bims-proteo 2023-10-01 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023–10–01
thirty-two papers selected by
Eric Chevet, INSERM

Small molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states.
Harnessing UBR5 for targeted protein degradation of key transcriptional regulators.
A CRISPR activation screen identifies FBXO22 as an E3 ligase supporting targeted protein degradation.
Signal sequence-triage is activated by translocon obstruction sensed by an ER stress sensor IRE1α.
Proteome census upon nutrient stress reveals Golgiphagy membrane receptors.
Znf598-mediated Rps10/eS10 ubiquitination contributes to the ribosome ubiquitination dynamics during zebrafish development.
p97/VCP promotes the recycling of endocytic cargo.
Early fate decision for mitochondrially encoded proteins by a molecular triage.
Proteotoxic stresses stimulate dissociation of UBL4A from the tail-anchored protein recognition complex.
Chemical Specification of E3 Ubiquitin Ligase Engagement by Cysteine-Reactive Chemistry.
ULK1 forms distinct oligomeric states and nanoscopic structures during autophagy initiation.
Discovery of Nedd4 auto-ubiquitination inhibitors.
Current and future directions of USP7 interactome in cancer study.
Autophagy Receptor-Inspired Antibody-Fusion Proteins for Targeted Intracellular Degradation.
The promise of targeted protein degradation approaches.
Induction of proteasomal activity in mammalian cells by lifespan-extending tRNA synthetase inhibitors.
Protein interaction network analysis of mTOR signaling reveals modular organization.
DBT is a metabolic switch for maintenance of proteostasis under proteasomal impairment.
Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer.
AKIR-1 regulates proteasome subcellular function in Caenorhabditis elegans.
Structure and activation of the RING E3 ubiquitin ligase TRIM72 on the membrane.
ISGylation-independent protection of cell growth by USP18 following interferon stimulation.
Light Activated BioID (LAB): an optically activated proximity labeling system to study protein-protein interactions.
Exploring the pan-cancer landscape of posttranscriptional regulation.
Mitochondrial proteome research: the road ahead.
Coordination between aminoacylation and editing to protect against proteotoxicity.
Protein-Folding Chaperones Predict Structure-Function Relationships and Cancer Risk in BRCA1 Mutation Carriers.
XBP1 promotes NRASG12D pre-B acute lymphoblastic leukaemia through IL-7 receptor signalling and provides a therapeutic vulnerability for oncogenic RAS.
The colocatome as a spatial -omic reveals shared microenvironment features between tumour-stroma assembloids and human lung cancer.
Protection of eIF2B from inhibitory phosphorylated eIF2: a viral strategy to maintain mRNA translation during the PKR-triggered integrated stress response.
Mechanically sensitive HSF1 is a key regulator of left-right symmetry breaking in zebrafish embryos.
Structure-based discovery of CFTR potentiators and inhibitors.

bioRxiv. 2023 Sep 16. pii: 2023.09.15.556420. [Epub ahead of print]

Small molecule correctors divert CFTR-F508del from ERAD by stabilizing sequential folding states.

Celeste Riepe, Magda Wąchalska, Kirandeep K Deol, Anais K Amaya, Matthew H Porteus, James A Olzmann, Ron R Kopito.

Over 80% of people with cystic fibrosis (CF) carry the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel at the apical plasma membrane (PM) of epithelial cells. F508del impairs CFTR folding causing it to be destroyed by endoplasmic reticulum associated degradation (ERAD). Small molecule correctors, which act as pharmacological chaperones to divert CFTR-F508del from ERAD, are the primary strategy for treating CF, yet corrector development continues with only a rudimentary understanding of how ERAD targets CFTR-F508del. We conducted genome-wide CRISPR/Cas9 knockout screens to systematically identify the molecular machinery that underlies CFTR-F508del ERAD. Although the ER-resident ubiquitin ligase, RNF5 was the top E3 hit, knocking out RNF5 only modestly reduced CFTR-F508del degradation. Sublibrary screens in an RNF5 knockout background identified RNF185 as a redundant ligase, demonstrating that CFTR-F508del ERAD is highly buffered. Gene-drug interaction experiments demonstrated that correctors tezacaftor (VX-661) and elexacaftor (VX-445) stabilize sequential, RNF5-resistant folding states. We propose that binding of correctors to nascent CFTR-F508del alters its folding landscape by stabilizing folding states that are not substrates for RNF5-mediated ubiquitylation.
SIGNIFICANCE STATEMENT: Clinically effective small molecule cystic fibrosis (CF) correctors divert mutant CFTR molecules from ER-associated degradation (ERAD). However, the mechanisms underlying CFTR ERAD are not well-understood.The authors used CRISPR knockout screens to identify ERAD machinery targeting CFTR-F508del and found that the pathway is highly buffered, with RNF185 serving as a redundant ubiquitin ligase for RNF5. Gene-drug interaction experiments demonstrated that correctors act synergistically by stabilizing sequential RNF5-resistant folding states.Inhibiting proteostasis machinery is a complementary approach for enhancing current CF corrector therapies.

DOI: https://doi.org/10.1101/2023.09.15.556420
Trends Pharmacol Sci. 2023 Sep 26. pii: S0165-6147(23)00199-2. [Epub ahead of print]

Harnessing UBR5 for targeted protein degradation of key transcriptional regulators.

Asad M Taherbhoy, Danette L Daniels.

  Targeted protein degradation (TPD) has opened the door for drugging transcriptional regulators, yet the number of proteins targeted and E3 ligases utilized remain limited. Here, we highlight UBR5 and propose multiple strategies by which this E3 ligase could be modulated to drive degradation of key transcriptional targets implicated in disease.

Keywords:  HECT E3 ligase; PROTACs; molecular glue; nuclear receptors; transcription factors; ubiquitination

DOI:  https://doi.org/10.1016/j.tips.2023.09.001
bioRxiv. 2023 Sep 16. pii: 2023.09.15.557708. [Epub ahead of print]

A CRISPR activation screen identifies FBXO22 as an E3 ligase supporting targeted protein degradation.

Ananya A Basu, Chenlu Zhang, Isabella A Riha, Assa Magassa, Felicia Ko, Xiaoyu Zhang.

Targeted protein degradation (TPD) represents a potent chemical biology paradigm that leverages the cellular degradation machinery to pharmacologically eliminate specific proteins of interest. Although multiple E3 ligases have been discovered to facilitate TPD, there exists a compelling requirement to diversify the pool of E3 ligases available for such applications. This expansion will broaden the scope of potential protein targets, accommodating those with varying subcellular localizations and expression patterns. In this study, we describe a CRISPR-based transcriptional activation screen focused on human E3 ligases, with the goal of identifying E3 ligases that can facilitate heterobifunctional compound-mediated target degradation. This approach allows us to address the limitations associated with investigating candidate degrader molecules in specific cell lines that either lack or have low levels of the desired E3 ligases. Through this approach, we identified a candidate proteolysis-targeting chimera (PROTAC), 22-SLF, that induces the degradation of FKBP12 when the FBXO22 gene transcription is activated. 22-SLF induced the degradation of endogenous FKBP12 in a FBXO22-dependent manner across multiple cancer cell lines. Subsequent mechanistic investigations revealed that 22-SLF interacts with C227 and/or C228 in FBXO22 to achieve the target degradation. Finally, we demonstrated the versatility of FBXO22-based PROTACs by effectively degrading another endogenous protein BRD4. This study uncovers FBXO22 as an E3 ligase capable of supporting ligand-induced protein degradation through electrophilic PROTACs. The platform we have developed can readily be applied to elucidate protein degradation pathways by identifying E3 ligases that facilitate either small molecule-induced or endogenous protein degradation.

DOI: https://doi.org/10.1101/2023.09.15.557708
Cell Struct Funct. 2023 Sep 28.

Signal sequence-triage is activated by translocon obstruction sensed by an ER stress sensor IRE1α.

Ashuei Sogawa, Ryota Komori, Kota Yanagitani, Miku Ohfurudono, Akio Tsuru, Koji Kadoi, Yukio Kimata, Hiderou Yoshida, Kenji Kohno.

  Secretory pathway proteins are cotranslationally translocated into the endoplasmic reticulum (ER) of metazoan cells through the protein channel, translocon. Given that there are far fewer translocons than ribosomes in a cell, it is essential that secretory protein-translating ribosomes only occupy translocons transiently. Therefore, if translocons are obstructed by ribosomes stalled or slowed in translational elongation, it possibly results in deleterious consequences to cellular function. Hence, we investigated how translocon clogging by stalled ribosomes affects mammalian cells. First, we constructed ER-destined translational arrest proteins (ER-TAP) as an artificial protein that clogged the translocon in the ER membrane. Here, we show that the translocon clogging by ER-TAP expression activates triage of signal sequences (SS) in which secretory pathway proteins harboring highly efficient SS are preferentially translocated into the ER lumen. Interestingly, the translocon obstructed status specifically activates inositol requiring enzyme 1α (IRE1α) but not protein kinase R-like ER kinase (PERK). Given that the IRE1α-XBP1 pathway mainly induces the translocon components, our discovery implies that lowered availability of translocon activates IRE1α, which induces translocon itself. This results in rebalance between protein influx into the ER and the cellular translocation capacity.Keywords: endoplasmic reticulum, translocation capacity, translocon clogging, IRE1, signal sequence.

Keywords:  IRE1; endoplasmic reticulum; signal sequence; translocation capacity; translocon clogging

DOI:  https://doi.org/10.1247/csf.23072
Nature. 2023 Sep 27.

Proteome census upon nutrient stress reveals Golgiphagy membrane receptors.

Kelsey L Hickey, Sharan Swarup, Ian R Smith, Julia C Paoli, Enya Miguel Whelan, Joao A Paulo, J Wade Harper.

During nutrient stress, macroautophagy degrades cellular macromolecules, thereby providing biosynthetic building blocks while simultaneously remodeling the proteome1,2. While machinery responsible for initiation of macroautophagy is well characterized3,4, our understanding of the extent to which individual proteins, protein complexes and organelles are selected for autophagic degradation, and the underlying targeting mechanisms is limited. Here, we use orthogonal proteomic strategies to provide a spatial proteome census of autophagic cargo during nutrient stress in mammalian cells. We find that macroautophagy has selectivity for recycling membrane-bound organelles (principally Golgi and ER). Through autophagic cargo prioritization, we identify a complex of membrane-embedded proteins, YIPF3 and YIPF4, as receptors for Golgiphagy. During nutrient stress, YIPF3 and YIPF4 interact with ATG8s via LIR motifs and are mobilized into autophagosomes that traffic to lysosomes in a process that requires the canonical autophagic machinery. Cells lacking YIPF3 or YIPF4 are selectively defective in elimination of a specific cohort of Golgi membrane proteins during nutrient stress. Moreover, YIPF3/4 play an analogous role in Golgi remodeling during programmed conversion of stem cells to the neuronal lineage in vitro. Collectively, this study reveals prioritization of membrane protein cargo during nutrient stress-dependent proteome remodeling and identifies an unanticipated Golgi remodeling pathway requiring membrane-embedded receptors.

DOI: https://doi.org/10.1038/s41586-023-06657-6
RNA. 2023 Sep 26. pii: rna.079633.123. [Epub ahead of print]

Znf598-mediated Rps10/eS10 ubiquitination contributes to the ribosome ubiquitination dynamics during zebrafish development.

Nozomi Ugajin, Koshi Imami, Hiraku Takada, Yasushi Ishihama, Shinobu Chiba, Yuichiro Mishima.

  Ribosome is a translational apparatus that comprises about 80 ribosomal proteins and four rRNAs. Recent studies reported that ribosome ubiquitination is crucial for translational regulation and ribosome-associated quality control (RQC). However, little is known about the dynamics of ribosome ubiquitination under complex biological processes of multicellular organisms. To explore ribosome ubiquitination during animal development, we generated a zebrafish strain that expresses a FLAG-tagged ribosomal protein Rpl36/eL36 from its endogenous locus. We examined ribosome ubiquitination during zebrafish development by combining affinity purification of ribosomes from rpl36-FLAG zebrafish embryos with immunoblotting analysis. Our findings showed that ubiquitination of ribosomal proteins dynamically changed as development proceeded. We also showed that during zebrafish development, the ribosome was ubiquitinated by Znf598, an E3 ubiquitin ligase that activates RQC. Ribosomal protein Rps10/eS10 was found to be a key ubiquitinated protein during development. Furthermore, we showed that Rps10/eS10 ubiquitination-site mutations reduced the overall ubiquitination pattern of ribosome. These results demonstrate the complexity and dynamics of ribosome ubiquitination during zebrafish development.

Keywords:  development; ribosome; ubiquitination; zebrafish

DOI:  https://doi.org/10.1261/rna.079633.123
Mol Biol Cell. 2023 Sep 27. mbcE23060237

p97/VCP promotes the recycling of endocytic cargo.

Mona Kawan, Maria Körner, Andreas Schlosser, Alexander Buchberger.

The endocytic pathway is of central importance for eukaryotic cells, as it enables uptake of extracellular materials, membrane protein quality control and recycling, as well as modulation of receptor signaling. While the ATPase p97 (VCP, Cdc48) has been found to be involved in the fusion of early endosomes and endolysosomal degradation, its role in endocytic trafficking is still incompletely characterized. Here, we identify myoferlin (MYOF), a ferlin family member with functions in membrane trafficking and repair, as a hitherto unknown p97 interactor. The interaction of MYOF with p97 depends on the cofactor PLAA previously linked to endosomal sorting. Besides PLAA, shared interactors of p97 and MYOF comprise several proteins involved in endosomal recycling pathways, including Rab11, Rab14 and the transferrin receptor CD71. Accordingly, a fraction of p97 and PLAA localizes to MYOF-, Rab11-, and Rab14-positive endosomal compartments. Pharmacological inhibition of p97 delays transferrin recycling, indicating that p97 promotes not only the lysosomal degradation, but also the recycling of endocytic cargo.

DOI: https://doi.org/10.1091/mbc.E23-06-0237
Mol Cell. 2023 Sep 21. pii: S1097-2765(23)00696-2. [Epub ahead of print]

Early fate decision for mitochondrially encoded proteins by a molecular triage.

Andreas Kohler, Andreas Carlström, Hendrik Nolte, Verena Kohler, Sung-Jun Jung, Sagar Sridhara, Takashi Tatsuta, Jens Berndtsson, Thomas Langer, Martin Ott.

  Folding of newly synthesized proteins poses challenges for a functional proteome. Dedicated protein quality control (PQC) systems either promote the folding of nascent polypeptides at ribosomes or, if this fails, ensure their degradation. Although well studied for cytosolic protein biogenesis, it is not understood how these processes work for mitochondrially encoded proteins, key subunits of the oxidative phosphorylation (OXPHOS) system. Here, we identify dedicated hubs in proximity to mitoribosomal tunnel exits coordinating mitochondrial protein biogenesis and quality control. Conserved prohibitin (PHB)/m-AAA protease supercomplexes and the availability of assembly chaperones determine the fate of newly synthesized proteins by molecular triaging. The localization of these competing activities in the vicinity of the mitoribosomal tunnel exit allows for a prompt decision on whether newly synthesized proteins are fed into OXPHOS assembly or are degraded.

Keywords:  assembly factors; complex assembly; m-AAA protease; mitochondria; mitoribosome; prohibitin; protein biogenesis; protein quality control; respiratory chain; translation

DOI:  https://doi.org/10.1016/j.molcel.2023.09.001
Biochem J. 2023 Sep 25. pii: BCJ20230267. [Epub ahead of print]

Proteotoxic stresses stimulate dissociation of UBL4A from the tail-anchored protein recognition complex.

Hiroyuki Kawahara, Takumi Hagiwara, Ryosuke Minami, Chizuru Ushio, Naoto Yokota.

  Inclusion body formation is associated with cytotoxicity in a number of neurodegenerative diseases. However, the molecular basis of the toxicity caused by the accumulation of aggregation-prone proteins remains controversial. In this study, we found that disease-associated inclusions induced by elongated polyglutamine chains disrupt the complex formation of BAG6 with UBL4A, a mammalian homologue of yeast Get5. UBL4A also dissociated from BAG6 in response to proteotoxic stresses such as proteasomal inhibition and mitochondrial depolarization. These findings imply that the cytotoxicity of pathological protein aggregates might be attributed in part to disruption of the BAG6-UBL4A complex that is required for the biogenesis of tail-anchored proteins.

Keywords:  BAG6; Polyglutamine disease; Proteasome; Protein quality control; Tail-anchored protein; UBL4A

DOI:  https://doi.org/10.1042/BCJ20230267
J Am Chem Soc. 2023 Sep 28.

Chemical Specification of E3 Ubiquitin Ligase Engagement by Cysteine-Reactive Chemistry.

Roman C Sarott, Inchul You, Yen-Der Li, Sean T Toenjes, Katherine A Donovan, Pooreum Seo, Martha Ordonez, Woong Sub Byun, Muhammad Murtaza Hassan, Franziska Wachter, Edward T Chouchani, Mikołaj Słabicki, Eric S Fischer, Benjamin L Ebert, Stephen M Hinshaw, Nathanael S Gray.

Targeted protein degradation relies on small molecules that induce new protein-protein interactions between targets and the cellular protein degradation machinery. Most of these small molecules feature specific ligands for ubiquitin ligases. Recently, the attachment of cysteine-reactive chemical groups to pre-existing small molecule inhibitors has been shown to drive specific target degradation. We demonstrate here that different cysteine-reactive groups can specify target degradation via distinct ubiquitin ligases. By focusing on the bromodomain ligand JQ1, we identify cysteine-reactive functional groups that drive BRD4 degradation by either DCAF16 or DCAF11. Unlike proteolysis-targeting chimeric molecules (PROTACs), the new compounds use a single small molecule ligand with a well-positioned cysteine-reactive group to induce protein degradation. The finding that nearly identical compounds can engage multiple ubiquitination pathways suggests that targeting cellular pathways that search for and eliminate chemically reactive proteins is a feasible avenue for converting existing small molecule drugs into protein degrader molecules.

DOI: https://doi.org/10.1021/jacs.3c06622
Sci Adv. 2023 Sep 29. 9(39): eadh4094

ULK1 forms distinct oligomeric states and nanoscopic structures during autophagy initiation.

Chiranjib Banerjee, Dushyant Mehra, Daihyun Song, Angel Mancebo, Ji-Man Park, Do-Hyung Kim, Elias M Puchner.

Autophagy induction involves extensive molecular and membrane reorganization. Despite substantial progress, the mechanism underlying autophagy initiation remains poorly understood. Here, we used quantitative photoactivated localization microscopy with single-molecule sensitivity to analyze the nanoscopic distribution of endogenous ULK1, the kinase that triggers autophagy. Under amino acid starvation, ULK1 formed large clusters containing up to 161 molecules at the endoplasmic reticulum. Cross-correlation analysis revealed that ULK1 clusters engaging in autophagosome formation require 30 or more molecules. The ULK1 structures with more than the threshold number contained varying levels of Atg13, Atg14, Atg16, LC3B, GEC1, and WIPI2. We found that ULK1 activity is dispensable for the initial clustering of ULK1, but necessary for the subsequent expansion of the clusters, which involves interaction with Atg14, Atg16, and LC3B and relies on Vps34 activity. This quantitative analysis at the single-molecule level has provided unprecedented insights into the behavior of ULK1 during autophagy initiation.

DOI: https://doi.org/10.1126/sciadv.adh4094
Sci Rep. 2023 09 25. 13(1): 16057

Discovery of Nedd4 auto-ubiquitination inhibitors.

Darren Yong, Stuart R Green, Pegah Ghiabi, Vijayaratnam Santhakumar, Masoud Vedadi.

E3 ubiquitin ligases are critical to the protein degradation pathway by catalyzing the final step in protein ubiquitination by mediating ubiquitin transfer from E2 enzymes to target proteins. Nedd4 is a HECT domain-containing E3 ubiquitin ligase with a wide range of protein targets, the dysregulation of which has been implicated in myriad pathologies, including cancer and Parkinson's disease. Towards the discovery of compounds disrupting the auto-ubiquitination activity of Nedd4, we developed and optimized a TR-FRET assay for high-throughput screening. Through selective screening of a library of potentially covalent compounds, compounds 25 and 81 demonstrated apparent IC50 values of 52 µM and 31 µM, respectively. Tandem mass spectrometry (MS/MS) analysis confirmed that 25 and 81 were covalently bound to Nedd4 cysteine residues (Cys182 and Cys867). In addition, 81 also adducted to Cys627. Auto-ubiquitination assays of Nedd4 mutants featuring alanine substitutions for each of these cysteines suggested that the mode of inhibition of these compounds occurs through blocking the catalytic Cys867. The discovery of these inhibitors could enable the development of therapeutics for various diseases caused by Nedd4 E3 ligase dysregulation.

DOI: https://doi.org/10.1038/s41598-023-42997-z
Biochim Biophys Acta Rev Cancer. 2023 Sep 27. pii: S0304-419X(23)00141-5. [Epub ahead of print] 188992

Current and future directions of USP7 interactome in cancer study.

Hong-Beom Park, Kwang-Hyun Baek.

  The ubiquitin-proteasome system (UPS) is an essential protein quality controller for regulating protein homeostasis and autophagy. Ubiquitination is a protein modification process that involves the binding of one or more ubiquitins to substrates through a series of enzymatic processes. These include ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). Conversely, deubiquitination is a reverse process that removes ubiquitin from substrates via deubiquitinating enzymes (DUBs). Dysregulation of ubiquitination-related enzymes can lead to various human diseases, including cancer, through the modulation of protein ubiquitination. The most structurally and functionally studied DUB is the ubiquitin-specific protease 7 (USP7). Both the TRAF and UBL domains of USP7 are known to bind to the [P/A/E]-X-X-S or K-X-X-X-K motif of substrates. USP7 has been shown to be involved in cancer pathogenesis by binding with numerous substrates. Recently, a novel substrate of USP7 was discovered through a systemic analysis of its binding motif. This review summarizes the currently discovered substrates and cellular functions of USP7 in cancer and suggests putative substrates of USP7 through a comprehensive systemic analysis.

Keywords:  Binding motif; Bioinformatics; Deubiquitination; Substrates; Ubiquitin-specific protease 7

DOI:  https://doi.org/10.1016/j.bbcan.2023.188992
J Am Chem Soc. 2023 Sep 25.

Autophagy Receptor-Inspired Antibody-Fusion Proteins for Targeted Intracellular Degradation.

Ziwen Jiang, Yu-Hsuan Kuo, Michelle R Arkin.

Autophagy is responsible for the degradation of large intracellular contents, such as unwanted protein aggregates and organelles. Impaired autophagy can therefore lead to the accumulation of pathological aggregates, correlating with aging and neurodegenerative diseases. However, a broadly applicable methodology is not available for the targeted degradation of protein aggregates or organelles in mammalian cells. Herein, we developed a series of autophagy receptor-inspired targeting chimeras (AceTACs) that can induce the targeted degradation of aggregation-prone proteins and protein aggregates (e.g., huntingtin, TDP-43, and FUS mutants), as well as organelles (e.g., mitochondria, peroxisomes, and endoplasmic reticulum). These antibody-fusion-based AceTAC degraders were designed to mimic the function of autophagy receptors, simultaneously binding with the cellular targets and the LC3 proteins on the autophagosomal membrane, eventually transporting the target to the autophagy-lysosomal process for degradation. The AceTAC degradation system provides design principles for antibody-based degradation through autophagy, largely expanding the scope of intracellular targeted degradation technologies.

DOI: https://doi.org/10.1021/jacs.3c05199
Trends Pharmacol Sci. 2023 Sep 27. pii: S0165-6147(23)00185-2. [Epub ahead of print]

The promise of targeted protein degradation approaches.

Danette Daniels, Gopal Sapkota, Lyn Jones.

DOI: https://doi.org/10.1016/j.tips.2023.08.015
Geroscience. 2023 Sep 25.

Induction of proteasomal activity in mammalian cells by lifespan-extending tRNA synthetase inhibitors.

Blaise L Mariner, Antonio S Rodriguez, Olivia C Heath, Mark A McCormick.

  We have recently shown that multiple tRNA synthetase inhibitors can greatly increase lifespan in multiple models by acting through the conserved transcription factor ATF4. Here, we show that these compounds, and several others of the same class, can greatly upregulate mammalian ATF4 in cells in vitro, in a dose dependent manner. Further, RNASeq analysis of these cells pointed toward changes in protein turnover. In subsequent experiments here we show that multiple tRNA synthetase inhibitors can greatly upregulate activity of the ubiquitin proteasome system (UPS) in cells in an ATF4-dependent manner. The UPS plays an important role in the turnover of many damaged or dysfunctional proteins in an organism. Increasing UPS activity has been shown to enhance the survival of Huntington's disease cell models, but there are few known pharmacological enhancers of the UPS. Additionally, we see separate ATF4 dependent upregulation of macroautophagy upon treatment with tRNA synthetase inhibitors. Protein degradation is an essential cellular process linked to many important human diseases of aging such as Alzheimer's disease and Huntington's disease. These drugs' ability to enhance proteostasis more broadly could have wide-ranging implications in the treatment of important age-related neurodegenerative diseases.

Keywords:  ATF-4; ATF4; Gcn4; Ubiquitin proteasome system; tRNA synthetase

DOI:  https://doi.org/10.1007/s11357-023-00938-8
J Biol Chem. 2023 Sep 21. pii: S0021-9258(23)02299-8. [Epub ahead of print] 105271

Protein interaction network analysis of mTOR signaling reveals modular organization.

Devin T Wehle, Carter S Bass, Josef Sulc, Ghayda Mirzaa, Stephen E P Smith.

  The mammalian target of rapamycin (mTOR) is a serine-threonine kinase that acts as a central mediator of translation, and plays important roles in cell growth, synaptic plasticity, cancer, and a wide range of developmental disorders. The signaling cascade linking lipid kinases (PI3Ks), protein kinases (AKT) and translation initiation complexes (EIFs) to mTOR has been extensively modeled, but does not fully describe mTOR system behavior. Here, we use quantitative multiplex co-immunoprecipitation to monitor a protein interaction network (PIN) composed of 300+ binary interactions among mTOR-related proteins. Using a simple model system of serum-deprived or fresh-media-fed mouse 3T3 fibroblasts, we observed extensive PIN remodeling involving 27+ individual protein interactions after one hour, despite phosphorylation changes observed after only five minutes. Using small molecule inhibitors of PI3K, AKT, mTOR, MEK and ERK, we define subsets of the PIN, termed 'modules', that respond differently to each inhibitor. Using primary fibroblasts from individuals with overgrowth disorders caused by pathogenic PIK3CA or MTOR variants, we find that hyperactivation of mTOR pathway components is reflected in a hyperactive PIN. Our data define a "modular" organization of the mTOR PIN in which coordinated groups of interactions respond to activation or inhibition of distinct nodes, and demonstrate that kinase inhibitors affect the modular network architecture in a complex manner, inconsistent with simple linear models of signal transduction.

Keywords:  Protein-protein interaction; mTOR; signal transduction

DOI:  https://doi.org/10.1016/j.jbc.2023.105271
bioRxiv. 2023 Sep 13. pii: 2023.09.12.556394. [Epub ahead of print]

DBT is a metabolic switch for maintenance of proteostasis under proteasomal impairment.

Ran-Der Hwang, Yu-Ning Lu, Qing Tang, Goran Periz, Giho Park, Xiangning Li, Yang Liu, Tao Zhang, Jiou Wang.

Proteotoxic stress impairs cellular homeostasis and underlies the pathogeneses of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). The proteasomal and autophagic degradation of proteins are two major pathways for protein quality control in the cell. Here, we report a genome-wide CRISPR screen uncovering a major regulator of cytotoxicity resulting from the inhibition of the proteasome. Dihydrolipoamide branched chain transacylase E2 (DBT) was found to be a robust suppressor, loss of which protects against proteasome inhibition-associated cell death through promoting clearance of ubiquitinated proteins. Loss of DBT altered the metabolic and energetic status of the cell and resulted in activation of autophagy in an AMP-activated protein kinase (AMPK)-dependent mechanism in the presence of the proteasomal inhibition. Loss of DBT protected against proteotoxicity induced by ALS-linked mutant TDP-43 in Drosophila and mammalian neurons. DBT is upregulated in tissues from ALS patients. These results demonstrate that DBT is a master switch in the metabolic control of protein quality control with implications in neurodegenerative diseases.

DOI: https://doi.org/10.1101/2023.09.12.556394
Autophagy. 2023 Sep 29. 1-20

Direct regulation of FNIP1 and FNIP2 by MEF2 sustains MTORC1 activation and tumor progression in pancreatic cancer.

Li Xia, Tiejian Nie, Fangfang Lu, Lu Huang, Xiaolong Shi, Dongni Ren, Jianjun Lu, Xiaobin Li, Tuo Xu, Bozhou Cui, Qing Wang, Guodong Gao, Qian Yang.

  MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) orchestrates diverse environmental signals to facilitate cell growth and is frequently activated in cancer. Translocation of MTORC1 from the cytosol to the lysosomal surface by the RRAG GTPases is the key step in MTORC1 activation. Here, we demonstrated that transcription factors MEF2A and MEF2D synergistically regulated MTORC1 activation via modulating its cyto-lysosome shutting. Mechanically, MEF2A and MEF2D controlled the transcription of FNIP1 and FNIP2, the components of the FLCN-FNIP1 or FNIP2 complex that acts as a RRAGC-RRAGD GTPase-activating element to promote the recruitment of MTORC1 to lysosome and its activation. Furthermore, we determined that the pro-oncogenic protein kinase SRC/c-Src directly phosphorylated MEF2D at three conserved tyrosine residues. The tyrosine phosphorylation enhanced MEF2D transcriptional activity and was indispensable for MTORC1 activation. Finally, both the protein and tyrosine phosphorylation levels of MEF2D are elevated in human pancreatic cancers, positively correlating with MTORC1 activity. Depletion of both MEF2A and MEF2D or expressing the unphosphorylatable MEF2D mutant suppressed tumor cell growth. Thus, our study revealed a transcriptional regulatory mechanism of MTORC1 that promoted cell anabolism and proliferation and uncovered its critical role in pancreatic cancer progression.Abbreviation: ACTB: actin beta; ChIP: chromatin immunoprecipitation; EGF: epidermal growth factor; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FLCN: folliculin; FNIP1: folliculin interacting protein 1; FNIP2: folliculin interacting protein 2; GAP: GTPase activator protein; GEF: guanine nucleotide exchange factors; GTPase: guanosine triphosphatase; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MEF2: myocyte enhancer factor 2; MEF2A: myocyte enhancer factor 2A; MEF2D: myocyte enhancer factor 2D; MEF2D-3YF: Y131F, Y333F, Y337F mutant; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NR4A1: nuclear receptor subfamily 4 group A member 1; RPTOR: regulatory associated protein of MTOR complex 1; RHEB: Ras homolog, mTORC1 binding; RPS6KB1: ribosomal protein S6 kinase B1; RRAG: Ras related GTP binding; RT-qPCR: real time-quantitative PCR; SRC: SRC proto-oncogene, non-receptor tyrosine kinase; TMEM192: transmembrane protein 192; WT: wild-type.

Keywords:  Autophagy; cell metabolism; oncogenesis; protein kinase; transcription factor

DOI:  https://doi.org/10.1080/15548627.2023.2259735
iScience. 2023 Oct 20. 26(10): 107886

AKIR-1 regulates proteasome subcellular function in Caenorhabditis elegans.

Johanna Pispa, Elisa Mikkonen, Leena Arpalahti, Congyu Jin, Carmen Martínez-Fernández, Julián Cerón, Carina I Holmberg.

  Polyubiquitinated proteins are primarily degraded by the ubiquitin-proteasome system (UPS). Proteasomes are present both in the cytoplasm and nucleus. Here, we investigated mechanisms coordinating proteasome subcellular localization and activity in a multicellular organism. We identified the nuclear protein-encoding gene akir-1 as a proteasome regulator in a genome-wide Caenorhabditis elegans RNAi screen. We demonstrate that depletion of akir-1 causes nuclear accumulation of endogenous polyubiquitinated proteins in intestinal cells, concomitant with slower in vivo proteasomal degradation in this subcellular compartment. Remarkably, akir-1 is essential for nuclear localization of proteasomes both in oocytes and intestinal cells but affects differentially the subcellular distribution of polyubiquitinated proteins. We further reveal that importin ima-3 genetically interacts with akir-1 and influences nuclear localization of a polyubiquitin-binding reporter. Our study shows that the conserved AKIR-1 is an important regulator of the subcellular function of proteasomes in a multicellular organism, suggesting a role for AKIR-1 in proteostasis maintenance.

Keywords:  Biochemistry; Cell biology; Genomics

DOI:  https://doi.org/10.1016/j.isci.2023.107886
Nat Struct Mol Biol. 2023 Sep 28.

Structure and activation of the RING E3 ubiquitin ligase TRIM72 on the membrane.

Si Hoon Park, Juhyun Han, Byung-Cheon Jeong, Ju Han Song, Se Hwan Jang, Hyeongseop Jeong, Bong Heon Kim, Young-Gyu Ko, Zee-Yong Park, Kyung Eun Lee, Jaekyung Hyun, Hyun Kyu Song.

Defects in plasma membrane repair can lead to muscle and heart diseases in humans. Tripartite motif-containing protein (TRIM)72 (mitsugumin 53; MG53) has been determined to rapidly nucleate vesicles at the site of membrane damage, but the underlying molecular mechanisms remain poorly understood. Here we present the structure of Mus musculus TRIM72, a complete model of a TRIM E3 ubiquitin ligase. We demonstrated that the interaction between TRIM72 and phosphatidylserine-enriched membranes is necessary for its oligomeric assembly and ubiquitination activity. Using cryogenic electron tomography and subtomogram averaging, we elucidated a higher-order model of TRIM72 assembly on the phospholipid bilayer. Combining structural and biochemical techniques, we developed a working molecular model of TRIM72, providing insights into the regulation of RING-type E3 ligases through the cooperation of multiple domains in higher-order assemblies. Our findings establish a fundamental basis for the study of TRIM E3 ligases and have therapeutic implications for diseases associated with membrane repair.

DOI: https://doi.org/10.1038/s41594-023-01111-7
Biochem J. 2023 Sep 26. pii: BCJ20230301. [Epub ahead of print]

ISGylation-independent protection of cell growth by USP18 following interferon stimulation.

Anne Clancy, Emma V Rusilowicz-Jones, Iona Wallace, Kirby Swatek, Sylvie Urbe, Michael J Clague.

  Type 1 interferon stimulation highly up-regulates all elements of a ubiquitin-like conjugation system that leads to ISGylation of target proteins. An ISG15-specific member of the deubiquitylase family, USP18, is up-regulated in a co-ordinated manner. USP18 can also provide a negative feedback by inhibiting JAK-STAT signaling through protein interactions independently of DUB activity. Here, we provide an acute example of this phenomenon, whereby the early expression of USP18, post-interferon treatment of HCT116 colon cancer cells is sufficient to fully suppress the expression of the ISG15 E1 enzyme, UBA7. Stimulation of lung adenocarcinoma A549 cells with interferon reduces their growth rate but they remain viable. In contrast, A549 USP18 knock-out cells show similar growth characteristics under basal conditions, but upon interferon stimulation, a profound inhibition of cell growth is observed. We show that this contingency on USP18 is independent of ISGylation, suggesting non-catalytic functions are required for viability. We also demonstrate that global deISGylation kinetics are very slow compared to deubiquitylation. This is not influenced by USP18 expression, suggesting that enhanced ISGylation in USP18 KOcells reflects increased conjugating activity.

Keywords:  ISG15; USP15; deubiquitylase; interferons; ubiquitin

DOI:  https://doi.org/10.1042/BCJ20230301
J Cell Sci. 2023 Sep 27. pii: jcs.261430. [Epub ahead of print]

Light Activated BioID (LAB): an optically activated proximity labeling system to study protein-protein interactions.

Omer Shafraz, Carolyn Marie Orduno Davis, Sanjeevi Sivasankar.

  Proximity labeling with genetically encoded enzymes are widely used to study protein-protein interactions in cells. However, the accuracy of proximity labeling is limited by a lack of control over the enzymatic labeling process. Here, we present a light-activated proximity labeling technology for mapping protein-protein interactions at the cell membrane with high accuracy and precision. Our technology, called Light Activated BioID (LAB), fuses the two halves of the split-TurboID proximity labeling enzyme to the photodimeric proteins CRY2 and CIB1. We demonstrate in multiple cell lines, that upon illumination with blue light, CRY2 and CIB1 dimerize, reconstitute split-TurboID, and initiate biotinylation. Turning off the light dissociates CRY2 and CIB1 and halts biotinylation. We benchmark LAB against the widely used TurboID proximity labeling method by measuring the proteome of E-cadherin, an essential cell-cell adhesion protein. We show that LAB can map E-cadherin binding partners with higher accuracy and significantly fewer false positives compared to TurboID.

Keywords:  E-cadherin; Membrane protein; Optogenetic; Proximity labeling; TurboID

DOI:  https://doi.org/10.1242/jcs.261430
Cell Rep. 2023 Sep 22. pii: S2211-1247(23)01184-1. [Epub ahead of print]42(10): 113172

Exploring the pan-cancer landscape of posttranscriptional regulation.

Umesh Ghoshdastider, Ataman Sendoel.

  Understanding the mechanisms underlying cancer gene expression is critical for precision oncology. Posttranscriptional regulation is a key determinant of protein abundance and cancer cell behavior. However, to what extent posttranscriptional regulatory mechanisms impact protein levels and cancer progression is an ongoing question. Here, we exploit cancer proteogenomics data to systematically compare mRNA-protein correlations across 14 different human cancer types. We identify two clusters of genes with particularly low mRNA-protein correlations across all cancer types, shed light on the role of posttranscriptional regulation of cancer driver genes and drug targets, and unveil a cohort of 55 mutations that alter systems-wide posttranscriptional regulation. Surprisingly, we find that decreased levels of posttranscriptional control in patients correlate with shorter overall survival across multiple cancer types, prompting further mechanistic studies into how posttranscriptional regulation affects patient outcomes. Our findings underscore the importance of a comprehensive understanding of the posttranscriptional regulatory landscape for predicting cancer progression.

Keywords:  CP: Cancer; CP: Genomics; proteogenomics; translational regulation

DOI:  https://doi.org/10.1016/j.celrep.2023.113172
Nat Rev Mol Cell Biol. 2023 Sep 29.

Mitochondrial proteome research: the road ahead.

Zakery N Baker, Patrick Forny, David J Pagliarini.

Mitochondria are multifaceted organelles with key roles in anabolic and catabolic metabolism, bioenergetics, cellular signalling and nutrient sensing, and programmed cell death processes. Their diverse functions are enabled by a sophisticated set of protein components encoded by the nuclear and mitochondrial genomes. The extent and complexity of the mitochondrial proteome remained unclear for decades. This began to change 20 years ago when, driven by the emergence of mass spectrometry-based proteomics, the first draft mitochondrial proteomes were established. In the ensuing decades, further technological and computational advances helped to refine these 'maps', with current estimates of the core mammalian mitochondrial proteome ranging from 1,000 to 1,500 proteins. The creation of these compendia provided a systemic view of an organelle previously studied primarily in a reductionist fashion and has accelerated both basic scientific discovery and the diagnosis and treatment of human disease. Yet numerous challenges remain in understanding mitochondrial biology and translating this knowledge into the medical context. In this Roadmap, we propose a path forward for refining the mitochondrial protein map to enhance its discovery and therapeutic potential. We discuss how emerging technologies can assist the detection of new mitochondrial proteins, reveal their patterns of expression across diverse tissues and cell types, and provide key information on proteoforms. We highlight the power of an enhanced map for systematically defining the functions of its members. Finally, we examine the utility of an expanded, functionally annotated mitochondrial proteome in a translational setting for aiding both diagnosis of mitochondrial disease and targeting of mitochondria for treatment.

DOI: https://doi.org/10.1038/s41580-023-00650-7
Nucleic Acids Res. 2023 Sep 23. pii: gkad778. [Epub ahead of print]

Coordination between aminoacylation and editing to protect against proteotoxicity.

Hong Zhang, Parker Murphy, Jason Yu, Sukyeong Lee, Francis T F Tsai, Ambro van Hoof, Jiqiang Ling.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that ligate amino acids to tRNAs, and often require editing to ensure accurate protein synthesis. Recessive mutations in aaRSs cause various neurological disorders in humans, yet the underlying mechanism remains poorly understood. Pathogenic aaRS mutations frequently cause protein destabilization and aminoacylation deficiency. In this study, we report that combined aminoacylation and editing defects cause severe proteotoxicity. We show that the ths1-C268A mutation in yeast threonyl-tRNA synthetase (ThrRS) abolishes editing and causes heat sensitivity. Surprisingly, experimental evolution of the mutant results in intragenic mutations that restore heat resistance but not editing. ths1-C268A destabilizes ThrRS and decreases overall Thr-tRNAThr synthesis, while the suppressor mutations in the evolved strains improve aminoacylation. We further show that deficiency in either ThrRS aminoacylation or editing is insufficient to cause heat sensitivity, and that ths1-C268A impairs ribosome-associated quality control. Our results suggest that aminoacylation deficiency predisposes cells to proteotoxic stress.

DOI: https://doi.org/10.1093/nar/gkad778
bioRxiv. 2023 Sep 15. pii: 2023.09.14.557795. [Epub ahead of print]

Protein-Folding Chaperones Predict Structure-Function Relationships and Cancer Risk in BRCA1 Mutation Carriers.

Brant Gracia, Patricia Montes, Angelica Maria Gutierrez, Banu Arun, Georgios Ioannis Karras.

Identifying pathogenic mutations and predicting their impact on protein structure, function and phenotype remain major challenges in genome sciences. Protein-folding chaperones participate in structure-function relationships by facilitating the folding of protein variants encoded by mutant genes. Here, we utilize a high-throughput protein-protein interaction assay to test HSP70 and HSP90 chaperone interactions as predictors of pathogenicity for variants in the tumor suppressor BRCA1. Chaperones bind 77% of pathogenic BRCA1-BRCT variants, most of which engaged HSP70 more than HSP90. Remarkably, the magnitude of chaperone binding to variants is proportional to the degree of structural and phenotypic defect induced by BRCA1 mutation. Quantitative chaperone interactions identified BRCA1-BRCT separation-of-function variants and hypomorphic alleles missed by pathogenicity prediction algorithms. Furthermore, increased chaperone binding signified greater cancer risk in human BRCA1 carriers. Altogether, our study showcases the utility of chaperones as quantitative cellular biosensors of variant folding and phenotypic severity.
HIGHLIGHTS: Chaperones detect an abundance of pathogenic folding variants of BRCA1-BRCT.Degree of chaperone binding reflects severity of structural and phenotypic defect.Chaperones identify separation-of-function and hypomorphic variants. Chaperone interactions indicate penetrance and expressivity of BRCA1 alleles.

DOI: https://doi.org/10.1101/2023.09.14.557795
J Cell Mol Med. 2023 Sep 27.

XBP1 promotes NRASG12D pre-B acute lymphoblastic leukaemia through IL-7 receptor signalling and provides a therapeutic vulnerability for oncogenic RAS.

Azam Salimi, Mirle Schemionek-Reinders, Michael Huber, Margherita Vieri, John B Patterson, Julia Alten, Tim H Brümmendorf, Behzad Kharabi Masouleh, Iris Appelmann.

  Activating point mutations of the RAS gene act as driver mutations for a subset of precursor-B cell acute lymphoblastic leukaemias (pre-B ALL) and represent an ambitious target for therapeutic approaches. The X box-binding protein 1 (XBP1), a key regulator of the unfolded protein response (UPR), is critical for pre-B ALL cell survival, and high expression of XBP1 confers poor prognosis in ALL patients. However, the mechanism of XBP1 activation has not yet been elucidated in RAS mutated pre-B ALL. Here, we demonstrate that XBP1 acts as a downstream linchpin of the IL-7 receptor signalling pathway and that pharmacological inhibition or genetic ablation of XBP1 selectively abrogates IL-7 receptor signalling via inhibition of its downstream effectors, JAK1 and STAT5. We show that XBP1 supports malignant cell growth of pre-B NRASG12D ALL cells and that genetic loss of XBP1 consequently leads to cell cycle arrest and apoptosis. Our findings reveal that active XBP1 prevents the cytotoxic effects of a dual PI3K/mTOR pathway inhibitor (BEZ235) in pre-B NRASG12D ALL cells. This implies targeting XBP1 in combination with BEZ235 as a promising new targeted strategy against the oncogenic RAS in NRASG12D -mutated pre-B ALL.

Keywords:  IL-7; NRASG12D; PI3K/mTOR; XBP1; acute lymphoblastic leukaemia

DOI:  https://doi.org/10.1111/jcmm.17904
bioRxiv. 2023 Sep 12. pii: 2023.09.11.557278. [Epub ahead of print]

The colocatome as a spatial -omic reveals shared microenvironment features between tumour-stroma assembloids and human lung cancer.

Gina Bouchard, Weiruo Zhang, Irene Li, Ilayda Ilerten, Asmita Bhattacharya, Yuanyuan Li, Winston Trope, Joseph B Shrager, Calvin Kuo, Lu Tian, Amato J Giaccia, Sylvia K Plevritis.

Computational frameworks to quantify and compare microenvironment spatial features of in-vitro patient-derived models and clinical specimens are needed. Here, we acquired and analysed multiplexed immunofluorescence images of human lung adenocarcinoma (LUAD) alongside tumour- stroma assembloids constructed with organoids and fibroblasts harvested from the leading edge (Tumour-Adjacent Fibroblasts;TAFs) or core (Tumour Core Fibroblasts;TCFs) of human LUAD. We introduce the concept of the "colocatome" as a spatial -omic dimension to catalogue all proximate and distant colocalisations between malignant and fibroblast subpopulations in both the assembloids and clinical specimens. The colocatome expands upon the colocalisation quotient (CLQ) through a nomalisation strategy that involves permutation analysis and thereby allows comparisons of CLQs under different conditions. Using colocatome analysis, we report that both TAFs and TCFs protected cancer cells from targeted oncogene treatment by uniquely reorganising the tumour-stroma cytoarchitecture, rather than by promoting cellular heterogeneity or selection. Moreover, we show that the assembloids' colocatome recapitulates the tumour-stroma cytoarchitecture defining the tumour microenvironment of LUAD clinical samples and thereby can serve as a functional spatial readout to guide translational discoveries.
Abstract Figure:

DOI: https://doi.org/10.1101/2023.09.11.557278
J Biol Chem. 2023 Sep 22. pii: S0021-9258(23)02315-3. [Epub ahead of print] 105287

Protection of eIF2B from inhibitory phosphorylated eIF2: a viral strategy to maintain mRNA translation during the PKR-triggered integrated stress response.

Takuiro Ito, Jennifer Deborah Wuerth, Friedemann Weber.

  The integrated stress response (ISR) protects cells from a variety of insults. Once elicited (e.g. by virus infections), it eventually leads to the block of mRNA translation. Central to the ISR are the interactions between translation initiation factors eIF2 and eIF2B. Under normal conditions, eIF2 drives the initiation of protein synthesis through hydrolysis of GTP, which becomes replenished when binding to the guanine nucleotide exchange factor (GEF) eIF2B. The antiviral branch of the ISR is activated by the RNA-activated kinase PKR which phosphorylates eIF2, thereby converting it into an eIF2B inhibitor. Here, we describe the recently solved structures of eIF2B in complex with eIF2, and a novel escape strategy used by viruses. While unphosphorylated eIF2 interacts with eIF2B in its "productive" conformation, phosphorylated eIF2 [eIF2(αP)] engages a different binding cavity on eIF2B and forces it into the "non-productive" conformation that prohibits GEF activity. It is well established that viruses express so-called PKR antagonists that interfere with double-strand RNA, PKR itself, or eIF2. However recently, three taxonomically unrelated viruses were reported to encode antagonists targeting eIF2B instead. For one antagonist, the S segment non-structural protein (NSs) of Sandfly fever Sicilian virus, atomic structures showed that it occupies the eIF2(αP)-binding cavity on eIF2B without imposing a switch to the non-productive conformation. NSs thus antagonizes the activity of PKR by protecting eIF2B from inhibition by eIF2(αP). As the ISR and specifically eIF2B are central to neuroprotection and a wide range of genetic and age-related diseases, these developments may open new possibilities for treatments.

Keywords:  NSs mechanism; NSs structure; PKR antagonist; Sandfly fever Sicilian phlebovirus; coronavirus AcP10; eIF2; eIF2B non-productive state; eIF2B productive state; integrated stress response; phlebovirus NSs; phosphorylated eIF2; picornavirus AiVL

DOI:  https://doi.org/10.1016/j.jbc.2023.105287
iScience. 2023 Oct 20. 26(10): 107864

Mechanically sensitive HSF1 is a key regulator of left-right symmetry breaking in zebrafish embryos.

Jing Du, Shu-Kai Li, Liu-Yuan Guan, Zheng Guo, Jiang-Fan Yin, Li Gao, Toru Kawanishi, Atsuko Shimada, Qiu-Ping Zhang, Li-Sha Zheng, Yi-Yao Liu, Xi-Qiao Feng, Lin Zhao, Dong-Yan Chen, Hiroyuki Takeda, Yu-Bo Fan.

  The left-right symmetry breaking of vertebrate embryos requires nodal flow. However, the molecular mechanisms that mediate the asymmetric gene expression regulation under nodal flow remain elusive. Here, we report that heat shock factor 1 (HSF1) is asymmetrically activated in the Kupffer's vesicle of zebrafish embryos in the presence of nodal flow. Deficiency in HSF1 expression caused a significant situs inversus and disrupted gene expression asymmetry of nodal signaling proteins in zebrafish embryos. Further studies demonstrated that HSF1 is a mechanosensitive protein. The mechanical sensation ability of HSF1 is conserved in a variety of mechanical stimuli in different cell types. Moreover, cilia and Ca2+-Akt signaling axis are essential for the activation of HSF1 under mechanical stress in vitro and in vivo. Considering the conserved expression of HSF1 in organisms, these findings unveil a fundamental mechanism of gene expression regulation by mechanical clues during embryonic development and other physiological and pathological transformations.

Keywords:  Cell biology; Developmental biology; Embryology; Molecular biology

DOI:  https://doi.org/10.1016/j.isci.2023.107864
bioRxiv. 2023 Sep 12. pii: 2023.09.09.557002. [Epub ahead of print]

Structure-based discovery of CFTR potentiators and inhibitors.

Fangyu Liu, Anat Levit Kaplan, Jesper Levring, Jürgen Einsiedel, Stephanie Tiedt, Katharina Distler, Natalie S Omattage, Ivan S Kondratov, Yurii S Moroz, Harlan L Pietz, John J Irwin, Peter Gmeiner, Brian K Shoichet, Jue Chen.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a crucial ion channel whose loss of function leads to cystic fibrosis, while its hyperactivation leads to secretory diarrhea. Small molecules that improve CFTR folding (correctors) or function (potentiators) are clinically available. However, the only potentiator, ivacaftor, has suboptimal pharmacokinetics and inhibitors have yet to be clinically developed. Here we combine molecular docking, electrophysiology, cryo-EM, and medicinal chemistry to identify novel CFTR modulators. We docked ∼155 million molecules into the potentiator site on CFTR, synthesized 53 test ligands, and used structure-based optimization to identify candidate modulators. This approach uncovered novel mid-nanomolar potentiators as well as inhibitors that bind to the same allosteric site. These molecules represent potential leads for the development of more effective drugs for cystic fibrosis and secretory diarrhea, demonstrating the feasibility of large-scale docking for ion channel drug discovery.

DOI: https://doi.org/10.1101/2023.09.09.557002