bims-proteo 2023-12-17 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023–12–17
35 papers selected by
Eric Chevet, INSERM

The formation of ubiquitin rich condensates triggers recruitment of the ATG9A lipid transfer complex to initiate basal autophagy.
Sequestration of translation initiation factors in p62 condensates.
The ER stress sensor IRE1 interacts with STIM1 to promote store-operated calcium entry, T cell activation, and muscular differentiation.
Bilayer tension-induced clustering of the UPR sensor IRE1.
LTN1 promotes RLR degradation to inhibit immune response to RNA virus through the ESCRT pathway.
Molecular determinants of the crosstalk between endosomal microautophagy and chaperone-mediated autophagy.
Biotin-based strategies to explore the world of ubiquitin and ubiquitin-like modifiers.
Nutrient deprivation alters the rate of COPII subunit recruitment at ER subdomains to tune secretory protein transport.
Mammalian phagophores with finger-like shapes emerge from recycling endosomes.
Mechanistic dissection of premature translation termination induced by acidic residues-enriched nascent peptide.
Role of a holo-insertase complex in the biogenesis of biophysically diverse ER membrane proteins.
Tripartite split-GFP for high throughput screening of small molecules: a powerful strategy for targeting transient/labile interactors like E2-E3 ubiquitination enzymes.
Extracellular targeted protein degradation: an emerging modality for drug discovery.
VCIP135 associates with both the N- and C-terminal regions of p97 ATPase.
The hookup model of the HOPS complex in autophagosome-lysosome fusion.
Characterization of nucleolar SUMO isopeptidases unveils a general p53-independent checkpoint of impaired ribosome biogenesis.
Synonymous codon usage regulates translation initiation.
GRAF1 integrates PINK1-Parkin signaling and actin dynamics to mediate cardiac mitochondrial homeostasis.
Codon optimality modulates protein output by tuning translation initiation.
Atypical K6-ubiquitin chains mobilize p97/VCP and the proteasome to resolve formaldehyde-induced RNA-protein crosslinks.
An integrated workflow for quantitative analysis of the newly synthesized proteome.
Parkinson's VPS35[D620N] mutation induces LRRK2-mediated lysosomal association of RILPL1 and TMEM55B.
UPF1 ATPase autoinhibition and activation modulate RNA binding kinetics and NMD efficiency.
Nuclear cGAS restricts L1 retrotransposition by promoting TRIM41-mediated ORF2p ubiquitination and degradation.
ORFeus: a computational method to detect programmed ribosomal frameshifts and other non-canonical translation events.
Enhanced SREBP2-driven cholesterol biosynthesis by PKCλ/ι deficiency in intestinal epithelial cells promotes aggressive serrated tumorigenesis.
GABA(A) receptor activation drives GABARAP-Nix mediated autophagy to radiation-sensitize primary and brain-metastatic lung adenocarcinoma tumors.
mRNA nuclear export: how mRNA identity features distinguish functional RNAs from junk transcripts.
Cell stretching activates an ATM mechano-transduction pathway that remodels cytoskeleton and chromatin.
Mint3-depletion-induced energy stress sensitizes triple-negative breast cancer to chemotherapy via HSF1 inactivation.
XAF1 promotes colorectal cancer metastasis via VCP-RNF114-JUP axis.
Translation Rates and Protein Folding.
Profiling nuclear cysteine ligandability and effects on nuclear localization using proximity labeling-coupled chemoproteomics.
Wild-type IDH1 maintains NSCLC stemness and chemoresistance through activation of the serine biosynthetic pathway.
Small molecules as modulators of the proteostasis machinery: Implication in cardiovascular diseases.

bioRxiv. 2023 Nov 29. pii: 2023.11.28.569058. [Epub ahead of print]

The formation of ubiquitin rich condensates triggers recruitment of the ATG9A lipid transfer complex to initiate basal autophagy.

D G Broadbent, C M McEwan, T M Tsang, D M Poole, B C Naylor, J C Price, J C Schmidt, J L Andersen.

Autophagy is an essential cellular recycling process that maintains protein and organelle homeostasis. ATG9A vesicle recruitment is a critical early step in autophagy to initiate autophagosome biogenesis. The mechanisms of ATG9A vesicle recruitment are best understood in the context of starvation-induced non-selective autophagy, whereas less is known about the signals driving ATG9A vesicle recruitment to autophagy initiation sites in the absence of nutrient stress. Here we demonstrate that loss of ATG9A or the lipid transfer protein ATG2 leads to the accumulation of phosphorylated p62 aggregates in the context of basal autophagy. Furthermore, we show that p62 degradation requires the lipid scramblase activity of ATG9A. Lastly, we present evidence that poly-ubiquitin is an essential signal that recruits ATG9A and mediates autophagy foci assembly in nutrient replete cells. Together, our data support a ubiquitin-driven model of ATG9A recruitment and autophagosome formation during basal autophagy.

DOI: https://doi.org/10.1101/2023.11.28.569058
Cell Rep. 2023 Dec 12. pii: S2211-1247(23)01595-4. [Epub ahead of print]42(12): 113583

Sequestration of translation initiation factors in p62 condensates.

Alberto Danieli, Georg Vucak, Manuela Baccarini, Sascha Martens.

  Selective autophagy mediates the removal of harmful material from the cytoplasm. This cargo material is selected by cargo receptors, which orchestrate its sequestration within double-membrane autophagosomes and subsequent lysosomal degradation. The cargo receptor p62/SQSTM1 is present in cytoplasmic condensates, and a fraction of them are constantly delivered into lysosomes. However, the molecular composition of the p62 condensates is incompletely understood. To obtain insights into their composition, we develop a method to isolate these condensates and find that p62 condensates are enriched in components of the translation machinery. Furthermore, p62 interacts with translation initiation factors, and eukaryotic initiation factor 2α (eIF2α) and eIF4E are degraded by autophagy in a p62-dependent manner. Thus, p62-mediated autophagy may in part be linked to down-regulation of translation initiation. The p62 condensate isolation protocol developed here may facilitate the study of their contribution to cellular quality control and their roles in health and disease.

Keywords:  CP: Cell biology; CP: Molecular biology; SQSTM1; autophagy; cargo receptor; translation

DOI:  https://doi.org/10.1016/j.celrep.2023.113583
Cell Rep. 2023 Dec 05. pii: S2211-1247(23)01552-8. [Epub ahead of print]42(12): 113540

The ER stress sensor IRE1 interacts with STIM1 to promote store-operated calcium entry, T cell activation, and muscular differentiation.

Amado Carreras-Sureda, Xin Zhang, Loann Laubry, Jessica Brunetti, Stéphane Koenig, Xiaoxia Wang, Cyril Castelbou, Claudio Hetz, Yong Liu, Maud Frieden, Nicolas Demaurex.

  Store-operated Ca2+ entry (SOCE) mediated by stromal interacting molecule (STIM)-gated ORAI channels at endoplasmic reticulum (ER) and plasma membrane (PM) contact sites maintains adequate levels of Ca2+ within the ER lumen during Ca2+ signaling. Disruption of ER Ca2+ homeostasis activates the unfolded protein response (UPR) to restore proteostasis. Here, we report that the UPR transducer inositol-requiring enzyme 1 (IRE1) interacts with STIM1, promotes ER-PM contact sites, and enhances SOCE. IRE1 deficiency reduces T cell activation and human myoblast differentiation. In turn, STIM1 deficiency reduces IRE1 signaling after store depletion. Using a CaMPARI2-based Ca2+ genome-wide screen, we identify CAMKG2 and slc105a as SOCE enhancers during ER stress. Our findings unveil a direct crosstalk between SOCE and UPR via IRE1, acting as key regulator of ER Ca2+ and proteostasis in T cells and muscles. Under ER stress, this IRE1-STIM1 axis boosts SOCE to preserve immune cell functions, a pathway that could be targeted for cancer immunotherapy.

Keywords:  CP: Cell biology; CP: Immunology; CRISPR screening; CaMPARI; ER stress; IRE1; SOCE; STIM1; T cells; calcium; muscle

DOI:  https://doi.org/10.1016/j.celrep.2023.113540
Biochim Biophys Acta Biomembr. 2023 Dec 09. pii: S0005-2736(23)00144-X. [Epub ahead of print] 184262

Bilayer tension-induced clustering of the UPR sensor IRE1.

Md Zobayer Hossain, Wylie Stroberg.

  The endoplasmic reticulum acts as a protein quality control center where a range of chaperones and foldases facilitates protein folding. IRE1 is a sensory transmembrane protein that transduces signals of proteotoxic stress by forming clusters and activating a cellular program called the unfolded protein response (UPR). Recently, membrane thickness variation due to membrane compositional changes have been shown to drive IRE1 cluster formation, activating the UPR even in the absence of proteotoxic stress. Here, we demonstrate a direct relationship between bilayer tension and UPR activation based on IRE1 dimer stability. The stability of the IRE1 dimer in a (50%DOPC-50%POPC) membrane at different applied bilayer tensions was analyzed via molecular dynamics simulations. The potential of mean force for IRE1 dimerization predicts a higher concentration of IRE1 dimers for both tensed and compressed ER membranes. This study shows that IRE1 may be a mechanosensitive membrane protein and establishes a direct biophysical relationship between bilayer tension and UPR activation.

Keywords:  IRE1; Lipid bilayer stress; Mechanosensitivity; Membrane tension; Molecular dynamics; Unfolded protein response

DOI:  https://doi.org/10.1016/j.bbamem.2023.184262
Autophagy. 2023 Dec 07. 1-16

LTN1 promotes RLR degradation to inhibit immune response to RNA virus through the ESCRT pathway.

Fei Qin, Baoshan Cai, Peng Wang, Runyu Cao, Yuling Zhang, Hongling Wen, Yi Zheng, Wei Zhao, Chengjiang Gao, Bingyu Liu.

  The excessive activation of immune responses will trigger autoimmune diseases or inflammatory injury. The endosomal sorting complexes required for transport (ESCRT) system can capture and mediate ubiquitinated protein degradation, which timely terminates signaling pathway hyperactivation. However, whether the ESCRT system participates in regulating RIGI-like receptor (RLR)-mediated antiviral responses remains unknown. In this study, we show that LTN1/listerin, a major component of RQC, can recruit E3 ubiquitin ligase TRIM27 to trigger K63-linked polyubiquitination of RIGI and IFIH1/MDA5. This K63-linked polyubiquitination facilitates the sorting and degradation of RIGI and IFIH1 proteins through the ESCRT-dependent pathway. Concordantly, LTN1 deficiency enhances the innate antiviral response to infection with RNA viruses. Thus, our work uncovers a new mechanism for RIGI and IFIH1 degradation and identifies the role of LTN1 in negatively regulating RLR-mediated antiviral innate immunity, which may provide new targets for the intervention of viral infection.Abbreviation: 5'-pppRNA: 5' triphosphate double stranded RNA; ATG5: autophagy related 5; ATG7: autophagy related 7; BafA1: bafilomycin A1; ESCRT: endosomal sorting complexes required for transport; CHX: cycloheximide; IFIH1/MDA5: interferon induced with helicase C domain 1; IFN: interferon; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; RIGI: RNA sensor RIG-I; RLR: RIGI-like receptors; RQC: ribosome-associated protein quality control; SeV: Sendai virus; TRIM27: tripartite motif-containing 27; VSV: vesicular stomatitis virus; VPS4: vacuolar protein sorting 4.

Keywords:  Antiviral innate immunity; ESCRT; LTN1; RLR; ubiquitination

DOI:  https://doi.org/10.1080/15548627.2023.2291939
Cell Rep. 2023 Dec 05. pii: S2211-1247(23)01541-3. [Epub ahead of print]42(12): 113529

Molecular determinants of the crosstalk between endosomal microautophagy and chaperone-mediated autophagy.

Gregory J Krause, Philipp Kirchner, Barbara Stiller, Kateryna Morozova, Antonio Diaz, Kuei-Ho Chen, Nevan J Krogan, Esperanza Agullo-Pascual, Cristina C Clement, Kristen Lindenau, Danielle L Swaney, Shilpa Dilipkumar, Jose Javier Bravo-Cordero, Laura Santambrogio, Ana Maria Cuervo.

  Chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI) are pathways for selective degradation of cytosolic proteins in lysosomes and late endosomes, respectively. These autophagic processes share as a first step the recognition of the same five-amino-acid motif in substrate proteins by the Hsc70 chaperone, raising the possibility of coordinated activity of both pathways. In this work, we show the existence of a compensatory relationship between CMA and eMI and identify a role for the chaperone protein Bag6 in triage and internalization of eMI substrates into late endosomes. Association and dynamics of Bag6 at the late endosome membrane change during starvation, a stressor that, contrary to other autophagic pathways, causes a decline in eMI activity. Collectively, these results show a coordinated function of eMI with CMA, identify the interchangeable subproteome degraded by these pathways, and start to elucidate the molecular mechanisms that facilitate the switch between them.

Keywords:  Bag6; CP: Molecular biology; autophagy; chaperone; late endosome; lysosome; microautophagy; protein degradation; protein targeting; proteostasis; starvation

DOI:  https://doi.org/10.1016/j.celrep.2023.113529
Chembiochem. 2023 Dec 11. e202300746

Biotin-based strategies to explore the world of ubiquitin and ubiquitin-like modifiers.

Laura Merino-Cacho, Orhi Barroso-Gomila, Sandra Hernández-Sánchez, Juanma Ramirez, Ugo Mayor, James D Sutherland, Rosa Barrio.

  A complex code of cellular signals is mediated by ubiquitin and ubiquitin-like (Ub/UbL) modifications on substrate proteins. The so-called Ubiquitin Code specifies protein fates, such as stability, subcellular localization, functional activation or suppression, and interactions. Hundreds of enzymes are involved in placing and removing Ub/UbL on thousands of substrates, while the consequences of modifications and the mechanisms of specificity are still poorly defined. Challenges include rapid and transient engagement of enzymes and Ub/UbL interactors, low stoichiometry of modified versus non-modified cellular substrates, and protease-mediated loss of Ub/UbL in lysates. To decipher this complexity and confront the challenges, many tools have been created to trap and identify substrates and interactors linked to Ub/UbL modification. This review focuses on an assortment of biotin-based tools we have developed for this purpose (for example BioUbLs, UbL-ID, BioE3), taking advantage of the strong affinity of biotin-streptavidin and the stringent lysis/washing approach allowed by it, paired with sensitive mass-spectrometry-based proteomic methods. Knowing how substrates change during development and disease, the consequences of substrate modification, and matching substrates to particular UbL-ligating enzymes will contribute new insights into how Ub/UbL signaling works and how it can be exploited for therapies.

Keywords:  Biotin; E3 ligase; Proximity proteomics; SUMO; Ubiquitin

DOI:  https://doi.org/10.1002/cbic.202300746
Nat Commun. 2023 Dec 08. 14(1): 8140

Nutrient deprivation alters the rate of COPII subunit recruitment at ER subdomains to tune secretory protein transport.

William Kasberg, Peter Luong, Kevin A Swift, Anjon Audhya.

Co-assembly of the multilayered coat protein complex II (COPII) with the Sar1 GTPase at subdomains of the endoplasmic reticulum (ER) enables secretory cargoes to be concentrated efficiently within nascent transport intermediates, which subsequently deliver their contents to ER-Golgi intermediate compartments. Here, we define the spatiotemporal accumulation of native COPII subunits and secretory cargoes at ER subdomains under differing nutrient availability conditions using a combination of CRISPR/Cas9-mediated genome editing and live cell imaging. Our findings demonstrate that the rate of inner COPII coat recruitment serves as a determinant for the pace of cargo export, irrespective of COPII subunit expression levels. Moreover, increasing inner COPII coat recruitment kinetics is sufficient to rescue cargo trafficking deficits caused by acute nutrient limitation. Our findings are consistent with a model in which the rate of inner COPII coat addition acts as an important control point to regulate cargo export from the ER.

DOI: https://doi.org/10.1038/s41467-023-44002-7
Autophagy. 2023 Dec 14.

Mammalian phagophores with finger-like shapes emerge from recycling endosomes.

Claudia Puri, David C Rubinsztein.

  Autophagosomes are double-membraned vesicles that engulf cytoplasmic contents, which are ultimately degraded after autophagosome-lysosome fusion. The prevailing view, largely inferred from EM-based studies, was that mammalian autophagosomes evolved from disc-shaped precursors that invaginated and then were closed at the single opening. Many site(s) of origin of these precursors have been proposed. Using superresolution structured illumination microscopy and electron microscopy, we find that mammalian autophagosomes derive from finger-like outgrowths from the recycling endosome. These "fingers" survey a large cell volume and then close into a "fist" and the openings are sealed in an ESCRT-dependent fashion, while the precursors are still attached to the recycling endosome. We call this transient recycling endosome-attached, closed, autophagic structure an "autophago-dome". DNM2-dependent scission of the autophago-dome from the recycling endosomes liberates free autophagosomes from this compartment. These data reveal unexpected morphologies of autophagosome precursors and raise new questions about the control of this process.

Keywords:  ESCRT; RAB11; autophagy; phagophore; recycling endosome

DOI:  https://doi.org/10.1080/15548627.2023.2293439
Cell Rep. 2023 Dec 09. pii: S2211-1247(23)01581-4. [Epub ahead of print]42(12): 113569

Mechanistic dissection of premature translation termination induced by acidic residues-enriched nascent peptide.

Yuhei Chadani, Takashi Kanamori, Tatsuya Niwa, Kazuya Ichihara, Keiichi I Nakayama, Akinobu Matsumoto, Hideki Taguchi.

  Ribosomes polymerize nascent peptides through repeated inter-subunit rearrangements between the classic and hybrid states. The peptidyl-tRNA, the intermediate species during translation elongation, stabilizes the translating ribosome to ensure robust continuity of elongation. However, the translation of acidic residue-rich sequences destabilizes the ribosome, leading to a stochastic premature translation cessation termed intrinsic ribosome destabilization (IRD), which is still ill-defined. Here, we dissect the molecular mechanisms underlying IRD in Escherichia coli. Reconstitution of the IRD event reveals that (1) the prolonged ribosome stalling enhances IRD-mediated translation discontinuation, (2) IRD depends on temperature, (3) the destabilized 70S ribosome complex is not necessarily split, and (4) the destabilized ribosome is subjected to peptidyl-tRNA hydrolase-mediated hydrolysis of the peptidyl-tRNA without subunit splitting or recycling factors-mediated subunit splitting. Collectively, our data indicate that the translation of acidic-rich sequences alters the conformation of the 70S ribosome to an aberrant state that allows the noncanonical premature termination.

Keywords:  CP: Molecular biology; EF-G; Pth; nascent polypeptide; peptidyl-tRNA; premature termination; ribosome; ribosome recycling; ribosome stalling; temperature; translation

DOI:  https://doi.org/10.1016/j.celrep.2023.113569
bioRxiv. 2023 Nov 29. pii: 2023.11.28.569054. [Epub ahead of print]

Role of a holo-insertase complex in the biogenesis of biophysically diverse ER membrane proteins.

Katharine R Page, Vy N Nguyen, Tino Pleiner, Giovani Pinton Tomaleri, Maxine L Wang, Alina Guna, Ting-Yu Wang, Tsui-Fen Chou, Rebecca M Voorhees.

Mammalian membrane proteins perform essential physiologic functions that rely on their accurate insertion and folding at the endoplasmic reticulum (ER). Using forward and arrayed genetic screens, we systematically studied the biogenesis of a panel of membrane proteins, including several G-protein coupled receptors (GPCRs). We observed a central role for the insertase, the ER membrane protein complex (EMC), and developed a dual-guide approach to identify genetic modifiers of the EMC. We found that the back of sec61 (BOS) complex, a component of the 'multipass translocon', was a physical and genetic interactor of the EMC. Functional and structural analysis of the EMC•BOS holocomplex showed that characteristics of a GPCR's soluble domain determine its biogenesis pathway. In contrast to prevailing models, no single insertase handles all substrates. We instead propose a unifying model for coordination between the EMC, multipass translocon, and Sec61 for biogenesis of diverse membrane proteins in human cells.

DOI: https://doi.org/10.1101/2023.11.28.569054
Chembiochem. 2023 Dec 13. e202300723

Tripartite split-GFP for high throughput screening of small molecules: a powerful strategy for targeting transient/labile interactors like E2-E3 ubiquitination enzymes.

Cécile Polge, Stéphanie Cabantous, Daniel Taillandier.

  The search for inhibitors of the Ubiquitin Proteasome System (UPS) is an expanding area, due to the crucial role of UPS enzymes in several diseases. The complexity of the UPS and the multiple protein-protein interactions (PPIs) involved, either between UPS proteins themselves or between UPS components and theirs targets, offer an incredibly wide field for the development of chemical compounds for specifically modulating or inhibiting metabolic pathways. However, numerous UPS PPIs are transient/labile, due the processivity of the system (Ubiquitin [Ub] chain elongation, Ub transfer, etc.). Among the different strategies that can be used either for deciphering UPS PPI or for identifying/characterizing small compounds inhibitors, the split-GFP approach offers several advantages notably for high throughput screening of drugs. Split-GFP is based on the principle of protein-fragment complementation assay (PCA). PCA allows addressing PPIs by coupling each protein of interest (POI) to fragments of a reporter protein whose reconstitution is linked to the interaction of the POI. Here, we review the evolution of the split-GFP approach from bipartite to tripartite Split-GFP and its recent applicability for screening chemical compounds targeting the UPS.

Keywords:  E2 ubiquitin-conjugating enzyme; E3-ubiquitin ligase; High throughput screening; Tripartite split-GFP; Ubiquitin Proteasome System

DOI:  https://doi.org/10.1002/cbic.202300723
Nat Rev Drug Discov. 2023 Dec 07.

Extracellular targeted protein degradation: an emerging modality for drug discovery.

James A Wells, Kaan Kumru.

Targeted protein degradation (TPD) has emerged in the past decade as a major new drug modality to remove intracellular proteins with bispecific small molecules that recruit the protein of interest (POI) to an E3 ligase for degradation in the proteasome. Unlike classic occupancy-based drugs, intracellular TPD (iTPD) eliminates the target and works catalytically, and so can be more effective and sustained, with lower dose requirements. Recently, this approach has been expanded to the extracellular proteome, including both secreted and membrane proteins. Extracellular targeted protein degradation (eTPD) uses bispecific antibodies, conjugates or small molecules to degrade extracellular POIs by trafficking them to the lysosome for degradation. Here, we focus on recent advances in eTPD, covering degrader systems, targets, molecular designs and parameters to advance them. Now almost any protein, intracellular or extracellular, is addressable in principle with TPD.

DOI: https://doi.org/10.1038/s41573-023-00833-z
J Biol Chem. 2023 Dec 08. pii: S0021-9258(23)02568-1. [Epub ahead of print] 105540

VCIP135 associates with both the N- and C-terminal regions of p97 ATPase.

Suzune Nakayama, Hisao Kondo.

  Two distinct p97ATPase-mediated membrane fusion pathways are required for Golgi and endoplasmic reticulum (ER) biogenesis; namely, the p97/p47 pathway and the p97/p37 pathway. p97(VCP)/p47 complex-interacting protein, p135 (VCIP135) is necessary for both of these pathways. Although VCIP135 is known to form a complex with p97 in the cytosol, the role of this complex in Golgi and ER biogenesis has remained unclear. In this study, we demonstrated that VCIP135 has two distinct p97-binding sites at its N- and C-terminal regions. In particular, the C-terminal binding site includes the SHP motif, which is also found in other p97-binding proteins, such as p47, p37 and Ufd1. We also clarified that VCIP135 binds to both the N- and C-terminal regions of p97; i.e., the N- and C-terminal binding sites in VCIP135 interact with the C- and N-terminal regions of p97, respectively. These two interactions within the complex are synchronously controlled by the nucleotide state of p97. We next generated VCIP135 mutants lacking each of the p97-binding sites to investigate their functions in living cells, and clarified that VCIP135 is involved in Golgi and ER biogenesis through its two distinct interactions with p97. VCIP135 is hence a unique p97-binding protein that functions by interacting with both the N-and C-terminal regions of p97, which strongly suggests that it plays crucial roles in p97-mediated events.

Keywords:  ATPases associated with diverse cellular activities (AAA); Golgi; SNARE; complex; endoplasmic reticulum (ER); membrane fusion

DOI:  https://doi.org/10.1016/j.jbc.2023.105540
Autophagy. 2023 Dec 11. 1-2

The hookup model of the HOPS complex in autophagosome-lysosome fusion.

Shen Zhang, Linsen Li, Xiaoxia Liu, Qing Zhong.

  Macroautophagy/autophagy is a highly conserved process that involves the degradation of proteins, damaged organelles, and other cytoplasmic macromolecules. Autophagosome-lysosome fusion is critical for successful substrate degradation and is mediated by SNARE proteins. The fusion process requires additional vesicle docking and tethering-regulating factors. Our recent work has uncovered a functional model of autophagosome-lysosome fusion. We demonstrated that the six-subunit homotypic fusion and vacuole protein sorting (HOPS) complex can be assembled by two subcomplexes, the VPS39-VPS11 subcomplex (HOPS-2) and the VPS41-VPS16-VPS18-VPS33A subcomplex (HOPS-4). VPS39 binds with RAB2 on the autophagosome and VPS41 binds with RAB39A on the lysosome, which then promotes membrane tethering and autophagic SNARE-mediated membrane fusion. Moreover, we have revealed that ALS- and FTD-related C9orf72 is a guanine exchange factor (GEF) for RAB39A. In this punctum, we discuss how the C9orf72-RAB39A-HOPS axis function regulates autophagosome-lysosome fusion.

Keywords:  Autophagosome-lysosome fusion; C9orf72; HOPS complex; RAB39A; membrane tethering

DOI:  https://doi.org/10.1080/15548627.2023.2291938
Nat Commun. 2023 Dec 08. 14(1): 8121

Characterization of nucleolar SUMO isopeptidases unveils a general p53-independent checkpoint of impaired ribosome biogenesis.

Judith Dönig, Hannah Mende, Jimena Davila Gallesio, Kristina Wagner, Paul Hotz, Kathrin Schunck, Tanja Piller, Soraya Hölper, Sara Uhan, Manuel Kaulich, Matthias Wirth, Ulrich Keller, Georg Tascher, Katherine E Bohnsack, Stefan Müller.

Ribosome biogenesis is a multi-step process, in which a network of trans-acting factors ensures the coordinated assembly of pre-ribosomal particles in order to generate functional ribosomes. Ribosome biogenesis is tightly coordinated with cell proliferation and its perturbation activates a p53-dependent cell-cycle checkpoint. How p53-independent signalling networks connect impaired ribosome biogenesis to the cell-cycle machinery has remained largely enigmatic. We demonstrate that inactivation of the nucleolar SUMO isopeptidases SENP3 and SENP5 disturbs distinct steps of 40S and 60S ribosomal subunit assembly pathways, thereby triggering the canonical p53-dependent impaired ribosome biogenesis checkpoint. However, inactivation of SENP3 or SENP5 also induces a p53-independent checkpoint that converges on the specific downregulation of the key cell-cycle regulator CDK6. We further reveal that impaired ribosome biogenesis generally triggers the downregulation of CDK6, independent of the cellular p53 status. Altogether, these data define the role of SUMO signalling in ribosome biogenesis and unveil a p53-independent checkpoint of impaired ribosome biogenesis.

DOI: https://doi.org/10.1038/s41467-023-43751-9
Cell Rep. 2023 Dec 12. pii: S2211-1247(23)01425-0. [Epub ahead of print]42(12): 113413

Synonymous codon usage regulates translation initiation.

Chloe L Barrington, Gabriel Galindo, Amanda L Koch, Emma R Horton, Evan J Morrison, Samantha Tisa, Timothy J Stasevich, Olivia S Rissland.

  Nonoptimal synonymous codons repress gene expression, but the underlying mechanisms are poorly understood. We and others have previously shown that nonoptimal codons slow translation elongation speeds and thereby trigger messenger RNA (mRNA) degradation. Nevertheless, transcript levels are often insufficient to explain protein levels, suggesting additional mechanisms by which codon usage regulates gene expression. Using reporters in human and Drosophila cells, we find that transcript levels account for less than half of the variation in protein abundance due to codon usage. This discrepancy is explained by translational differences whereby nonoptimal codons repress translation initiation. Nonoptimal transcripts are also less bound by the translation initiation factors eIF4E and eIF4G1, providing a mechanistic explanation for their reduced initiation rates. Importantly, translational repression can occur without mRNA decay and deadenylation, and it does not depend on the known nonoptimality sensor, CNOT3. Our results reveal a potent mechanism of regulation by codon usage where nonoptimal codons repress further rounds of translation.

Keywords:  CNOT3; CP: Molecular biology; codon optimality; deadenylation; eIF4E; gene regulation; mRNA decay; ribosome; translation initiation; translational repression

DOI:  https://doi.org/10.1016/j.celrep.2023.113413
Nat Commun. 2023 Dec 11. 14(1): 8187

GRAF1 integrates PINK1-Parkin signaling and actin dynamics to mediate cardiac mitochondrial homeostasis.

Qiang Zhu, Matthew E Combs, Juan Liu, Xue Bai, Wenbo B Wang, Laura E Herring, Jiandong Liu, Jason W Locasale, Dawn E Bowles, Ryan T Gross, Michelle Mendiola Pla, Christopher P Mack, Joan M Taylor.

The serine/threonine kinase, PINK1, and the E3 ubiquitin ligase, Parkin, are known to facilitate LC3-dependent autophagosomal encasement and lysosomal clearance of dysfunctional mitochondria, and defects in this process contribute to a variety of cardiometabolic and neurological diseases. Although recent evidence indicates that dynamic actin remodeling plays an important role in PINK1/Parkin-mediated mitochondrial autophagy (mitophagy), the underlying signaling mechanisms remain unknown. Here, we identify the RhoGAP GRAF1 (Arhgap26) as a PINK1 substrate that regulates mitophagy. GRAF1 promotes the release of damaged mitochondria from F-actin anchors, regulates mitochondrial-associated Arp2/3-mediated actin remodeling and facilitates Parkin-LC3 interactions to enhance mitochondria capture by autophagosomes. Graf1 phosphorylation on PINK1-dependent sites is dysregulated in human heart failure, and cardiomyocyte-restricted Graf1 depletion in mice blunts mitochondrial clearance and attenuates compensatory metabolic adaptations to stress. Overall, we identify GRAF1 as an enzyme that coordinates cytoskeletal and metabolic remodeling to promote cardioprotection.

DOI: https://doi.org/10.1038/s41467-023-43889-6
bioRxiv. 2023 Nov 28. pii: 2023.11.27.568910. [Epub ahead of print]

Codon optimality modulates protein output by tuning translation initiation.

Elijah F Lyons, Lou C Devanneaux, Ryan Y Muller, Anna V Freitas, Zuriah A Meacham, Maria V McSharry, Van N Trinh, Anna J Rogers, Nicholas T Ingolia, Liana F Lareau.

The impact of synonymous codon choice on protein output has important implications for understanding endogenous gene expression and design of synthetic mRNAs. Previously, we used a neural network model to design a series of synonymous fluorescent reporters whose protein output in yeast spanned a seven-fold range corresponding to their predicted translation speed. Here, we show that this effect is not due primarily to the established impact of slow elongation on mRNA stability, but rather, that an active mechanism further decreases the number of proteins made per mRNA. We combine simulations and careful experiments on fluorescent reporters to argue that translation initiation is limited on non-optimally encoded transcripts. Using a genome-wide CRISPRi screen to discover factors modulating the output from non-optimal transcripts, we identify a set of translation initiation factors including multiple subunits of eIF3 whose depletion restored protein output of a non-optimal reporter. Our results show that codon usage can directly limit protein production, across the full range of endogenous variability in codon usage, by limiting translation initiation.

DOI: https://doi.org/10.1101/2023.11.27.568910
Mol Cell. 2023 Dec 07. pii: S1097-2765(23)00954-1. [Epub ahead of print]83(23): 4197-4199

Atypical K6-ubiquitin chains mobilize p97/VCP and the proteasome to resolve formaldehyde-induced RNA-protein crosslinks.

Wei Song, Kristijan Ramadan.

In this issue of Molecular Cell, Rahmanto et al.1 and Zhao et al.2 demonstrate that RNA-protein crosslinks contribute to formaldehyde toxicity by blocking protein synthesis. Furthermore, they identify a ubiquitin-mediated degradation system for RNA-protein crosslink resolution in eukaryotes.

DOI: https://doi.org/10.1016/j.molcel.2023.11.009
Nat Commun. 2023 Dec 12. 14(1): 8237

An integrated workflow for quantitative analysis of the newly synthesized proteome.

Toman Borteçen, Torsten Müller, Jeroen Krijgsveld.

The analysis of proteins that are newly synthesized upon a cellular perturbation can provide detailed insight into the proteomic response that is elicited by specific cues. This can be investigated by pulse-labeling of cells with clickable and stable-isotope-coded amino acids for the enrichment and mass spectrometric characterization of newly synthesized proteins (NSPs), however convoluted protocols prohibit their routine application. Here we report the optimization of multiple steps in sample preparation, mass spectrometry and data analysis, and we integrate them into a semi-automated workflow for the quantitative analysis of the newly synthesized proteome (QuaNPA). Reduced input requirements and data-independent acquisition (DIA) enable the analysis of triple-SILAC-labeled NSP samples, with enhanced throughput while featuring high quantitative accuracy. We apply QuaNPA to investigate the time-resolved cellular response to interferon-gamma (IFNg), observing rapid induction of targets 2 h after IFNg treatment. QuaNPA provides a powerful approach for large-scale investigation of NSPs to gain insight into complex cellular processes.

DOI: https://doi.org/10.1038/s41467-023-43919-3
Sci Adv. 2023 Dec 15. 9(50): eadj1205

Parkinson's VPS35[D620N] mutation induces LRRK2-mediated lysosomal association of RILPL1 and TMEM55B.

Prosenjit Pal, Matthew Taylor, Pui Yiu Lam, Francesca Tonelli, Chloe A Hecht, Pawel Lis, Raja S Nirujogi, Toan K Phung, Wondwossen M Yeshaw, Ebsy Jaimon, Rotimi Fasimoye, Emily A Dickie, Melanie Wightman, Thomas Macartney, Suzanne R Pfeffer, Dario R Alessi.

We demonstrate that the Parkinson's VPS35[D620N] mutation alters the expression of ~220 lysosomal proteins and stimulates recruitment and phosphorylation of Rab proteins at the lysosome. This recruits the phospho-Rab effector protein RILPL1 to the lysosome where it binds to the lysosomal integral membrane protein TMEM55B. We identify highly conserved regions of RILPL1 and TMEM55B that interact and design mutations that block binding. In mouse fibroblasts, brain, and lung, we demonstrate that the VPS35[D620N] mutation reduces RILPL1 levels, in a manner reversed by LRRK2 inhibition and proteasome inhibitors. Knockout of RILPL1 enhances phosphorylation of Rab substrates, and knockout of TMEM55B increases RILPL1 levels. The lysosomotropic agent LLOMe also induced LRRK2 kinase-mediated association of RILPL1 to the lysosome, but to a lower extent than the D620N mutation. Our study uncovers a pathway through which dysfunctional lysosomes resulting from the VPS35[D620N] mutation recruit and activate LRRK2 on the lysosomal surface, driving assembly of the RILPL1-TMEM55B complex.

DOI: https://doi.org/10.1126/sciadv.adj1205
bioRxiv. 2023 Nov 03. pii: 2023.11.03.565554. [Epub ahead of print]

UPF1 ATPase autoinhibition and activation modulate RNA binding kinetics and NMD efficiency.

Joseph H Chapman, Alice M Youle, Acadia L Grimme, Keir C Neuman, J Robert Hogg.

The RNA helicase UPF1 interacts with mRNAs, mRNA decay machinery, and the terminating ribosome to promote nonsense-mediated mRNA decay (NMD). Structural and biochemical data have revealed that UPF1 exists in an enzymatically autoinhibited "closed" state. Upon binding the NMD protein UPF2, UPF1 undergoes an extensive conformational change into a more enzymatically active "open" state, which exhibits enhanced ATPase and helicase activity. However, mechanically deficient UPF1 mutants can support efficient NMD, bringing into question the roles of UPF1 enzymatic autoinhibition and activation in NMD. Here, we identify two additional important features of the activated open state: slower nucleic acid binding kinetics and enhanced ATP-stimulated nucleic acid dissociation kinetics. Computational modeling based on empirical measurements of UPF1, UPF2, and RNA interaction kinetics predicts that the majority of UPF1-RNA binding and dissociation events in cells occur independently of UPF2 binding. We find that UPF1 mutants with either reduced or accelerated dissociation from RNA have NMD defects, whereas UPF1 mutants that are more dependent on UPF2 for catalytic activity remain active on well-established NMD targets. These findings support a model in which the kinetics of UPF1-mRNA interactions are important determinants of cellular NMD efficiency.

DOI: https://doi.org/10.1101/2023.11.03.565554
Nat Commun. 2023 Dec 12. 14(1): 8217

Nuclear cGAS restricts L1 retrotransposition by promoting TRIM41-mediated ORF2p ubiquitination and degradation.

Zhengyi Zhen, Yu Chen, Haiyan Wang, Huanyin Tang, Haiping Zhang, Haipeng Liu, Ying Jiang, Zhiyong Mao.

Cyclic GMP-AMP synthase (cGAS), initially identified as a cytosolic DNA sensor, detects DNA fragments to trigger an innate immune response. Recently, accumulating evidence reveals the presence of cGAS within the nucleus. However, the biological functions of nuclear cGAS are not fully understood. Here, we demonstrate that nuclear cGAS represses LINE-1 (L1) retrotransposition to preserve genome integrity in human cells. Mechanistically, the E3 ligase TRIM41 interacts with and ubiquitinates ORF2p to influence its stability, and cGAS enhances the association of ORF2p with TRIM41, thereby promoting TRIM41-mediated ORF2p degradation and the suppression of L1 retrotransposition. In response to DNA damage, cGAS is phosphorylated at serine residues 120 and 305 by CHK2, which promotes cGAS-TRIM41 association, facilitating TRIM41-mediated ORF2p degradation. Moreover, we show that nuclear cGAS mediates the repression of L1 retrotransposition in senescent cells induced by DNA damage agents. We also identify several cancer-associated cGAS mutations that abolish the suppressive effect on L1 retrotransposition by disrupting the CHK2-cGAS-TRIM41-ORF2p regulatory axis. Together, these findings indicate that nuclear cGAS exhibits an inhibitory function in L1 retrotransposition which could provide avenues for future interventions in both aging and tumorigenesis.

DOI: https://doi.org/10.1038/s41467-023-43001-y
BMC Bioinformatics. 2023 Dec 13. 24(1): 471

ORFeus: a computational method to detect programmed ribosomal frameshifts and other non-canonical translation events.

Mary O Richardson, Sean R Eddy.

   BACKGROUND: In canonical protein translation, ribosomes initiate translation at a specific start codon, maintain a single reading frame throughout elongation, and terminate at the first in-frame stop codon. However, ribosomal behavior can deviate at each of these steps, sometimes in a programmed manner. Certain mRNAs contain sequence and structural elements that cause ribosomes to begin translation at alternative start codons, shift reading frame, read through stop codons, or reinitiate on the same mRNA. These processes represent important translational control mechanisms that can allow an mRNA to encode multiple functional protein products or regulate protein expression. The prevalence of these events remains uncertain, due to the difficulty of systematic detection.
RESULTS: We have developed a computational model to infer non-canonical translation events from ribosome profiling data.
CONCLUSION: ORFeus identifies known examples of alternative open reading frames and recoding events across different organisms and enables transcriptome-wide searches for novel events.

Keywords:  Alternative ORF; HMM; Non-canonical ORF; Ribosome profiling

DOI:  https://doi.org/10.1186/s12859-023-05602-8
Nat Commun. 2023 Dec 13. 14(1): 8075

Enhanced SREBP2-driven cholesterol biosynthesis by PKCλ/ι deficiency in intestinal epithelial cells promotes aggressive serrated tumorigenesis.

Yu Muta, Juan F Linares, Anxo Martinez-Ordoñez, Angeles Duran, Tania Cid-Diaz, Hiroto Kinoshita, Xiao Zhang, Qixiu Han, Yuki Nakanishi, Naoko Nakanishi, Thekla Cordes, Gurpreet K Arora, Marc Ruiz-Martinez, Miguel Reina-Campos, Hiroaki Kasashima, Masakazu Yashiro, Kiyoshi Maeda, Ana Albaladejo-Gonzalez, Daniel Torres-Moreno, José García-Solano, Pablo Conesa-Zamora, Giorgio Inghirami, Christian M Metallo, Timothy F Osborne, Maria T Diaz-Meco, Jorge Moscat.

The metabolic and signaling pathways regulating aggressive mesenchymal colorectal cancer (CRC) initiation and progression through the serrated route are largely unknown. Although relatively well characterized as BRAF mutant cancers, their poor response to current targeted therapy, difficult preneoplastic detection, and challenging endoscopic resection make the identification of their metabolic requirements a priority. Here, we demonstrate that the phosphorylation of SCAP by the atypical PKC (aPKC), PKCλ/ι promotes its degradation and inhibits the processing and activation of SREBP2, the master regulator of cholesterol biosynthesis. We show that the upregulation of SREBP2 and cholesterol by reduced aPKC levels is essential for controlling metaplasia and generating the most aggressive cell subpopulation in serrated tumors in mice and humans. Since these alterations are also detected prior to neoplastic transformation, together with the sensitivity of these tumors to cholesterol metabolism inhibitors, our data indicate that targeting cholesterol biosynthesis is a potential mechanism for serrated chemoprevention.

DOI: https://doi.org/10.1038/s41467-023-43690-5
bioRxiv. 2023 Dec 01. pii: 2023.11.29.569295. [Epub ahead of print]

GABA(A) receptor activation drives GABARAP-Nix mediated autophagy to radiation-sensitize primary and brain-metastatic lung adenocarcinoma tumors.

Debanjan Bhattacharya, Riccardo Barille, Donatien Kamdem Toukam, Vaibhavkumar S Gawali, Laura Kallay, Taukir Ahmed, Hawley Brown, Sepideh Rezvanian, Aniruddha Karve, Pankaj B Desai, Mario Medvedovic, Kyle Wang, Dan Ionascu, Nusrat Harun, Chenran Wang, Andrew M Baschnagel, Joshua A Kritzer, James M Cook, Daniel A Pomeranz Krummel, Soma Sengupta.

In non-small cell lung cancer (NSCLC) treatment, targeted therapies benefit only a subset of NSCLC, while radiotherapy responses are not durable and toxicity limits therapy. We find that a GABA(A) receptor activator, AM-101, impairs viability and clonogenicity of NSCLC primary and brain metastatic cells. Employing an ex vivo 'chip', AM-101 is as efficacious as the chemotherapeutic docetaxel, which is used with radiotherapy for advanced-stage NSCLC. In vivo , AM-101 potentiates radiation, including conferring a survival benefit to mice bearing NSCLC intracranial tumors. GABA(A) receptor activation stimulates a selective-autophagic response via multimerization of GABA(A) Receptor-Associated Protein (GABARAP), stabilization of mitochondrial receptor Nix, and utilization of ubiquitin-binding protein p62. A targeted-peptide disrupting Nix binding to GABARAP inhibits AM-101 cytotoxicity. This supports a model of GABA(A) receptor activation driving a GABARAP-Nix multimerization axis triggering autophagy. In patients receiving radiotherapy, GABA(A) receptor activation may improve tumor control while allowing radiation dose de-intensification to reduce toxicity.
Highlights: Activating GABA(A) receptors intrinsic to lung primary and metastatic brain cancer cells triggers a cytotoxic response. GABA(A) receptor activation works as well as chemotherapeutic docetaxel in impairing lung cancer viability ex vivo . GABA(A) receptor activation increases survival of mice bearing lung metastatic brain tumors.A selective-autophagic response is stimulated by GABA(A) receptor activation that includes multimerization of GABARAP and Nix.Employing a new nanomolar affinity peptide that abrogates autophagosome formation inhibits cytotoxicity elicited by GABA(A) receptor activation.

DOI: https://doi.org/10.1101/2023.11.29.569295
RNA Biol. 2024 Jan;21(1): 1-12

mRNA nuclear export: how mRNA identity features distinguish functional RNAs from junk transcripts.

Alexander F Palazzo, Yi Qiu, Yoon Mo Kang.

  The division of the cellular space into nucleoplasm and cytoplasm promotes quality control mechanisms that prevent misprocessed mRNAs and junk RNAs from gaining access to the translational machinery. Here, we explore how properly processed mRNAs are distinguished from both misprocessed mRNAs and junk RNAs by the presence or absence of various 'identity features'.

Keywords:  GC-content; mRNA nuclear export; mRNA quality control; nuclear retention; splicing

DOI:  https://doi.org/10.1080/15476286.2023.2293339
Cell Rep. 2023 Dec 11. pii: S2211-1247(23)01567-X. [Epub ahead of print]42(12): 113555

Cell stretching activates an ATM mechano-transduction pathway that remodels cytoskeleton and chromatin.

Giulia Bastianello, Giancarlo Porcella, Galina V Beznoussenko, Gururaj Kidiyoor, Flora Ascione, Qingsen Li, Angela Cattaneo, Vittoria Matafora, Andrea Disanza, Micaela Quarto, Alexander A Mironov, Amanda Oldani, Sara Barozzi, Angela Bachi, Vincenzo Costanzo, Giorgio Scita, Marco Foiani.

  Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) kinases contain elastic domains. ATM also responds to reactive oxygen species (ROS) and ATR to nuclear mechanical stress. Mre11 mediates ATM activation following DNA damage; ATM mutations cause ataxia telangiectasia (A-T). Here, using in vivo imaging, electron microscopy, proteomic, and mechano-biology approaches, we study how ATM responds to mechanical stress. We report that cytoskeleton and ROS, but not Mre11, mediate ATM activation following cell deformation. ATM deficiency causes hyper-stiffness, stress fiber accumulation, Yes-associated protein (YAP) nuclear enrichment, plasma and nuclear membrane alterations during interstitial migration, and H3 hyper-methylation. ATM locates to the actin cytoskeleton and, following cytoskeleton stress, promotes phosphorylation of key cytoskeleton and chromatin regulators. Our data contribute to explain some clinical features of patients with A-T and pinpoint the existence of an integrated mechano-response in which ATM and ATR have distinct roles unrelated to their canonical DDR functions.

Keywords:  ATM and ATR; CP: Cell biology; CP: Molecular biology; DNA damage response; ROS; ataxia telangiectasia; cell stretching; checkpoints; chromatin; cytoskeleton; interstitial migration; mechanical stress

DOI:  https://doi.org/10.1016/j.celrep.2023.113555
Cell Death Dis. 2023 Dec 11. 14(12): 815

Mint3-depletion-induced energy stress sensitizes triple-negative breast cancer to chemotherapy via HSF1 inactivation.

Noritaka Tanaka, Hikari Okada, Kiyoshi Yamaguchi, Masahide Seki, Daisuke Matsubara, Noriko Gotoh, Yutaka Suzuki, Yoichi Furukawa, Taro Yamashita, Jun-Ichiro Inoue, Shuichi Kaneko, Takeharu Sakamoto.

Given the lack of therapeutic targets, the conventional approach for managing triple-negative breast cancer (TNBC) involves the utilization of cytotoxic chemotherapeutic agents. However, most TNBCs acquire resistance to chemotherapy, thereby lowering the therapeutic outcome. In addition to oncogenic mutations in TNBC, microenvironment-induced mechanisms render chemoresistance more complex and robust in vivo. Here, we aimed to analyze whether depletion of Munc18-1 interacting protein 3 (Mint3), which activates hypoxia-inducible factor 1 (HIF-1) during normoxia, sensitizes TNBC to chemotherapy. We found that Mint3 promotes the chemoresistance of TNBC in vivo. Mint3 depletion did not affect the sensitivity of human TNBC cell lines to doxorubicin and paclitaxel in vitro but sensitized tumors of these cells to chemotherapy in vivo. Transcriptome analyses revealed that the Mint3-HIF-1 axis enhanced heat shock protein 70 (HSP70) expression in tumors of TNBC cells. Administering an HSP70 inhibitor enhanced the antitumor activity of doxorubicin in TNBC tumors, similar to Mint3 depletion. Mint3 expression was also correlated with HSP70 expression in human TNBC specimens. Mechanistically, Mint3 depletion induces glycolytic maladaptation to the tumor microenvironment in TNBC tumors, resulting in energy stress. This energy stress by Mint3 depletion inactivated heat shock factor 1 (HSF-1), the master regulator of HSP expression, via the AMP-activated protein kinase/mechanistic target of the rapamycin pathway following attenuated HSP70 expression. In conclusion, Mint3 is a unique regulator of TNBC chemoresistance in vivo via metabolic adaptation to the tumor microenvironment, and a combination of Mint3 inhibition and chemotherapy may be a good strategy for TNBC treatment.

DOI: https://doi.org/10.1038/s41419-023-06352-4
J Cell Biol. 2024 Feb 05. pii: e202303015. [Epub ahead of print]223(2):

XAF1 promotes colorectal cancer metastasis via VCP-RNF114-JUP axis.

Ji Xia, Ning Ma, Qian Shi, Qin-Cheng Liu, Wei Zhang, Hui-Jun Cao, Yi-Kang Wang, Qian-Wen Zheng, Qian-Zhi Ni, Sheng Xu, Bing Zhu, Xiao-Song Qiu, Kai Ding, Jing-Yi Huang, Xin Liang, Yu Chen, Yan-Jun Xiang, Xi-Ran Zhang, Lin Qiu, Wei Chen, Dong Xie, Xiang Wang, Lingyun Long, Jing-Jing Li.

Metastasis is the main cause of colorectal cancer (CRC)-related death, and the 5-year relative survival rate for CRC patients with distant metastasis is only 14%. X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a zinc-rich protein belonging to the interferon (IFN)-induced gene family. Here, we report a metastasis-promoting role of XAF1 in CRC by acting as a novel adaptor of valosin-containing protein (VCP). XAF1 facilitates VCP-mediated deubiquitination of the E3 ligase RING finger protein 114 (RNF114), which promotes K48-linked ubiquitination and subsequent degradation of junction plakoglobin (JUP). The XAF1-VCP-RNF114-JUP axis is critical for the migration and metastasis of CRC cells. Moreover, we observe correlations between the protein levels of XAF1, RNF114, and JUP in clinical samples. Collectively, our findings reveal an oncogenic function of XAF1 in mCRC and suggest that the XAF1-VCP-RNF114-JUP axis is a potential therapeutic target for CRC treatment.

DOI: https://doi.org/10.1083/jcb.202303015
J Mol Biol. 2023 Dec 06. pii: S0022-2836(23)00495-3. [Epub ahead of print] 168384

Translation Rates and Protein Folding.

Anton A Komar, Ekaterina Samatova, Marina V Rodnina.

The mRNA coding sequence defines not only the amino acid sequence of the protein, but also the speed at which the ribosome move along the mRNA while making the protein. The non-uniform local kinetics - denoted as translational rhythm - is similar among mRNAs coding for related protein folds. Deviations from this conserved rhythm can result in protein misfolding. In this review we summarize the experimental evidence demonstrating how local translation rates affect cotranslational protein folding, with the focus on the synonymous codons and patches of charged residues in the nascent peptide as best-studied examples. Alterations in nascent protein conformation due to disturbed translational rhythm can persist off the ribosome, as demonstrated by the effects of synonymous codon variants of several disease-related proteins. Charged amino acid patches in nascent chains also modulate translation and cotranslational protein folding, but can abrogate translation when placed at the N-terminus of the nascent peptide. During cotranslational folding, incomplete nascent chains navigate through a unique conformational landscape in which earlier intermediate states become inaccessible as the nascent peptide grows. Precisely tuned local translation rates, as well as interactions with the ribosome, guide the folding pathway towards the native structure, whereas deviations from the natural translation rhythm may favor pathways leading to trapped misfolded states. Deciphering the 'folding code' of the mRNA will contribute to understanding the diseases caused by protein misfolding and to rational protein design.

DOI: https://doi.org/10.1016/j.jmb.2023.168384
Cell Chem Biol. 2023 Nov 30. pii: S2451-9456(23)00425-7. [Epub ahead of print]

Profiling nuclear cysteine ligandability and effects on nuclear localization using proximity labeling-coupled chemoproteomics.

Qianni Peng, Eranthie Weerapana.

  The nucleus controls cell growth and division through coordinated interactions between nuclear proteins and chromatin. Mutations that impair nuclear protein association with chromatin are implicated in numerous diseases. Covalent ligands are a promising strategy to pharmacologically target nuclear proteins, such as transcription factors, which lack ordered small-molecule binding pockets. To identify nuclear cysteines that are susceptible to covalent liganding, we couple proximity labeling (PL), using a histone H3.3-TurboID (His-TID) construct, with chemoproteomics. Using covalent scout fragments, KB02 and KB05, we identified ligandable cysteines on proteins involved in spindle assembly, DNA repair, and transcriptional regulation, such as Cys101 of histone acetyltransferase 1 (HAT1). Furthermore, we show that covalent fragments can affect the abundance, localization, and chromatin association of nuclear proteins. Notably, the Parkinson disease protein 7 (PARK7) showed increased nuclear localization and chromatin association upon KB02 modification at Cys106. Together, this platform provides insights into targeting nuclear cysteines with covalent ligands.

Keywords:  PARK7; TurboID; chromatin association; nuclear cysteine ligandability; proximity labeling

DOI:  https://doi.org/10.1016/j.chembiol.2023.11.010
Sci Transl Med. 2023 Dec 13. 15(726): eade4113

Wild-type IDH1 maintains NSCLC stemness and chemoresistance through activation of the serine biosynthetic pathway.

Cheng Zhang, Jiao-Jiao Yu, Chen Yang, Zhen-Long Yuan, Hui Zeng, Jun-Jian Wang, Shuang Shang, Xiao-Xi Lv, Xiao-Tong Liu, Jing Liu, Qi Xue, Bing Cui, Feng-Wei Tan, Fang Hua.

Tumor-initiating cells (TICs) reprogram their metabolic features to meet their bioenergetic, biosynthetic, and redox demands. Our previous study established a role for wild-type isocitrate dehydrogenase 1 (IDH1WT) as a potential diagnostic and prognostic biomarker for non-small cell lung cancer (NSCLC), but how IDH1WT modulates NSCLC progression remains elusive. Here, we report that IDH1WT activates serine biosynthesis by enhancing the expression of phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase 1 (PSAT1), the first and second enzymes of de novo serine synthetic pathway. Augmented serine synthesis leads to GSH/ROS imbalance and supports pyrimidine biosynthesis, maintaining tumor initiation capacity and enhancing gemcitabine chemoresistance. Mechanistically, we identify that IDH1WT interacts with and stabilizes PHGDH and fragile X-related protein-1 (FXR1) by impeding their association with the E3 ubiquitin ligase parkin by coimmunoprecipitation assay and proximity ligation assay. Subsequently, stabilized FXR1 supports PSAT1 mRNA stability and translation, as determined by actinomycin D chase experiment and in vitro translation assay. Disrupting IDH1WT-PHGDH and IDH1WT-FXR1 interactions synergistically reduces NSCLC stemness and sensitizes NSCLC cells to gemcitabine and serine/glycine-depleted diet therapy in lung cancer xenograft models. Collectively, our findings offer insights into the role of IDH1WT in serine metabolism, highlighting IDH1WT as a potential therapeutic target for eradicating TICs and overcoming gemcitabine chemoresistance in NSCLC.

DOI: https://doi.org/10.1126/scitranslmed.ade4113
Eur J Med Chem. 2023 Dec 07. pii: S0223-5234(23)00997-2. [Epub ahead of print]264 116030

Small molecules as modulators of the proteostasis machinery: Implication in cardiovascular diseases.

Zhiheng Yang, Yu Cao, Limin Kong, Jianjun Xi, Shourong Liu, Jiankang Zhang, Weiyan Cheng.

  With the escalating prevalence of cardiovascular diseases, the substantial socioeconomic burden on healthcare systems is intensifying. Accumulating empirical evidence underscores the pivotal role of the proteostasis network in regulating cardiac homeostasis and function. Disruptions in proteostasis may contribute to the loss of protein function or the acquisition of toxic functions, which are intricately linked to the development of cardiovascular ailments such as atrial fibrillation, heart failure, atherosclerosis, and cardiac aging. It is widely acknowledged that the proteostasis network encompasses molecular chaperones, autophagy, and the ubiquitin proteasome system (UPS). Consequently, the proteostasis network emerges as an appealing target for therapeutic interventions in cardiovascular diseases. Numerous small molecules, acting as modulators of the proteostasis machinery, have exhibited therapeutic efficacy in managing cardiovascular diseases. This review centers on elucidating the role of the proteostasis network in various cardiovascular diseases and explores the potential of small molecules as therapeutic agents.

Keywords:  Autophagy; Cardiovascular diseases; Molecular chaperones; Proteostasis; Small molecules; Ubiquitin proteasome system

DOI:  https://doi.org/10.1016/j.ejmech.2023.116030