins from degradation in stressed proteome.
  21. Activation of ACLY by SEC63 deploys metabolic reprogramming to facilitate hepatocellular carcinoma metastasis upon endoplasmic reticulum stress.
  22. The ribosome-associated protein RACK1 represses Kir4.1 translation in astrocytes and influences neuronal activity.
  23. Investigation of cellular response to the HSP90 inhibition in human cells through thermal proteome profiling.
  24. Dissecting the effects of GTPase and kinase domain mutations on LRRK2 endosomal localization and activity.
  25. Mitochondrial protein import clogging as a mechanism of disease.
  26. The role of eIF4F-driven mRNA translation in regulating the tumour microenvironment.
  27. Intracellular trafficking of HLA-E and its regulation.
  28. Sirtuin 6 is required for the integrated stress response and resistance to inhibition of transcriptional cyclin-dependent kinases.
  29. Sirtuin4 impacts mitochondrial homeostasis in pancreatic cancer cells by reducing the stability of AlkB homolog 1 via deacetylation of the HRD1-SEL1L complex.
  30. Protocol to screen for Sorafenib resistance regulators using pooled lentiviral shRNA library and a Sorafenib-resistant hepatocellular carcinoma cell model.
  31. Molecular basis of translation termination at noncanonical stop codons in human mitochondria.
  32. The mitochondrial unfolded protein response regulates hippocampal neural stem cell aging.
  1. Autophagy. 2023 May 01. 1-2
    Ufmylation bridges autophagy and ER homeostasis in plants.
    Baiying Li, Liwen Jiang.
      The autophagic machinery is highly conserved in eukaryotes. Plants, as sessile organisms, are more susceptible to environmental stresses than animals. Autophagy plays a pivotal role in plant stress responses, but the regulation of autophagic flux in plants remains enigmatic with few autophagic receptors identified. We recently characterized an E3 ligase, the ubiquitin-fold modifier 1 (Ufm1) ligase 1 (Ufl1), as well as its small modifier protein Ufm1, as interactors of the core autophagy-related (ATG) proteins. Mutants of these ufmylation system components are hypersensitive to salt stress and trigger the upregulation of endoplasmic reticulum (ER) stress-responsive genes, as well as the accumulation of ER sheets caused by a defect in reticulophagy. Increased expression of Ufl1, Ufm1 and Ufm1-conjugating enzyme 1 (Ufc1) are also triggered by salt stress in plants. This study identified and demonstrated the participation of ufmylation components in maintaining ER homeostasis by regulating reticulophagy under salt stress in plants.Abbreviations: ATG, autophagy-related; ER, endoplasmic reticulum; LIR, LC3-interacting region; ROS, reactive oxygen species; CDK5RAP3/C53, CDK5 regulatory subunit-associated protein 3; Uba5, Ufm1-activating enzyme 5; Ufc1, Ufm1-conjugating enzyme 1; Ufl1, Ufm1 ligase 1; Ufm1, ubiquitin-fold modifier 1; UPR, unfolded protein response.
    Keywords:  Arabidopsis; ER homeostasis; ER stress; autophagy; reticulophagy; salt stress
    DOI:  https://doi.org/10.1080/15548627.2023.2203985
  2. bioRxiv. 2023 Apr 21. pii: 2023.04.20.537623. [Epub ahead of print]
    Structural remodeling of the endoplasmic reticulum in response to extracellular signals requires αTAT1-induced microtubule acetylation.
    Hannah R Ortiz, Paola Cruz Flores, Aaron Ramonett, Tasmia Ahmed, Nathan A Ellis, Paul R Langlais, Karthikeyan Mythreye, Nam Y Lee.
      Dynamic changes in the endoplasmic reticulum (ER) morphology are central to maintaining cellular homeostasis. Microtubules (MT) facilitate the continuous remodeling of the ER network into sheets and tubules by coordinating with many ER-shaping protein complexes, although how this process is controlled by extracellular signals remains unknown. Here we report that TAK1, a kinase responsive to numerous growth factors and cytokines including TGF-β and TNF-α, triggers ER tubulation by activating αTAT1, an MT-acetylating enzyme that enhances ER-sliding. We show that this TAK1/αTAT-dependent ER remodeling promotes cell survival by actively downregulating BOK, an ER membrane-associated proapoptotic effector. While BOK is normally protected from degradation when complexed with IP3R, it is rapidly degraded upon their dissociation during the ER sheets-to-tubules conversion. These findings demonstrate a distinct mechanism of ligand-induced ER remodeling and suggest that the TAK1/αTAT pathway may be a key target in ER stress and dysfunction.
    DOI:  https://doi.org/10.1101/2023.04.20.537623
  3. Nat Commun. 2023 04 29. 14(1): 2474
    Recognition of an Ala-rich C-degron by the E3 ligase Pirh2.
    Xiaolu Wang, Yao Li, Xiaojie Yan, Qing Yang, Bing Zhang, Ying Zhang, Xinxin Yuan, Chenhao Jiang, Dongxing Chen, Quanyan Liu, Tong Liu, Wenyi Mi, Ying Yu, Cheng Dong.
      The ribosome-associated quality-control (RQC) pathway degrades aberrant nascent polypeptides arising from ribosome stalling during translation. In mammals, the E3 ligase Pirh2 mediates the degradation of aberrant nascent polypeptides by targeting the C-terminal polyalanine degrons (polyAla/C-degrons). Here, we present the crystal structure of Pirh2 bound to the polyAla/C-degron, which shows that the N-terminal domain and the RING domain of Pirh2 form a narrow groove encapsulating the alanine residues of the polyAla/C-degron. Affinity measurements in vitro and global protein stability assays in cells further demonstrate that Pirh2 recognizes a C-terminal A/S-X-A-A motif for substrate degradation. Taken together, our study provides the molecular basis underlying polyAla/C-degron recognition by Pirh2 and expands the substrate recognition spectrum of Pirh2.
    DOI:  https://doi.org/10.1038/s41467-023-38173-6
  4. PLoS Genet. 2023 May 01. 19(5): e1010734
    Substrate-specific effects of natural genetic variation on proteasome activity.
    Mahlon A Collins, Randi Avery, Frank W Albert.
      Protein degradation is an essential biological process that regulates protein abundance and removes misfolded and damaged proteins from cells. In eukaryotes, most protein degradation occurs through the stepwise actions of two functionally distinct entities, the ubiquitin system and the proteasome. Ubiquitin system enzymes attach ubiquitin to cellular proteins, targeting them for degradation. The proteasome then selectively binds and degrades ubiquitinated substrate proteins. Genetic variation in ubiquitin system genes creates heritable differences in the degradation of their substrates. However, the challenges of measuring the degradative activity of the proteasome independently of the ubiquitin system in large samples have limited our understanding of genetic influences on the proteasome. Here, using the yeast Saccharomyces cerevisiae, we built and characterized reporters that provide high-throughput, ubiquitin system-independent measurements of proteasome activity. Using single-cell measurements of proteasome activity from millions of genetically diverse yeast cells, we mapped 15 loci across the genome that influence proteasomal protein degradation. Twelve of these 15 loci exerted specific effects on the degradation of two distinct proteasome substrates, revealing a high degree of substrate-specificity in the genetics of proteasome activity. Using CRISPR-Cas9-based allelic engineering, we resolved a locus to a causal variant in the promoter of RPT6, a gene that encodes a subunit of the proteasome's 19S regulatory particle. The variant increases RPT6 expression, which we show results in increased proteasome activity. Our results reveal the complex genetic architecture of proteasome activity and suggest that genetic influences on the proteasome may be an important source of variation in the many cellular and organismal traits shaped by protein degradation.
    DOI:  https://doi.org/10.1371/journal.pgen.1010734
  5. Nat Commun. 2023 May 05. 14(1): 2617
    Oligomeric scaffolding for curvature generation by ER tubule-forming proteins.
    Yun Xiang, Rui Lyu, Junjie Hu.
      The reticulons and receptor expression-enhancing proteins (REEPs) in the endoplasmic reticulum (ER) are necessary and sufficient for generating ER tubules. However, the mechanism of curvature generation remains elusive. Here, we systematically analyze components of the REEP family based on AI-predicted structures. In yeast REEP Yop1p, TM1/2 and TM3/4 form hairpins and TM2-4 exist as a bundle. Site-directed cross-linking reveals that TM2 and TM4 individually mediate homotypic dimerization, allowing further assembly into a curved shape. Truncated Yop1p lacking TM1 (equivalent to REEP1) retains the curvature-generating capability, undermining the role of the intrinsic wedge. Unexpectedly, both REEP1 and REEP5 fail to replace Yop1p in the maintenance of ER morphology, mostly due to a subtle difference in oligomerization tendency, which involves not only the TM domains, but also the TM-connecting cytosolic loop and previously neglected C-terminal helix. Several hereditary spastic paraplegia-causing mutations in REEP1 appear at the oligomeric interfaces identified here, suggesting compromised self-association of REEP as a pathogenic mechanism. These results indicate that membrane curvature stabilization by integral membrane proteins is dominantly achieved by curved, oligomeric scaffolding.
    DOI:  https://doi.org/10.1038/s41467-023-38294-y
  6. Cell Rep. 2023 May 03. pii: S2211-1247(23)00474-6. [Epub ahead of print]42(5): 112463
    Ubiquitin proteomics identifies RNA polymerase I as a target of the Smc5/6 complex.
    Eva Ibars, Joan Codina-Fabra, Gemma Bellí, Celia Casas, Marc Tarrés, Roger Solé-Soler, Neus P Lorite, Pilar Ximénez-Embún, Javier Muñoz, Neus Colomina, Jordi Torres-Rosell.
      Ubiquitination controls numerous cellular processes, and its deregulation is associated with many pathologies. The Nse1 subunit in the Smc5/6 complex contains a RING domain with ubiquitin E3 ligase activity and essential functions in genome integrity. However, Nse1-dependent ubiquitin targets remain elusive. Here, we use label-free quantitative proteomics to analyze the nuclear ubiquitinome of nse1-C274A RING mutant cells. Our results show that Nse1 impacts the ubiquitination of several proteins involved in ribosome biogenesis and metabolism that, importantly, extend beyond canonical functions of Smc5/6. In addition, our analysis suggests a connection between Nse1 and RNA polymerase I (RNA Pol I) ubiquitination. Specifically, Nse1 and the Smc5/6 complex promote ubiquitination of K408 and K410 in the clamp domain of Rpa190, a modification that induces its degradation in response to blocks in transcriptional elongation. We propose that this mechanism contributes to Smc5/6-dependent segregation of the rDNA array, the locus transcribed by RNA Pol I.
    Keywords:  CP: Molecular biology; ubiquitin, Nse1, Smc5/6, Rpa190, Ubp10, Pol I, rDNA, BMH-21, DNA damage
    DOI:  https://doi.org/10.1016/j.celrep.2023.112463
  7. Nat Cell Biol. 2023 May 04.
    SEL1L-HRD1 endoplasmic reticulum-associated degradation controls STING-mediated innate immunity by limiting the size of the activable STING pool.
    Yewei Ji, Yuan Luo, Yating Wu, Yao Sun, Lianfeng Zhao, Zhen Xue, Mengqi Sun, Xiaoqiong Wei, Zinan He, Shuangcheng Alivia Wu, Liangguang Leo Lin, You Lu, Lei Chang, Fei Chen, Siyu Chen, Wei Qian, Xiaoxi Xu, Shengnuo Chen, Dongli Pan, Zhangsen Zhou, Sheng Xia, Chih-Chi Andrew Hu, Tingbo Liang, Ling Qi.
      Stimulator of interferon genes (STING) orchestrates the production of proinflammatory cytokines in response to cytosolic double-stranded DNA; however, the pathophysiological significance and molecular mechanism underlying the folding and maturation of nascent STING protein at the endoplasmic reticulum (ER) remain unknown. Here we report that the SEL1L-HRD1 protein complex-the most conserved branch of ER-associated degradation (ERAD)-is a negative regulator of the STING innate immunity by ubiquitinating and targeting nascent STING protein for proteasomal degradation in the basal state. SEL1L or HRD1 deficiency in macrophages specifically amplifies STING signalling and immunity against viral infection and tumour growth. Mechanistically, nascent STING protein is a bona fide substrate of SEL1L-HRD1 in the basal state, uncoupled from ER stress or its sensor inositol-requiring enzyme 1α. Hence, our study not only establishes a key role of SEL1L-HRD1 ERAD in innate immunity by limiting the size of the activable STING pool, but identifies a regulatory mechanism and therapeutic approach to targeting STING.
    DOI:  https://doi.org/10.1038/s41556-023-01138-4
  8. Biochem Soc Trans. 2023 May 04. pii: BST20221304. [Epub ahead of print]
    Redox-regulated chaperones in cell stress responses.
    Kathrin Ulrich.
      Proteostasis and redox homeostasis are tightly interconnected and most protein quality control pathways are under direct redox regulation which allow cells to immediately respond to oxidative stress conditions. The activation of ATP-independent chaperones serves as a first line of defense to counteract oxidative unfolding and aggregation of proteins. Conserved cysteine residues evolved as redox-sensitive switches which upon reversible oxidation induce substantial conformational rearrangements and the formation of chaperone-active complexes. In addition to harnessing unfolding proteins, these chaperone holdases interact with ATP-dependent chaperone systems to facilitate client refolding and restoring proteostasis during stress recovery. This minireview gives an insight into highly orchestrated mechanisms regulating the stress-specific activation and inactivation of redox-regulated chaperones and their role in cell stress responses.
    Keywords:  molecular chaperones; oxidative stress; protein aggregation; proteostasis; redox signalling
    DOI:  https://doi.org/10.1042/BST20221304
  9. EMBO J. 2023 May 05. e113609
    Ubx5-Cdc48 assists the protease Wss1 at DNA-protein crosslink sites in yeast.
    Audrey Noireterre, Nataliia Serbyn, Ivona Bagdiul, Françoise Stutz.
      DNA-protein crosslinks (DPCs) pose a serious threat to genome stability. The yeast proteases Wss1, 26S proteasome, and Ddi1 are safeguards of genome integrity by acting on a plethora of DNA-bound proteins in different cellular contexts. The AAA ATPase Cdc48/p97 is known to assist Wss1/SPRTN in clearing DNA-bound complexes; however, its contribution to DPC proteolysis remains unclear. Here, we show that the Cdc48 adaptor Ubx5 is detrimental in yeast mutants defective in DPC processing. Using an inducible site-specific crosslink, we show that Ubx5 accumulates at persistent DPC lesions in the absence of Wss1, which prevents their efficient removal from the DNA. Abolishing Cdc48 binding or complete loss of Ubx5 suppresses sensitivity of wss1∆ cells to DPC-inducing agents by favoring alternate repair pathways. We provide evidence for cooperation of Ubx5-Cdc48 and Wss1 in the genotoxin-induced degradation of RNA polymerase II (RNAPII), a described candidate substrate of Wss1. We propose that Ubx5-Cdc48 assists Wss1 for proteolysis of a subset of DNA-bound proteins. Together, our findings reveal a central role for Ubx5 in DPC clearance and repair.
    Keywords:  Cdc48/p97; DNA-protein crosslink; UBX protein; protease; yeast
    DOI:  https://doi.org/10.15252/embj.2023113609
  10. Cell. 2023 Apr 27. pii: S0092-8674(23)00404-X. [Epub ahead of print]
    Clp-targeting BacPROTACs impair mycobacterial proteostasis and survival.
    David M Hoi, Sabryna Junker, Lukas Junk, Kristin Schwechel, Katharina Fischel, David Podlesainski, Paige M E Hawkins, Lasse van Geelen, Farnusch Kaschani, Julia Leodolter, Francesca Ester Morreale, Stefan Kleine, Somraj Guha, Klaus Rumpel, Volker M Schmiedel, Harald Weinstabl, Anton Meinhart, Richard J Payne, Markus Kaiser, Markus Hartl, Guido Boehmelt, Uli Kazmaier, Rainer Kalscheuer, Tim Clausen.
      The ClpC1:ClpP1P2 protease is a core component of the proteostasis system in mycobacteria. To improve the efficacy of antitubercular agents targeting the Clp protease, we characterized the mechanism of the antibiotics cyclomarin A and ecumicin. Quantitative proteomics revealed that the antibiotics cause massive proteome imbalances, including upregulation of two unannotated yet conserved stress response factors, ClpC2 and ClpC3. These proteins likely protect the Clp protease from excessive amounts of misfolded proteins or from cyclomarin A, which we show to mimic damaged proteins. To overcome the Clp security system, we developed a BacPROTAC that induces degradation of ClpC1 together with its ClpC2 caretaker. The dual Clp degrader, built from linked cyclomarin A heads, was highly efficient in killing pathogenic Mycobacterium tuberculosis, with >100-fold increased potency over the parent antibiotic. Together, our data reveal Clp scavenger proteins as important proteostasis safeguards and highlight the potential of BacPROTACs as future antibiotics.
    Keywords:  BacPROTAC; Clp protease; ClpC1; Mycobacterium tuberculosis; antibiotics; cyclomarin A; ecumicin; protein quality control; small-molecule degrader; targeted protein degradation
    DOI:  https://doi.org/10.1016/j.cell.2023.04.009
  11. Infect Immun. 2023 Apr 27. e0013023
    The Brucella abortus Type IV Effector BspA Inhibits MARCH6-Dependent ERAD To Promote Intracellular Growth.
    Stanimir Kambarev, Elizabeth Borghesan, Cheryl N Miller, Sebenzile Myeni, Jean Celli.
      Brucella abortus, the intracellular causative agent of brucellosis, relies on type IV secretion system (T4SS) effector-mediated modulation of host cell functions to establish a replicative niche, the Brucella-containing vacuole (BCV). Brucella exploits the host's endocytic, secretory, and autophagic pathways to modulate the nature and function of its vacuole from an endocytic BCV (eBCV) to an endoplasmic reticulum (ER)-derived replicative BCV (rBCV) to an autophagic egress BCV (aBCV). A role for the host ER-associated degradation pathway (ERAD) in the B. abortus intracellular cycle was recently uncovered, as it is enhanced by the T4SS effector BspL to control the timing of aBCV-mediated egress. Here, we show that the T4SS effector BspA also interferes with ERAD, yet to promote B. abortus intracellular proliferation. BspA was required for B. abortus replication in bone marrow-derived macrophages and interacts with membrane-associated RING-CH-type finger 6 (MARCH6), a host E3 ubiquitin ligase involved in ERAD. Pharmacological inhibition of ERAD and small interfering RNA (siRNA) depletion of MARCH6 did not affect the replication of wild-type B. abortus but rescued the replication defect of a bspA deletion mutant, while depletion of the ERAD component UbxD8 affected replication of B. abortus and rescued the replication defect of the bspA mutant. BspA affected the degradation of ERAD substrates and destabilized the MARCH6 E3 ligase complex. Taken together, these findings indicate that BspA inhibits the host ERAD pathway via targeting of MARCH6 to promote B. abortus intracellular growth. Our data reveal that targeting ERAD components by type IV effectors emerges as a multifaceted theme in Brucella pathogenesis.
    Keywords:  Brucella; BspA; ERAD; MARCH6; endoplasmic reticulum; type IV secretion
    DOI:  https://doi.org/10.1128/iai.00130-23
  12. Trends Cell Biol. 2023 Apr 27. pii: S0962-8924(23)00071-5. [Epub ahead of print]
    Ubiquitination of non-protein substrates.
    Jun-Ichi Sakamaki, Noboru Mizushima.
      The covalent attachment of ubiquitin is a common regulatory mechanism in various proteins. Although it has long been thought that the substrates of ubiquitination are limited to proteins, recent studies have changed this view: ubiquitin can be conjugated to lipids, sugars, and nucleotides. Ubiquitin is linked to these substrates by the action of different classes of ubiquitin ligases that have distinct catalytic mechanisms. Ubiquitination of non-protein substrates likely serves as a signal for the recruitment of other proteins to bring about specific effects. These discoveries have expanded the concept of ubiquitination and have advanced our insight into the biology and chemistry of this well-established modification process. In this review we describe the molecular mechanisms and roles of non-protein ubiquitination and discuss the current limitations.
    Keywords:  non-canonical ubiquitination; phospholipids; ubiquitin; ubiquitin-like proteins
    DOI:  https://doi.org/10.1016/j.tcb.2023.03.014
  13. Biochim Biophys Acta Gene Regul Mech. 2023 Apr 28. pii: S1874-9399(23)00035-4. [Epub ahead of print] 194940
    Proteomic approaches to study ubiquitinomics.
    Indrajit Sahu, He Zhu, Sara J Buhrlage, Jarrod A Marto.
      As originally described some 40 years ago, protein ubiquitination was thought to serve primarily as a static mark for protein degradation. In the ensuing years, it has become clear that 'ubiquitination' is a structurally diverse and dynamic post-translational modification and is intricately involved in a myriad of signaling pathways in all eukaryote cells. And like other key pathways in the functional proteome, ubiquitin signaling is often disrupted, sometimes severely so, in human pathophysiology. As a result of its central role in normal physiology and human disease, the ubiquitination field is now represented across the full landscape of biomedical research from fundamental structural and biochemical studies to translational and clinical research. In recent years, mass spectrometry has emerged as a powerful technology for the detection and characterization of protein ubiquitination. Herein we detail qualitative and quantitative proteomic methods using a compare/contrast approach to highlight their strengths and weaknesses.
    Keywords:  KεGG; Mass-spectrometry; Middle-down; Proteomics; Ubiquitin; diGly peptides
    DOI:  https://doi.org/10.1016/j.bbagrm.2023.194940
  14. Cell Death Differ. 2023 May 04.
    Actionable cancer vulnerability due to translational arrest, p53 aggregation and ribosome biogenesis stress evoked by the disulfiram metabolite CuET.
    Dimitris C Kanellis, Asimina Zisi, Zdenek Skrott, Bennie Lemmens, Jaime A Espinoza, Martin Kosar, Andrea Björkman, Xuexin Li, Stefanos Arampatzis, Jirina Bartkova, Miguel Andújar-Sánchez, Oscar Fernandez-Capetillo, Martin Mistrik, Mikael S Lindström, Jiri Bartek.
      Drug repurposing is a versatile strategy to improve current therapies. Disulfiram has long been used in the treatment of alcohol dependency and multiple clinical trials to evaluate its clinical value in oncology are ongoing. We have recently reported that the disulfiram metabolite diethyldithiocarbamate, when combined with copper (CuET), targets the NPL4 adapter of the p97VCP segregase to suppress the growth of a spectrum of cancer cell lines and xenograft models in vivo. CuET induces proteotoxic stress and genotoxic effects, however important issues concerning the full range of the CuET-evoked tumor cell phenotypes, their temporal order, and mechanistic basis have remained largely unexplored. Here, we have addressed these outstanding questions and show that in diverse human cancer cell models, CuET causes a very early translational arrest through the integrated stress response (ISR), later followed by features of nucleolar stress. Furthermore, we report that CuET entraps p53 in NPL4-rich aggregates leading to elevated p53 protein and its functional inhibition, consistent with the possibility of CuET-triggered cell death being p53-independent. Our transcriptomics profiling revealed activation of pro-survival adaptive pathways of ribosomal biogenesis (RiBi) and autophagy upon prolonged exposure to CuET, indicating potential feedback responses to CuET treatment. The latter concept was validated here by simultaneous pharmacological inhibition of RiBi and/or autophagy that further enhanced CuET's tumor cytotoxicity, using both cell culture and zebrafish in vivo preclinical models. Overall, these findings expand the mechanistic repertoire of CuET's anti-cancer activity, inform about the temporal order of responses and identify an unorthodox new mechanism of targeting p53. Our results are discussed in light of cancer-associated endogenous stresses as exploitable tumor vulnerabilities and may inspire future clinical applications of CuET in oncology, including combinatorial treatments and focus on potential advantages of using certain validated drug metabolites, rather than old, approved drugs with their, often complex, metabolic profiles.
    DOI:  https://doi.org/10.1038/s41418-023-01167-4
  15. Biochem Soc Trans. 2023 May 04. pii: BST20221468. [Epub ahead of print]
    The multiple ubiquitination mechanisms in CFTR peripheral quality control.
    Shogo Taniguchi, Ryosuke Fukuda, Tsukasa Okiyoneda.
      The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated anion channel, which is expressed on the apical plasma membrane (PM) of epithelial cells. Mutations in the CFTR gene cause cystic fibrosis (CF), one of the most common genetic diseases among Caucasians. Most CF-associated mutations result in misfolded CFTR proteins that are degraded by the endoplasmic reticulum quality control (ERQC) mechanism. However, the mutant CFTR reaching the PM through therapeutic agents is still ubiquitinated and degraded by the peripheral protein quality control (PeriQC) mechanism, resulting in reduced therapeutic efficacy. Moreover, certain CFTR mutants that can reach the PM under physiological conditions are degraded by PeriQC. Thus, it may be beneficial to counteract the selective ubiquitination in PeriQC to enhance therapeutic outcomes for CF. Recently, the molecular mechanisms of CFTR PeriQC have been revealed, and several ubiquitination mechanisms, including both chaperone-dependent and -independent pathways, have been identified. In this review, we will discuss the latest findings related to CFTR PeriQC and propose potential novel therapeutic strategies for CF.
    Keywords:  CFTR; CFTR modulators; RFFL; RNF34; cystic fibrosis; ubiquitination
    DOI:  https://doi.org/10.1042/BST20221468
  16. J Mol Biol. 2023 May 03. pii: S0022-2836(23)00217-6. [Epub ahead of print] 168139
    Dynamics and composition of small heat shock protein condensates and aggregates.
    Joep Joosten, Bob van Sluijs, Wilma Vree Egberts, Martin Emmaneel, Pascal W T C Jansen, Michiel Vermeulen, Wilbert Boelens, Kimberly M Bonger, Evan Spruijt.
      Small heat shock proteins (sHSPs) are essential ATP-independent chaperones that protect the cellular proteome. These proteins assemble into polydisperse oligomeric structures, the composition of which dramatically affects their chaperone activity. The biomolecular consequences of variations in sHSP ratios, especially inside living cells, remain elusive. Here, we study the consequences of altering the relative expression levels of HspB2 and HspB3 in HEK293T cells. These chaperones are partners in a hetero-oligomeric complex, and genetic mutations that abolish their mutual interaction are associated with myopathic disorders. HspB2 displays three distinct phenotypes when co-expressed with HspB3 at varying ratios. Expression of HspB2 alone leads to formation of liquid nuclear condensates, while shifting the stoichiometry towards HspB3 resulted in the formation of large solid-like aggregates. Only cells co-expressing HspB2 with a limited amount of HspB3 formed fully soluble complexes that were distributed homogeneously throughout the nucleus. Strikingly, both condensates and aggregates were reversible, as shifting the HspB2:HspB3 balance in situ resulted in dissolution of these structures. To uncover the molecular composition of HspB2 condensates and aggregates, we used APEX-mediated proximity labelling. Most proteins interact transiently with the condensates and were neither enriched nor depleted in these cells. In contrast, we found that HspB2:HspB3 aggregates sequestered several disordered proteins and autophagy factors, suggesting that the cell is actively attempting to clear these aggregates. This study presents a striking example of how changes in the relative expression levels of interacting proteins affects their phase behavior. Our approach could be applied to study the role of protein stoichiometry and the influence of client binding on phase behavior in other biomolecular condensates and aggregates.
    Keywords:  HspB2; HspB3; liquid-liquid phase separation; molecular chaperone; proximity labeling
    DOI:  https://doi.org/10.1016/j.jmb.2023.168139
  17. Transgenic Res. 2023 May 03.
    Atransgenic mouse line for assaying tissue-specific changes in endoplasmic reticulum proteostasis.
    Reinis Svarcbahs, Sarah M Blossom, Helena S Baffoe-Bonnie, Kathleen A Trychta, Lacey K Greer, James Pickel, Mark J Henderson, Brandon K Harvey.
      Maintenance of calcium homeostasis is important for proper endoplasmic reticulum (ER) function. When cellular stress conditions deplete the high concentration of calcium in the ER, ER-resident proteins are secreted into the extracellular space in a process called exodosis. Monitoring exodosis provides insight into changes in ER homeostasis and proteostasis resulting from cellular stress associated with ER calcium dysregulation. To monitor cell-type specific exodosis in the intact animal, we created a transgenic mouse line with a Gaussia luciferase (GLuc)-based, secreted ER calcium-modulated protein, SERCaMP, preceded by a LoxP-STOP-LoxP (LSL) sequence. The Cre-dependent LSL-SERCaMP mice were crossed with albumin (Alb)-Cre and dopamine transporter (DAT)-Cre mouse lines. GLuc-SERCaMP expression was characterized in mouse organs and extracellular fluids, and the secretion of GLuc-SERCaMP in response to cellular stress was monitored following pharmacological depletion of ER calcium. In LSL-SERCaMP × Alb-Cre mice, robust GLuc activity was observed only in the liver and blood, whereas in LSL-SERCaMP × DAT-Cre mice, GLuc activity was seen in midbrain dopaminergic neurons and tissue samples innervated by dopaminergic projections. After calcium depletion, we saw increased GLuc signal in the plasma and cerebrospinal fluid collected from the Alb-Cre and DAT-Cre crosses, respectively. This mouse model can be used to investigate the secretion of ER-resident proteins from specific cell and tissue types during disease pathogenesis and may aid in the identification of therapeutics and biomarkers of disease.
    Keywords:  ER calcium sensor; ER stress; Exodosis; Gaussia luciferase; SERCaMP; Secreted ER-resident proteins
    DOI:  https://doi.org/10.1007/s11248-023-00349-7
  18. EMBO J. 2023 May 04. e112712
    Termination of STING responses is mediated via ESCRT-dependent degradation.
    Katherine R Balka, Rajan Venkatraman, Tahnee L Saunders, Angus Shoppee, Ee Shan Pang, Zoe Magill, Jihane Homman-Ludiye, Cheng Huang, Rachael M Lane, Harrison M York, Peck Tan, Ralf B Schittenhelm, Senthil Arumugam, Benjamin T Kile, Meredith O'Keeffe, Dominic De Nardo.
      cGAS-STING signalling is induced by detection of foreign or mislocalised host double-stranded (ds)DNA within the cytosol. STING acts as the major signalling hub, where it controls production of type I interferons and inflammatory cytokines. Basally, STING resides on the ER membrane. Following activation STING traffics to the Golgi to initiate downstream signalling and subsequently to endolysosomal compartments for degradation and termination of signalling. While STING is known to be degraded within lysosomes, the mechanisms controlling its delivery remain poorly defined. Here we utilised a proteomics-based approach to assess phosphorylation changes in primary murine macrophages following STING activation. This identified numerous phosphorylation events in proteins involved in intracellular and vesicular transport. We utilised high-temporal microscopy to track STING vesicular transport in live macrophages. We subsequently identified that the endosomal complexes required for transport (ESCRT) pathway detects ubiquitinated STING on vesicles, which facilitates the degradation of STING in murine macrophages. Disruption of ESCRT functionality greatly enhanced STING signalling and cytokine production, thus characterising a mechanism controlling effective termination of STING signalling.
    Keywords:  ESCRT; cGAS-STING; innate immunity; lysosomal degradation; vesicular trafficking
    DOI:  https://doi.org/10.15252/embj.2022112712
  19. bioRxiv. 2023 Apr 19. pii: 2023.04.18.537383. [Epub ahead of print]
    Pharmacological chaperones restore proteostasis of epilepsy-associated GABA A receptor variants.
    Ya-Juan Wang, Hailey Seibert, Lucie Y Ahn, Ashleigh E Schaffer, Ting-Wei Mu.
      Recent advances in genetic diagnosis identified variants in genes encoding GABA A receptors as causative for genetic epilepsy. Here, we selected eight disease-associated variants in the α1 subunit of GABA A receptors causing mild to severe clinical phenotypes and showed that they are loss of function, mainly by reducing the folding and surface trafficking of the α1 protein. Furthermore, we sought client protein-specific pharmacological chaperones to restore the function of pathogenic receptors. Applications of positive allosteric modulators, including Hispidulin and TP003, increase the functional surface expression of the α1 variants. Mechanism of action study demonstrated that they enhance the folding and assembly and reduce the degradation of GABA A variants without activating the unfolded protein response in HEK293T cells and human iPSC-derived neurons. Since these compounds cross the blood-brain barrier, such a pharmacological chaperoning strategy holds great promise to treat genetic epilepsy in a GABA A receptor-specific manner.
    DOI:  https://doi.org/10.1101/2023.04.18.537383
  20. bioRxiv. 2023 Apr 21. pii: 2023.04.19.537522. [Epub ahead of print]
    Nuclear bodies protect phase separated proteins from degradation in stressed proteome.
    Kwan Ho Jung, Jiarui Sun, Chia-Heng Hsiung, Xiaojun Lance Lian, Yu Liu, Xin Zhang.
      RNA-binding proteins (RBPs) containing intrinsically disordered domains undergo liquid-liquid phase separation to form nuclear bodies under stress conditions. This process is also connected to the misfolding and aggregation of RBPs, which are associated with a series of neurodegenerative diseases. However, it remains elusive how folding states of RBPs changes upon the formation and maturation of nuclear bodies. Here, we describe SNAP-tag based imaging methods to visualize the folding states of RBPs in live cells via time-resolved quantitative microscopic analyses of their micropolarity and microviscosity. Using these imaging methods in conjunction with immunofluorescence imaging, we demonstrate that RBPs, represented by TDP-43, initially enters the PML nuclear bodies in its native state upon transient proteostasis stress, albeit it begins to misfolded during prolonged stress. Furthermore, we show that heat shock protein 70 co-enters the PML nuclear bodies to prevent the degradation of TDP-43 from the proteotoxic stress, thus revealing a previously unappreciated protective role of the PML nuclear bodies in the prevention of stress-induced degradation of TDP-43. In summary, our imaging methods described in the manuscript, for the first time, reveal the folding states of RBPs, which were previously challenging to study with conventional methods in nuclear bodies of live cells. This study uncovers the mechanistic correlations between the folding states of a protein and functions of nuclear bodies, in particular PML bodies. We envision that the imaging methods can be generally applied to elucidating the structural aspects of other proteins that exhibit granular structures under biological stimulus.
    DOI:  https://doi.org/10.1101/2023.04.19.537522
  21. J Exp Clin Cancer Res. 2023 May 01. 42(1): 108
    Activation of ACLY by SEC63 deploys metabolic reprogramming to facilitate hepatocellular carcinoma metastasis upon endoplasmic reticulum stress.
    Chenyu Hu, Zechang Xin, Xiaoyan Sun, Yang Hu, Chunfeng Zhang, Rui Yan, Yuying Wang, Min Lu, Jing Huang, Xiaojuan Du, Baocai Xing, Xiaofeng Liu.
      BACKGROUND: Tumor cells display augmented capability to maintain endoplasmic reticulum (ER) homeostasis and hijack ER stress pathway for malignant phenotypes under microenvironmental stimuli. Metabolic reprogramming is a well-known hallmark for tumor cells to provide specific adaptive traits to the microenvironmental alterations. However, it's unknown how tumor cells orchestrate metabolic reprogramming and tumor progression in response to ER stress. Herein, we aimed to explore the pivotal roles of SEC63-mediated metabolic remodeling in hepatocellular carcinoma (HCC) cell metastasis after ER stress.METHODS: The expression levels of SEC63 in HCC tissues and adjacent non-cancerous tissues were determined by immunohistochemistry and western blot. The regulatory roles of SEC63 in HCC metastasis were investigated both in vitro and in vivo by RNA-sequencing, metabolites detection, immunofluorescence, and transwell migration/invasion analyses. GST pull-down, immunoprecipitation/mass spectrometry and in vivo ubiquitination/phosphorylation assay were conducted to elucidate the underlying molecular mechanisms.
    RESULTS: We identified SEC63 as a new regulator of HCC cell metabolism. Upon ER stress, the phosphorylation of SEC63 at T537 by IRE1α pathway contributed to SEC63 activation. Then, the stability of ACLY was upregulated by SEC63 to increase the supply of acetyl-CoA and lipid biosynthesis, which are beneficial for improving ER capacity. Meanwhile, SEC63 also entered into nucleus for increasing nuclear acetyl-CoA production to upregulate unfolded protein response targets to improve ER homeostasis. Importantly, SEC63 coordinated with ACLY to epigenetically modulate expression of Snail1 in the nucleus. Consequently, SEC63 promoted HCC cell metastasis and these effects were reversed by ACLY inhibition. Clinically, SEC63 expression was significantly upregulated in HCC tissue specimens and was positively correlated with ACLY expression. Importantly, high expression of SEC63 predicted unfavorable prognosis of HCC patients.
    CONCLUSIONS: Our findings revealed that SEC63-mediated metabolic reprogramming plays important roles in keeping ER homeostasis upon stimuli in HCC cells. Meanwhile, SEC63 coordinates with ACLY to upregulate the expression of Snail1, which further promotes HCC metastasis. Metastasis is crucial for helping cancer cells seek new settlements upon microenvironmental stimuli. Taken together, our findings highlight a cancer selective adaption to ER stress as well as reveal the potential roles of the IRE1α-SEC63-ACLY axis in HCC treatment.
    Keywords:  ACLY; ER stress; Hepatocellular carcinoma; Metabolic reprogramming; Metastasis; SEC63
    DOI:  https://doi.org/10.1186/s13046-023-02656-7
  22. Cell Rep. 2023 Apr 30. pii: S2211-1247(23)00467-9. [Epub ahead of print]42(5): 112456
    The ribosome-associated protein RACK1 represses Kir4.1 translation in astrocytes and influences neuronal activity.
    Marc Oudart, Katia Avila-Gutierrez, Clara Moch, Elena Dossi, Giampaolo Milior, Anne-Cécile Boulay, Mathis Gaudey, Julien Moulard, Bérangère Lombard, Damarys Loew, Alexis-Pierre Bemelmans, Nathalie Rouach, Clément Chapat, Martine Cohen-Salmon.
      The regulation of translation in astrocytes, the main glial cells in the brain, remains poorly characterized. We developed a high-throughput proteomics screen for polysome-associated proteins in astrocytes and focused on ribosomal protein receptor of activated protein C kinase 1 (RACK1), a critical factor in translational regulation. In astrocyte somata and perisynaptic astrocytic processes (PAPs), RACK1 preferentially binds to a number of mRNAs, including Kcnj10, encoding the inward-rectifying potassium (K+) channel Kir4.1. By developing an astrocyte-specific, conditional RACK1 knockout mouse model, we show that RACK1 represses production of Kir4.1 in hippocampal astrocytes and PAPs. Upregulation of Kir4.1 in the absence of RACK1 increases astrocytic Kir4.1-mediated K+ currents and volume. It also modifies neuronal activity attenuating burst frequency and duration. Reporter-based assays reveal that RACK1 controls Kcnj10 translation through the transcript's 5' untranslated region. Hence, translational regulation by RACK1 in astrocytes represses Kir4.1 expression and influences neuronal activity.
    Keywords:  CP: Neuroscience; K(+)current; Kir4.1; RACK1; astrocytes; neuroglial interactions; neurotransmission; ribosome; translation
    DOI:  https://doi.org/10.1016/j.celrep.2023.112456
  23. Mol Cell Proteomics. 2023 Apr 27. pii: S1535-9476(23)00070-1. [Epub ahead of print] 100560
    Investigation of cellular response to the HSP90 inhibition in human cells through thermal proteome profiling.
    Kejun Yin, Ronghu Wu.
      Heat shock proteins are chaperones and they are responsible for protein folding in cells. HSP90 is one of the most important chaperones in human cells, and its inhibition is promising for cancer therapy. However, despite the development of multiple HSP90 inhibitors, none of them has been approved for disease treatment due to unexpected cellular toxicity and side-effects. Hence, a more comprehensive investigation of cellular response to HSP90 inhibitors can aid in a better understanding of the molecular mechanisms of the cytotoxicity and side effects of these inhibitors. The thermal stability shifts of proteins, which represent protein structure and interaction alterations, can provide valuable information complementary to the results obtained from commonly used abundance-based proteomics analysis. Here, we systematically investigated cell response to different HSP90 inhibitors through global quantification of protein thermal stability changes using thermal proteome profiling, together with measurement of protein abundance changes. Besides the targets and potential off-targets of the drugs, proteins with significant thermal stability changes under the HSP90 inhibition are found to be involved in cell stress responses and the translation process. Moreover, proteins with thermal stability shifts under the inhibition are upstream of those with altered expression. These findings indicate that the HSP90 inhibition perturbs cell transcription and translation processes. The current study provides a different perspective for achieving a better understanding of cellular response to the chaperone inhibition.
    Keywords:  HSP90 inhibitors; Thermal proteome profiling; and protein abundance change; cell response; heat shock protein 90; protein thermal stability shift
    DOI:  https://doi.org/10.1016/j.mcpro.2023.100560
  24. Cell Rep. 2023 May 02. pii: S2211-1247(23)00458-8. [Epub ahead of print]42(5): 112447
    Dissecting the effects of GTPase and kinase domain mutations on LRRK2 endosomal localization and activity.
    Capria Rinaldi, Christopher S Waters, Zizheng Li, Karl Kumbier, Lee Rao, R Jeremy Nichols, Matthew P Jacobson, Lani F Wu, Steven J Altschuler.
      Parkinson's disease-causing leucine-rich repeat kinase 2 (LRRK2) mutations lead to varying degrees of Rab GTPase hyperphosphorylation. Puzzlingly, LRRK2 GTPase-inactivating mutations-which do not affect intrinsic kinase activity-lead to higher levels of cellular Rab phosphorylation than kinase-activating mutations. Here, we investigate whether mutation-dependent differences in LRRK2 cellular localization could explain this discrepancy. We discover that blocking endosomal maturation leads to the rapid formation of mutant LRRK2+ endosomes on which LRRK2 phosphorylates substrate Rabs. LRRK2+ endosomes are maintained through positive feedback, which mutually reinforces membrane localization of LRRK2 and phosphorylated Rab substrates. Furthermore, across a panel of mutants, cells expressing GTPase-inactivating mutants form strikingly more LRRK2+ endosomes than cells expressing kinase-activating mutants, resulting in higher total cellular levels of phosphorylated Rabs. Our study suggests that the increased probability that LRRK2 GTPase-inactivating mutants are retained on intracellular membranes compared to kinase-activating mutants leads to higher substrate phosphorylation.
    Keywords:  CP: Cell biology; LRRK2; Parkinson's disease; Rab phosphorylation; VPS34 inhibition; endosomal localization; positive feedback
    DOI:  https://doi.org/10.1016/j.celrep.2023.112447
  25. Elife. 2023 May 02. pii: e84330. [Epub ahead of print]12
    Mitochondrial protein import clogging as a mechanism of disease.
    Liam P Coyne, Xiaowen Wang, Jiyao Song, Ebbing de Jong, Karin Schneider, Paul T Massa, Frank A Middleton, Thomas Becker, Xin Jie Chen.
      Mitochondrial biogenesis requires the import of >1,000 mitochondrial preproteins from the cytosol. Most studies on mitochondrial protein import are focused on the core import machinery. Whether and how the biophysical properties of substrate preproteins affect overall import efficiency is underexplored. Here, we show that protein traffic into mitochondria can be disrupted by amino acid substitutions in a single substrate preprotein. Pathogenic missense mutations in ADP/ATP translocase 1 (ANT1), and its yeast homolog Aac2, cause the protein to accumulate along the protein import pathway, thereby obstructing general protein translocation into mitochondria. This impairs mitochondrial respiration, cytosolic proteostasis and cell viability independent of ANT1's nucleotide transport activity. The mutations act synergistically, as double mutant Aac2/ANT1 cause severe clogging primarily at the Translocase of the Outer Membrane (TOM) complex. This confers extreme toxicity in yeast. In mice, expression of a super-clogger ANT1 variant led to neurodegeneration and an age-dependent dominant myopathy that phenocopy ANT1-induced human disease, suggesting clogging as a mechanism of disease. More broadly, this work implies the existence of uncharacterized amino acid requirements for mitochondrial carrier proteins to avoid clogging and subsequent disease.
    Keywords:  S. cerevisiae; biochemistry; chemical biology; mouse
    DOI:  https://doi.org/10.7554/eLife.84330
  26. Nat Rev Cancer. 2023 May 04.
    The role of eIF4F-driven mRNA translation in regulating the tumour microenvironment.
    Margarita Bartish, Madelyn J Abraham, Christophe Gonçalves, Ola Larsson, Charlotte Rolny, Sonia V Del Rincón.
      Cells can rapidly adjust their proteomes in dynamic environments by regulating mRNA translation. There is mounting evidence that dysregulation of mRNA translation supports the survival and adaptation of cancer cells, which has stimulated clinical interest in targeting elements of the translation machinery and, in particular, components of the eukaryotic initiation factor 4F (eIF4F) complex such as eIF4E. However, the effect of targeting mRNA translation on infiltrating immune cells and stromal cells in the tumour microenvironment (TME) has, until recently, remained unexplored. In this Perspective article, we discuss how eIF4F-sensitive mRNA translation controls the phenotypes of key non-transformed cells in the TME, with an emphasis on the underlying therapeutic implications of targeting eIF4F in cancer. As eIF4F-targeting agents are in clinical trials, we propose that a broader understanding of their effect on gene expression in the TME will reveal unappreciated therapeutic vulnerabilities that could be used to improve the efficacy of existing cancer therapies.
    DOI:  https://doi.org/10.1038/s41568-023-00567-5
  27. J Exp Med. 2023 Aug 07. pii: e20221941. [Epub ahead of print]220(8):
    Intracellular trafficking of HLA-E and its regulation.
    Wanlin He, Ester Gea-Mallorquí, Huw Colin-York, Marco Fritzsche, Geraldine M Gillespie, Simon Brackenridge, Persephone Borrow, Andrew J McMichael.
      Interest in MHC-E-restricted CD8+ T cell responses has been aroused by the discovery of their efficacy in controlling simian immunodeficiency virus (SIV) infection in a vaccine model. The development of vaccines and immunotherapies utilizing human MHC-E (HLA-E)-restricted CD8+ T cell response requires an understanding of the pathway(s) of HLA-E transport and antigen presentation, which have not been clearly defined previously. We show here that, unlike classical HLA class I, which rapidly exits the endoplasmic reticulum (ER) after synthesis, HLA-E is largely retained because of a limited supply of high-affinity peptides, with further fine-tuning by its cytoplasmic tail. Once at the cell surface, HLA-E is unstable and is rapidly internalized. The cytoplasmic tail plays a crucial role in facilitating HLA-E internalization, which results in its enrichment in late and recycling endosomes. Our data reveal distinctive transport patterns and delicate regulatory mechanisms of HLA-E, which help to explain its unusual immunological functions.
    DOI:  https://doi.org/10.1084/jem.20221941
  28. Sci Transl Med. 2023 May 03. 15(694): eabn9674
    Sirtuin 6 is required for the integrated stress response and resistance to inhibition of transcriptional cyclin-dependent kinases.
    Nithya Kartha, Jessica E Gianopulos, Zachary Schrank, Sarah M Cavender, Stephanie Dobersch, Bryan D Kynnap, Adrianne Wallace-Povirk, Cynthia L Wladyka, Juan F Santana, Jaeseung C Kim, Angela Yu, Caroline M Bridgwater, Kathrin Fuchs, Sarah Dysinger, Aaron E Lampano, Faiyaz Notta, David H Price, Andrew C Hsieh, Sunil R Hingorani, Sita Kugel.
      Pancreatic ductal adenocarcinoma (PDAC) is classified into two key subtypes, classical and basal, with basal PDAC predicting worse survival. Using in vitro drug assays, genetic manipulation experiments, and in vivo drug studies in human patient-derived xenografts (PDXs) of PDAC, we found that basal PDACs were uniquely sensitive to transcriptional inhibition by targeting cyclin-dependent kinase 7 (CDK7) and CDK9, and this sensitivity was recapitulated in the basal subtype of breast cancer. We showed in cell lines, PDXs, and publicly available patient datasets that basal PDAC was characterized by inactivation of the integrated stress response (ISR), which leads to a higher rate of global mRNA translation. Moreover, we identified the histone deacetylase sirtuin 6 (SIRT6) as a critical regulator of a constitutively active ISR. Using expression analysis, polysome sequencing, immunofluorescence, and cycloheximide chase experiments, we found that SIRT6 regulated protein stability by binding activating transcription factor 4 (ATF4) in nuclear speckles and protecting it from proteasomal degradation. In human PDAC cell lines and organoids as well as in murine PDAC genetically engineered mouse models where SIRT6 was deleted or down-regulated, we demonstrated that SIRT6 loss both defined the basal PDAC subtype and led to reduced ATF4 protein stability and a nonfunctional ISR, causing a marked vulnerability to CDK7 and CDK9 inhibitors. Thus, we have uncovered an important mechanism regulating a stress-induced transcriptional program that may be exploited with targeted therapies in particularly aggressive PDAC.
    DOI:  https://doi.org/10.1126/scitranslmed.abn9674
  29. Biochim Biophys Acta Gene Regul Mech. 2023 May 03. pii: S1874-9399(23)00036-6. [Epub ahead of print] 194941
    Sirtuin4 impacts mitochondrial homeostasis in pancreatic cancer cells by reducing the stability of AlkB homolog 1 via deacetylation of the HRD1-SEL1L complex.
    Dongnan Ping, Xiaofan Pu, Guoping Ding, Chaolei Zhang, Junbin Jin, Chengjie Xu, Jiazheng Liu, Shengnan Jia, Liping Cao.
      Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with a poor prognosis. As a tumor inhibitor, the specific tumor suppressor mechanism of Sirtuin4(SIRT4) in PDAC remains elusive. In this study, SIRT4 was found to inhibit PDAC by impacting mitochondrial homeostasis. SIRT4 deacetylated lysine 547 of SEL1L and increased the protein level of an E3 ubiquitin ligase HRD1. As a central member of ER-associated protein degradation (ERAD), HRD1-SEL1L complex is recently reported to regulate the mitochondria, though the mechanism is not fully delineated. Here, we found the increase in SEL1L-HRD1 complex decreased the stability of a mitochondrial protein, ALKBH1. Downregulation of ALKBH1 subsequently blocked the transcription of mitochondrial DNA-coded genes, and resulted in mitochondrial damage. Lastly, a putative SIRT4 stimulator, Entinostat, was identified, which upregulated the expression of SIRT4 and effectively inhibited pancreatic cancer in vivo and in vitro.
    Keywords:  ALKBH1; HRD1-SEL1L; Mitochondrial function; Pancreatic cancer; SIRT4
    DOI:  https://doi.org/10.1016/j.bbagrm.2023.194941
  30. STAR Protoc. 2023 Apr 30. pii: S2666-1667(23)00231-9. [Epub ahead of print]4(2): 102273
    Protocol to screen for Sorafenib resistance regulators using pooled lentiviral shRNA library and a Sorafenib-resistant hepatocellular carcinoma cell model.
    Ruize Gao, Fengyuan Tang, Gerhard Christofori.
      Approaches to study therapy resistance in HCC (hepatocellular carcinoma) are limited, especially when using HCC models in vitro. Here, we present a protocol to establish an in vitro Sorafenib-resistant human HCC cell model and conduct an shRNA-mediated synthetic lethal screen in established Sorafenib-resistant HCC cell lines to identify critical regulators of Sorafenib resistance. We describe steps for RNA sequencing and functional analysis to reveal the mode of action of potential candidates in conferring therapy resistance to HCC cells. For complete details on the use and execution of this protocol, please refer to Gao et al. (2021a)1 and Gao et al. (2021b).2.
    Keywords:  Cancer; Cell Biology; High Throughput Screening; Molecular Biology; RNAseq; Sequencing
    DOI:  https://doi.org/10.1016/j.xpro.2023.102273
  31. Science. 2023 May 05. 380(6644): 531-536
    Molecular basis of translation termination at noncanonical stop codons in human mitochondria.
    Martin Saurer, Marc Leibundgut, Hima Priyanka Nadimpalli, Alain Scaiola, Tanja Schönhut, Richard G Lee, Stefan J Siira, Oliver Rackham, René Dreos, Tea Lenarčič, Eva Kummer, David Gatfield, Aleksandra Filipovska, Nenad Ban.
      The genetic code that specifies the identity of amino acids incorporated into proteins during protein synthesis is almost universally conserved. Mitochondrial genomes feature deviations from the standard genetic code, including the reassignment of two arginine codons to stop codons. The protein required for translation termination at these noncanonical stop codons to release the newly synthesized polypeptides is not currently known. In this study, we used gene editing and ribosomal profiling in combination with cryo-electron microscopy to establish that mitochondrial release factor 1 (mtRF1) detects noncanonical stop codons in human mitochondria by a previously unknown mechanism of codon recognition. We discovered that binding of mtRF1 to the decoding center of the ribosome stabilizes a highly unusual conformation in the messenger RNA in which the ribosomal RNA participates in specific recognition of the noncanonical stop codons.
    DOI:  https://doi.org/10.1126/science.adf9890
  32. Cell Metab. 2023 Apr 28. pii: S1550-4131(23)00139-0. [Epub ahead of print]
    The mitochondrial unfolded protein response regulates hippocampal neural stem cell aging.
    Chih-Ling Wang, Rika Ohkubo, Wei-Chieh Mu, Wei Chen, Jiang Lan Fan, Zehan Song, Ayane Maruichi, Peter H Sudmant, Angela O Pisco, Dena B Dubal, Na Ji, Danica Chen.
      Aging results in a decline in neural stem cells (NSCs), neurogenesis, and cognitive function, and evidence is emerging to demonstrate disrupted adult neurogenesis in the hippocampus of patients with several neurodegenerative disorders. Here, single-cell RNA sequencing of the dentate gyrus of young and old mice shows that the mitochondrial protein folding stress is prominent in activated NSCs/neural progenitors (NPCs) among the neurogenic niche, and it increases with aging accompanying dysregulated cell cycle and mitochondrial activity in activated NSCs/NPCs in the dentate gyrus. Increasing mitochondrial protein folding stress results in compromised NSC maintenance and reduced neurogenesis in the dentate gyrus, neural hyperactivity, and impaired cognitive function. Reducing mitochondrial protein folding stress in the dentate gyrus of old mice improves neurogenesis and cognitive function. These results establish the mitochondrial protein folding stress as a driver of NSC aging and suggest approaches to improve aging-associated cognitive decline.
    Keywords:  SIRT1; SIRT2; SIRT3; SIRT6; SIRT7; cognitive aging; mitochondrial unfolded protein response; neural stem cell aging; sirtuin; stem cell aging
    DOI:  https://doi.org/10.1016/j.cmet.2023.04.012
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