bims-proteo 2024-02-18 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–02–18
thirty-one papers selected by
Eric Chevet, INSERM

Direct observation of autoubiquitination for an integral membrane ubiquitin ligase in ERAD.
Mechanisms of translation-coupled quality control.
Reticulophagy mediated by the V-ATPase-ATG16L1-LC3C axis.
Ribosomal collision is not a prerequisite for ZNF598-mediated ribosome ubiquitination and disassembly of ribosomal complexes by ASCC.
Mono-ubiquitination empowers ubiquitin chain elongation.
Structure-guided engineering enables E3 ligase-free and versatile protein ubiquitination via UBE2E1.
Chp1 is a dedicated chaperone at the ribosome that safeguards eEF1A biogenesis.
Activation of XBP1s attenuates disease severity in models of proteotoxic Charcot-Marie-Tooth type 1B.
SEL1L-HRD1 interaction is required to form a functional HRD1 ERAD complex.
The HRI branch of the integrated stress response selectively triggers mitophagy.
Visualization of translation reorganization upon persistent ribosome collision stress in mammalian cells.
The role of ATG9 vesicles in autophagosome biogenesis.
Human YKT6 forms priming complex with STX17 and SNAP29 to facilitate autophagosome-lysosome fusion.
Breaking the K48-chain: linking ubiquitin beyond protein degradation.
Shared and distinct mechanisms of UBA1 inactivation across different diseases.
Mapping Stress-Responsive Signaling Pathways Induced by Mitochondrial Proteostasis Perturbations.
Alternative mechanisms of Notch activation by partitioning into distinct endosomal domains.
Structural insights into the ubiquitylation strategy of the oligomeric CRL2FEM1B E3 ubiquitin ligase.
Ca 2+ -sensor ALG-2 engages ESCRTs to enhance lysosomal membrane resilience to osmotic stress.
Phylogeny-linked occurrence of ribosome stalling on the mRNAs of Arabidopsis unfolded protein response factor bZIP60 orthologs in divergent plant species.
DGAT2 inhibition blocks SREBP-1 cleavage and improves hepatic steatosis by increasing phosphatidylethanolamine in the ER.
Modulation of Cullin-RING E3 ubiquitin ligase-dependent ubiquitination by small molecule compounds.
Loss of Preproinsulin Interaction with Signal Recognition Particle Activates Protein Quality Control, Decreasing mRNA Stability.
A new family of bacterial ribosome hibernation factors.
Synonymous mutations can alter protein dimerization through localized interface misfolding involving self-entanglements.
ER stress impedes regulated secretion by governing key exocytotic and granulogenic molecular switches.
Nuclear RNA-related processes modulate the assembly of cytoplasmic RNA granules.
Understanding species-specific and conserved RNA-protein interactions in vivo and in vitro.
Komagataella phaffii Erp41 is a protein disulfide isomerase with unprecedented disulfide bond catalyzing activity when coupled to glutathione.
Radiotherapy induces persistent innate immune reprogramming of microglia into a primed state.
DAMPening tumor immune escape: the role of ER chaperones in immunogenic chemotherapy.

Nat Commun. 2024 Feb 13. 15(1): 1340

Direct observation of autoubiquitination for an integral membrane ubiquitin ligase in ERAD.

Basila Moochickal Assainar, Kaushik Ragunathan, Ryan D Baldridge.

The endoplasmic reticulum associated degradation (ERAD) pathway regulates protein quality control at the endoplasmic reticulum. ERAD of lumenal and membrane proteins requires a conserved E3 ubiquitin ligase, called Hrd1. We do not understand the molecular configurations of Hrd1 that enable autoubiquitination and the subsequent retrotranslocation of misfolded protein substrates from the ER to the cytosol. Here, we have established a generalizable, single-molecule platform that enables high-efficiency labeling, stoichiometry determination, and functional assays for any integral membrane protein. Using this approach, we directly count Hrd1 proteins reconstituted into individual proteoliposomes. We report that Hrd1 assembles in different oligomeric configurations with mostly monomers and dimers detected at limiting dilution. By correlating oligomeric states with ubiquitination in vitro, we conclude that Hrd1 monomers are inefficient in autoubiquitination while dimers efficiently assemble polyubiquitin chains. Therefore, our results reveal the minimal composition of a Hrd1 oligomer that is capable of autoubiquitination. Our methods are broadly applicable to studying other complex membrane protein functions using reconstituted bilayer systems.

DOI: https://doi.org/10.1038/s41467-024-45541-3
J Mol Biol. 2024 Feb 14. pii: S0022-2836(24)00068-8. [Epub ahead of print] 168496

Mechanisms of translation-coupled quality control.

Toshifumi Inada, Roland Beckmann.

Stalling of ribosomes engaged in protein synthesis can lead to significant defects in the function of newly synthesized proteins and thereby impair protein homeostasis. Consequently, partially synthesized polypeptides resulting from translation stalling are recognized and eliminated by several quality control mechanisms. First, if translation elongation reactions are halted prematurely, a quality control mechanism called ribosome-associated quality control (RQC) initiates the ubiquitination of the nascent polypeptide chain and subsequent proteasomal degradation. Additionally, when ribosomes with defective codon recognition or peptide-bond formation stall during translation, a quality control mechanism known as non-functional ribosomal RNA decay (NRD) leads to the degradation of malfunctioning ribosomes. In both of these quality control mechanisms, E3 ubiquitin ligases selectively recognize ribosomes in distinct translation-stalling states and ubiquitinate specific ribosomal proteins. Significant efforts have been devoted to characterize E3 ubiquitin ligase sensing of ribosome 'collision' or 'stalling' and subsequent ribosome is rescued. This article provides an overview of our current understanding of the molecular mechanisms and physiological functions of ribosome dynamics control and quality control of abnormal translation.

DOI: https://doi.org/10.1016/j.jmb.2024.168496
Autophagy. 2024 Feb 15. 1-2

Reticulophagy mediated by the V-ATPase-ATG16L1-LC3C axis.

Yiwei Sun, Xi Wang.

  The lysosomal degradation of the endoplasmic reticulum (ER), known as "reticulophagy", is important for protein quality control and organelle turnover. Here we present a noncanonical reticulophagy occurring at ER exit sites (ERESs) induced by the misfolded SERPINA1/α1-antitrypsin (AAT) mutant, Z-AAT. The accumulation of Z-AAT arrests ER-to-Golgi transport, and recruits V-ATPase and ATG16L1 to mediate LC3C decoration of ERESs. Consequently, the receptor RETREG1/FAM134B-2 is recruited by lipidated LC3C to initiate reticulophagy. Furthermore, the blockade of ER export acts as a universal signal to activate reticulophagy mediated by the V-ATPase-ATG16L1-LC3C axis. This study sheds light on the mechanism of how ERESs switch from ER export to reticulophagy for quality control.

Keywords:  ATG16L1; COPII; ER exit sites; SEC24C; V-ATPase; endoplasmic reticulum; protein quality control; reticulophagy; α1-antitrypsin

DOI:  https://doi.org/10.1080/15548627.2024.2317116
Nucleic Acids Res. 2024 Feb 15. pii: gkae087. [Epub ahead of print]

Ribosomal collision is not a prerequisite for ZNF598-mediated ribosome ubiquitination and disassembly of ribosomal complexes by ASCC.

Anna Miścicka, Alexander G Bulakhov, Kazushige Kuroha, Alexandra Zinoviev, Christopher U T Hellen, Tatyana V Pestova.

Ribosomal stalling induces the ribosome-associated quality control (RQC) pathway targeting aberrant polypeptides. RQC is initiated by K63-polyubiquitination of ribosomal protein uS10 located at the mRNA entrance of stalled ribosomes by the E3 ubiquitin ligase ZNF598 (Hel2 in yeast). Ubiquitinated ribosomes are dissociated by the ASC-1 complex (ASCC) (RQC-Trigger (RQT) complex in yeast). A cryo-EM structure of the ribosome-bound RQT complex suggested the dissociation mechanism, in which the RNA helicase Slh1 subunit of RQT (ASCC3 in mammals) applies a pulling force on the mRNA, inducing destabilizing conformational changes in the 40S subunit, whereas the collided ribosome acts as a wedge, promoting subunit dissociation. Here, using an in vitro reconstitution approach, we found that ribosomal collision is not a strict prerequisite for ribosomal ubiquitination by ZNF598 or for ASCC-mediated ribosome release. Following ubiquitination by ZNF598, ASCC efficiently dissociated all polysomal ribosomes in a stalled queue, monosomes assembled in RRL, in vitro reconstituted 80S elongation complexes in pre- and post-translocated states, and 48S initiation complexes, as long as such complexes contained ≥ 30-35 3'-terminal mRNA nt. downstream from the P site and sufficiently long ubiquitin chains. Dissociation of polysomes and monosomes both involved ribosomal splitting, enabling Listerin-mediated ubiquitination of 60S-associated nascent chains.

DOI: https://doi.org/10.1093/nar/gkae087
J Biol Chem. 2024 Feb 12. pii: S0021-9258(24)00129-7. [Epub ahead of print] 105753

Mono-ubiquitination empowers ubiquitin chain elongation.

Kenneth Wu, Robert J DeVita, Zhen-Qiang Pan.

  Ubiquitination often generates lysine 48-linked polyubiquitin chains that signal proteolytic destruction of the protein target. A significant subset of ubiquitination proceeds by a priming/extending mechanism, in which a substrate is first mono-ubiquitinated with a priming E2 conjugating enzyme or a set of E3 ARIH/E2 enzymes specific for priming. This is then followed by ubiquitin chain extension catalyzed by an E2 enzyme capable of elongation. This report provides further insights into the priming/extending mechanism. We employed reconstituted ubiquitination systems of substrates CK1α and β-catenin by Cullin-RING E3 ubiquitin ligases (CRLs) CRL4CRBN and CRL1βTrCP, respectively, in the presence of priming E2 UbcH5c and elongating E2 Cdc34b. We have established a new "Apyrase chase" strategy that uncouples priming from chain elongation, which allows accurate measurement of the decay rates of the ubiquitinated substrate with a defined chain length. Our work has revealed highly robust turnover of mono-ubiquitinated β-catenin that empowers efficient polyubiquitination. The results of competition experiments suggest that the interactions between the ubiquitinated β-catenin and CRL1βTrCP are highly dynamic. Moreover, ubiquitination of the ubiquitin-modified β-catenin appeared more resistant to inhibition by competitors than the unmodified substrate, suggesting tighter binding with CRL1βTrCP. These findings support a role for conjugated ubiquitin in enhancing interactions with E3.

Keywords:  Apyrase chase; Cullin-RING E3 ubiquitin ligase; Priming; Ubiquitin chain elongation; Ubiquitination

DOI:  https://doi.org/10.1016/j.jbc.2024.105753
Nat Commun. 2024 Feb 10. 15(1): 1266

Structure-guided engineering enables E3 ligase-free and versatile protein ubiquitination via UBE2E1.

Xiangwei Wu, Yunxiang Du, Lu-Jun Liang, Ruichao Ding, Tianyi Zhang, Hongyi Cai, Xiaolin Tian, Man Pan, Lei Liu.

Ubiquitination, catalyzed usually by a three-enzyme cascade (E1, E2, E3), regulates various eukaryotic cellular processes. E3 ligases are the most critical components of this catalytic cascade, determining both substrate specificity and polyubiquitination linkage specificity. Here, we reveal the mechanism of a naturally occurring E3-independent ubiquitination reaction of a unique human E2 enzyme UBE2E1 by solving the structure of UBE2E1 in complex with substrate SETDB1-derived peptide. Guided by this peptide sequence-dependent ubiquitination mechanism, we developed an E3-free enzymatic strategy SUE1 (sequence-dependent ubiquitination using UBE2E1) to efficiently generate ubiquitinated proteins with customized ubiquitinated sites, ubiquitin chain linkages and lengths. Notably, this strategy can also be used to generate site-specific branched ubiquitin chains or even NEDD8-modified proteins. Our work not only deepens the understanding of how an E3-free substrate ubiquitination reaction occurs in human cells, but also provides a practical approach for obtaining ubiquitinated proteins to dissect the biochemical functions of ubiquitination.

DOI: https://doi.org/10.1038/s41467-024-45635-y
Nat Commun. 2024 Feb 15. 15(1): 1382

Chp1 is a dedicated chaperone at the ribosome that safeguards eEF1A biogenesis.

Melania Minoia, Jany Quintana-Cordero, Katharina Jetzinger, Ilgin Eser Kotan, Kathryn Jane Turnbull, Michela Ciccarelli, Anna E Masser, Dorina Liebers, Eloïse Gouarin, Marius Czech, Vasili Hauryliuk, Bernd Bukau, Günter Kramer, Claes Andréasson.

Cotranslational protein folding depends on general chaperones that engage highly diverse nascent chains at the ribosomes. Here we discover a dedicated ribosome-associated chaperone, Chp1, that rewires the cotranslational folding machinery to assist in the challenging biogenesis of abundantly expressed eukaryotic translation elongation factor 1A (eEF1A). Our results indicate that during eEF1A synthesis, Chp1 is recruited to the ribosome with the help of the nascent polypeptide-associated complex (NAC), where it safeguards eEF1A biogenesis. Aberrant eEF1A production in the absence of Chp1 triggers instant proteolysis, widespread protein aggregation, activation of Hsf1 stress transcription and compromises cellular fitness. The expression of pathogenic eEF1A2 variants linked to epileptic-dyskinetic encephalopathy is protected by Chp1. Thus, eEF1A is a difficult-to-fold protein that necessitates a biogenesis pathway starting with dedicated folding factor Chp1 at the ribosome to protect the eukaryotic cell from proteostasis collapse.

DOI: https://doi.org/10.1038/s41467-024-45645-w
bioRxiv. 2024 Feb 02. pii: 2024.01.31.577760. [Epub ahead of print]

Activation of XBP1s attenuates disease severity in models of proteotoxic Charcot-Marie-Tooth type 1B.

Thierry Touvier, Francesca A Veneri, Anke Claessens, Cinzia Ferri, Rosa Mastrangelo, Noémie Sorgiati, Francesca Bianchi, Serena Valenzano, Ubaldo Del Carro, Cristina Rivellini, Phu Duong, Michael E Shy, Jeffery W Kelly, John Svaren, R Luke Wiseman, Maurizio D'Antonio.

Mutations in myelin protein zero (MPZ) are generally associated with Charcot-Marie-Tooth type 1B (CMT1B) disease, one of the most common forms of demyelinating neuropathy. Pathogenesis of some MPZ mutants, such as S63del and R98C, involves the misfolding and retention of MPZ in the endoplasmic reticulum (ER) of myelinating Schwann cells. To cope with proteotoxic ER-stress, Schwann cells mount an unfolded protein response (UPR) characterized by activation of the PERK, ATF6 and IRE1α/XBP1 pathways. Previous results showed that targeting the PERK UPR pathway mitigates neuropathy in mouse models of CMT1B; however, the contributions of other UPR pathways in disease pathogenesis remains poorly understood. Here, we probe the importance of the IRE1α/XBP1 signalling during normal myelination and in CMT1B. In response to ER stress, IRE1α is activated to stimulate the non-canonical splicing of Xbp1 mRNA to generate spliced Xbp1 ( Xbp1s ). This results in the increased expression of the adaptive transcription factor XBP1s, which regulates the expression of genes involved in diverse pathways including ER proteostasis. We generated mouse models where Xbp1 is deleted specifically in Schwann cells, preventing XBP1s activation in these cells. We observed that Xbp1 is dispensable for normal developmental myelination, myelin maintenance and remyelination after injury. However, Xbp1 deletion dramatically worsens the hypomyelination and the electrophysiological and locomotor parameters observed in young and adult CMT1B neuropathic animals. RNAseq analysis suggested that XBP1s exerts its adaptive function in CMT1B mouse models in large part via the induction of ER proteostasis genes. Accordingly, the exacerbation of the neuropathy in Xbp1 deficient mice was accompanied by upregulation of ER-stress pathways and of IRE1-mediated RIDD signaling in Schwann cells, suggesting that the activation of XBP1s via IRE1 plays a critical role in limiting mutant protein toxicity and that this toxicity cannot be compensated by other stress responses. Schwann cell specific overexpression of XBP1s partially re-established Schwann cell proteostasis and attenuated CMT1B severity in both the S63del and R98C mouse models. In addition, the selective, pharmacologic activation of IRE1α/XBP1 signaling ameliorated myelination in S63del dorsal root ganglia explants. Collectively, these data show that XBP1 has an essential adaptive role in different models of proteotoxic CMT1B neuropathy and suggest that activation of the IRE1α/XBP1 pathway may represent a therapeutic avenue in CMT1B and possibly for other neuropathies characterized by UPR activation.

DOI: https://doi.org/10.1101/2024.01.31.577760
Nat Commun. 2024 Feb 16. 15(1): 1440

SEL1L-HRD1 interaction is required to form a functional HRD1 ERAD complex.

Liangguang Leo Lin, Huilun Helen Wang, Brent Pederson, Xiaoqiong Wei, Mauricio Torres, You Lu, Zexin Jason Li, Xiaodan Liu, Hancheng Mao, Hui Wang, Linyao Elina Zhou, Zhen Zhao, Shengyi Sun, Ling Qi.

The SEL1L-HRD1 protein complex represents the most conserved branch of endoplasmic reticulum (ER)-associated degradation (ERAD). Despite recent advances in both mouse models and humans, in vivo evidence for the importance of SEL1L in the ERAD complex formation and its (patho-)physiological relevance in mammals remains limited. Here we report that SEL1L variant p.Ser658Pro (SEL1LS658P) is a pathogenic hypomorphic mutation, causing partial embryonic lethality, developmental delay, and early-onset cerebellar ataxia in homozygous mice carrying the bi-allelic variant. Biochemical analyses reveal that SEL1LS658P variant not only reduces the protein stability of SEL1L, but attenuates the SEL1L-HRD1 interaction, likely via electrostatic repulsion between SEL1L F668 and HRD1 Y30 residues. Proteomic screens of SEL1L and HRD1 interactomes reveal that SEL1L-HRD1 interaction is a prerequisite for the formation of a functional HRD1 ERAD complex, as SEL1L is required for the recruitment of E2 enzyme UBE2J1 as well as DERLIN to HRD1. These data not only establish the disease relevance of SEL1L-HRD1 ERAD, but also provide additional insight into the formation of a functional HRD1 ERAD complex.

DOI: https://doi.org/10.1038/s41467-024-45633-0
Mol Cell. 2024 Feb 06. pii: S1097-2765(24)00052-2. [Epub ahead of print]

The HRI branch of the integrated stress response selectively triggers mitophagy.

Yogaditya Chakrabarty, Zheng Yang, Hsiuchen Chen, David C Chan.

  To maintain mitochondrial homeostasis, damaged or excessive mitochondria are culled in coordination with the physiological state of the cell. The integrated stress response (ISR) is a signaling network that recognizes diverse cellular stresses, including mitochondrial dysfunction. Because the four ISR branches converge to common outputs, it is unclear whether mitochondrial stress detected by this network can regulate mitophagy, the autophagic degradation of mitochondria. Using a whole-genome screen, we show that the heme-regulated inhibitor (HRI) branch of the ISR selectively induces mitophagy. Activation of the HRI branch results in mitochondrial localization of phosphorylated eukaryotic initiation factor 2, which we show is sufficient to induce mitophagy. The HRI mitophagy pathway operates in parallel with the mitophagy pathway controlled by the Parkinson's disease related genes PINK1 and PARKIN and is mechanistically distinct. Therefore, HRI repurposes machinery that is normally used for translational initiation to trigger mitophagy in response to mitochondrial damage.

Keywords:  autophagy; integrated stress response; iron metabolism; mitochondria; mitophagy; organelle quality control

DOI:  https://doi.org/10.1016/j.molcel.2024.01.016
Mol Cell. 2024 Feb 02. pii: S1097-2765(24)00051-0. [Epub ahead of print]

Visualization of translation reorganization upon persistent ribosome collision stress in mammalian cells.

Juliette Fedry, Joana Silva, Mihajlo Vanevic, Stanley Fronik, Yves Mechulam, Emmanuelle Schmitt, Amédée des Georges, William James Faller, Friedrich Förster.

  Aberrantly slow ribosomes incur collisions, a sentinel of stress that triggers quality control, signaling, and translation attenuation. Although each collision response has been studied in isolation, the net consequences of their collective actions in reshaping translation in cells is poorly understood. Here, we apply cryoelectron tomography to visualize the translation machinery in mammalian cells during persistent collision stress. We find that polysomes are compressed, with up to 30% of ribosomes in helical polysomes or collided disomes, some of which are bound to the stress effector GCN1. The native collision interface extends beyond the in vitro-characterized 40S and includes the L1 stalk and eEF2, possibly contributing to translocation inhibition. The accumulation of unresolved tRNA-bound 80S and 60S and aberrant 40S configurations identifies potentially limiting steps in collision responses. Our work provides a global view of the translation machinery in response to persistent collisions and a framework for quantitative analysis of translation dynamics in situ.

Keywords:  cryoelectron tomography; initiation; polysome; ribosome collision; ribosome quality control; translation regulation

DOI:  https://doi.org/10.1016/j.molcel.2024.01.015
J Mol Biol. 2024 Feb 09. pii: S0022-2836(24)00061-5. [Epub ahead of print] 168489

The role of ATG9 vesicles in autophagosome biogenesis.

Elisabeth Holzer, Sascha Martens, Susanna Tulli.

  Autophagy mediates the degradation and recycling of cellular material in the lysosomal system. Dysfunctional autophagy is associated with a plethora of diseases including uncontrolled infections, cancer and neurodegeneration. In macroautophagy (hereafter autophagy) this material is encapsulated in double membrane vesicles, the autophagosomes, which form upon induction of autophagy. The precursors to autophagosomes, referred to as phagophores, first appear as small flattened membrane cisternae, which gradually enclose the cargo material as they grow. The assembly of phagophores during autophagy initiation has been a major subject of investigation over the past decades. A special focus has been ATG9, the only conserved transmembrane protein among the core machinery. The majority of ATG9 localizes to small Golgi-derived vesicles. Here we review the recent advances and breakthroughs regarding our understanding of how ATG9 and the vesicles it resides in serve to assemble the autophagy machinery and to establish membrane contact sites for autophagosome biogenesis. We also highlight open questions in the field that need to be addressed in the years to come.

Keywords:  ATG2; ATG9; Macroautophagy; endoplasmic reticulum; lipid synthesis; lipid transfer; membrane contact site; membrane traffic

DOI:  https://doi.org/10.1016/j.jmb.2024.168489
Cell Rep. 2024 Feb 09. pii: S2211-1247(24)00088-3. [Epub ahead of print]43(2): 113760

Human YKT6 forms priming complex with STX17 and SNAP29 to facilitate autophagosome-lysosome fusion.

Denghao Zheng, Mindan Tong, Shen Zhang, Yi Pan, Yanxiang Zhao, Qing Zhong, Xiaoxia Liu.

  Autophagy is crucial for degrading and recycling cellular components. Fusion between autophagosomes and lysosomes is pivotal, directing autophagic cargo to degradation. This process is driven by STX17-SNAP29-VAMP8 and STX7-SNAP29-YKT6 in mammalian cells. However, the interaction between STX17 and YKT6 and its significance remain to be revealed. In this study, we challenge the notion that STX17 and YKT6 function independently in autophagosome-lysosome fusion. YKT6, through its SNARE domain, forms a complex with STX17 and SNAP29 on autophagosomes, enhancing autophagy flux. VAMP8 displaces YKT6 from this complex, leading to the formation of the fusogenic complex STX17-SNAP29-VAMP8. We demonstrated that the YKT6-SNAP29-STX17 complex facilitates both lipid and content mixing driven by STX17-SNAP29-VAMP8, suggesting a priming role of YKT6 for efficient membrane fusion. Our results provide a potential regulation mechanism of autophagosome-lysosome fusion, highlighting the importance of YKT6 and its interactions with STX17 and SNAP29 in promoting autophagy flux.

Keywords:  CP: Cell biology; SNAREs; SXT17; YKT6; autophagosome-lysosome fusion; autophagy; autophagy flux; priming complex

DOI:  https://doi.org/10.1016/j.celrep.2024.113760
Nat Struct Mol Biol. 2024 Feb;31(2): 216-218

Breaking the K48-chain: linking ubiquitin beyond protein degradation.

Sanim Rahman, Cynthia Wolberger.

The discovery of ubiquitin conjugation to lysines and the role of K48-linked polyubiquitin in targeting substrates for proteasomal degradation was followed by revelation of non-degradative roles of ubiquitination and, more recently, of non-canonical covalent ubiquitin linkages. Here we summarize findings of the ever-expanding array of ubiquitin signals and their biological roles.

DOI: https://doi.org/10.1038/s41594-024-01221-w
EMBO J. 2024 Feb 15.

Shared and distinct mechanisms of UBA1 inactivation across different diseases.

Jason C Collins, Samuel J Magaziner, Maya English, Bakar Hassan, Xiang Chen, Nicholas Balanda, Meghan Anderson, Athena Lam, Sebastian Fernandez-Pol, Bernice Kwong, Peter L Greenberg, Benjamin Terrier, Mary E Likhite, Olivier Kosmider, Yan Wang, Nadine L Samara, Kylie J Walters, David B Beck, Achim Werner.

  Most cellular ubiquitin signaling is initiated by UBA1, which activates and transfers ubiquitin to tens of E2 enzymes. Clonally acquired UBA1 missense mutations cause an inflammatory-hematologic overlap disease called VEXAS (vacuoles, E1, X-linked, autoinflammatory, somatic) syndrome. Despite extensive clinical investigation into this lethal disease, little is known about the underlying molecular mechanisms. Here, by dissecting VEXAS-causing UBA1 mutations, we discovered that p.Met41 mutations alter cytoplasmic isoform expression, whereas other mutations reduce catalytic activity of nuclear and cytoplasmic isoforms by diverse mechanisms, including aberrant oxyester formation. Strikingly, non-p.Met41 mutations most prominently affect transthioesterification, revealing ubiquitin transfer to cytoplasmic E2 enzymes as a shared property of pathogenesis amongst different VEXAS syndrome genotypes. A similar E2 charging bottleneck exists in some lung cancer-associated UBA1 mutations, but not in spinal muscular atrophy-causing UBA1 mutations, which instead, render UBA1 thermolabile. Collectively, our results highlight the precision of conformational changes required for faithful ubiquitin transfer, define distinct and shared mechanisms of UBA1 inactivation in diverse diseases, and suggest that specific E1-E2 modules control different aspects of tissue differentiation and maintenance.

Keywords:  Lung Cancer in Never Smokers (LCINS); Spinal Muscular Atrophy (SMA); UBA1; Ubiquitin; VEXAS Syndrome

DOI:  https://doi.org/10.1038/s44318-024-00046-z
bioRxiv. 2024 Feb 01. pii: 2024.01.30.577830. [Epub ahead of print]

Mapping Stress-Responsive Signaling Pathways Induced by Mitochondrial Proteostasis Perturbations.

Nicole Madrazo, Zinia Khattar, Evan T Powers, Jessica D Rosarda, R Luke Wiseman.

Imbalances in mitochondrial proteostasis are associated with pathologic mitochondrial dysfunction implicated in etiologically-diverse diseases. This has led to considerable interest in defining the biological mechanisms responsible for regulating mitochondria in response to mitochondrial stress. Numerous stress responsive signaling pathways have been suggested to regulate mitochondria in response to proteotoxic stress, including the integrated stress response (ISR), the heat shock response (HSR), and the oxidative stress response (OSR). Here, we define the specific stress signaling pathways activated in response to mitochondrial proteostasis stress by monitoring the expression of sets of genes regulated downstream of each of these signaling pathways in published Perturb-seq datasets from K562 cells CRISPRi-depleted of individual mitochondrial proteostasis factors. Interestingly, we find that the ISR is preferentially activated in response to mitochondrial proteostasis stress, with no other pathway showing significant activation. Further expanding this study, we show that broad depletion of mitochondria-localized proteins similarly shows preferential activation of the ISR relative to other stress-responsive signaling pathways. These results both establish our gene set profiling approach as a viable strategy to probe stress responsive signaling pathways induced by perturbations to specific organelles and identify the ISR as the predominant stress-responsive signaling pathway activated in response to mitochondrial proteostasis disruption.

DOI: https://doi.org/10.1101/2024.01.30.577830
J Cell Biol. 2024 May 06. pii: e202211041. [Epub ahead of print]223(5):

Alternative mechanisms of Notch activation by partitioning into distinct endosomal domains.

Hideyuki Shimizu, Samira Hosseini-Alghaderi, Simon A Woodcock, Martin Baron.

Different membrane microdomain compositions provide unique environments that can regulate signaling receptor function. We identify microdomains on the endosome membrane of Drosophila endosomes, enriched in lipid-raft or clathrin/ESCRT-0, which are associated with Notch activation by distinct, ligand-independent mechanisms. Transfer of Notch between microdomains is regulated by Deltex and Suppressor of deltex ubiquitin ligases and is limited by a gate-keeper role for ESCRT complexes. Ubiquitination of Notch by Deltex recruits it to the clathrin/ESCRT-0 microdomain and enhances Notch activation by an ADAM10-independent/TRPML-dependent mechanism. This requirement for Deltex is bypassed by the downregulation of ESCRT-III. In contrast, while ESCRT-I depletion also activates Notch, it does so by an ADAM10-dependent/TRPML-independent mechanism and Notch is retained in the lipid raft-like microdomain. In the absence of such endosomal perturbation, different activating Notch mutations also localize to different microdomains and are activated by different mechanisms. Our findings demonstrate the interplay between Notch regulators, endosomal trafficking components, and Notch genetics, which defines membrane locations and activation mechanisms.

DOI: https://doi.org/10.1083/jcb.202211041
EMBO J. 2024 Feb 15.

Structural insights into the ubiquitylation strategy of the oligomeric CRL2FEM1B E3 ubiquitin ligase.

Zonglin Dai, Ling Liang, Weize Wang, Peng Zuo, Shang Yu, Yaqi Liu, Xuyang Zhao, Yishuo Lu, Yan Jin, Fangting Zhang, Dian Ding, Weiwei Deng, Yuxin Yin.

  Cullin-RING E3 ubiquitin ligase (CRL) family members play critical roles in numerous biological processes and diseases including cancer and Alzheimer's disease. Oligomerization of CRLs has been reported to be crucial for the regulation of their activities. However, the structural basis for its regulation and mechanism of its oligomerization are not fully known. Here, we present cryo-EM structures of oligomeric CRL2FEM1B in its unneddylated state, neddylated state in complex with BEX2 as well as neddylated state in complex with FNIP1/FLCN. These structures reveal that asymmetric dimerization of N8-CRL2FEM1B is critical for the ubiquitylation of BEX2 while FNIP1/FLCN is ubiquitylated by monomeric CRL2FEM1B. Our data present an example of the asymmetric homo-dimerization of CRL. Taken together, this study sheds light on the ubiquitylation strategy of oligomeric CRL2FEM1B according to substrates with different scales.

Keywords:  Cryo-EM; Cullin-RING E3 Ubiquitin Ligase; Oligomerization; Ubiquitylation

DOI:  https://doi.org/10.1038/s44318-024-00047-y
bioRxiv. 2024 Feb 04. pii: 2024.02.04.578682. [Epub ahead of print]

Ca 2+ -sensor ALG-2 engages ESCRTs to enhance lysosomal membrane resilience to osmotic stress.

Wei Chen, Madeline M Motsinger, Jiaqian Li, Kevin P Bohannon, Phyllis I Hanson.

Lysosomes are central players in cellular catabolism, signaling, and metabolic regulation. Cellular and environmental stresses that damage lysosomal membranes can compromise their function and release toxic content into the cytoplasm. Here, we examine how cells respond to osmotic stress within lysosomes. Using sensitive assays of lysosomal leakage and rupture, we examine acute effects of the cathepsin C-metabolized osmotic disruptant glycyl-L-phenylalanine 2-naphthylamide (GPN). Our findings reveal that widely used concentrations of GPN rupture only a small fraction of lysosomes, but surprisingly trigger Ca 2+ release from nearly all. Chelating cytoplasmic Ca 2+ using BAPTA makes lysosomes more likely to rupture under GPN-induced stress, suggesting that Ca 2+ plays a role in protecting or rapidly repairing lysosomal membranes. Mechanistically, we establish that GPN causes the Ca 2+ -sensitive protein Apoptosis Linked Gene-2 (ALG-2) and interacting ESCRT proteins to redistribute onto lysosomes, improving their resistance to membrane stress created by GPN as well as the lysosomotropic drug chlorpromazine. Furthermore, we show that activating the cation channel TRPML1, with or without blocking the endoplasmic reticulum Ca 2+ pump, creates local Ca 2+ signals that protect lysosomes from rupture by recruiting ALG-2 and ESCRTs without any membrane damage. These findings reveal that Ca 2+ , through ALG-2, helps bring ESCRTs to lysosomes to enhance their resilience and maintain organelle integrity in the face of osmotic stress.
SIGNIFICANCE: As the degradative hub of the cell, lysosomes are full of toxic content that can spill into the cytoplasm. There has been much recent interest in how cells sense and repair lysosomal membrane damage using ESCRTs and cholesterol to rapidly fix "nanoscale damage". Here, we extend understanding of how ESCRTs contribute by uncovering a preventative role of the ESCRT machinery. We show that ESCRTs, when recruited by the Ca 2+ -sensor ALG-2, play a critical role in stabilizing the lysosomal membrane against osmotically-induced rupture. This finding suggests that cells have mechanisms not just for repairing but also for actively protecting lysosomes from stress-induced membrane damage.

DOI: https://doi.org/10.1101/2024.02.04.578682
Nucleic Acids Res. 2024 Feb 16. pii: gkae101. [Epub ahead of print]

Phylogeny-linked occurrence of ribosome stalling on the mRNAs of Arabidopsis unfolded protein response factor bZIP60 orthologs in divergent plant species.

Tomoya Imamichi, Nao Kusumoto, Haruka Aoyama, Seidai Takamatsu, Yugo Honda, Shiori Muraoka, Yuka Hagiwara-Komoda, Yukako Chiba, Hitoshi Onouchi, Yui Yamashita, Satoshi Naito.

The bZIP60, XBP1 and HAC1 mRNAs encode transcription factors that mediate the unfolded protein response (UPR) in plants, animals and yeasts, respectively. Upon UPR, these mRNAs undergo unconventional cytoplasmic splicing on the endoplasmic reticulum (ER) to produce active transcription factors. Although cytoplasmic splicing is conserved, the ER targeting mechanism differs between XBP1 and HAC1. The ER targeting of HAC1 mRNA occurs before translation, whereas that of XBP1 mRNA involves a ribosome-nascent chain complex that is stalled when a hydrophobic peptide emerges from the ribosome; the corresponding mechanism is unknown for bZIP60. Here, we analyzed ribosome stalling on bZIP60 orthologs of plants. Using a cell-free translation system, we detected nascent peptide-mediated ribosome stalling during the translation elongation of the mRNAs of Arabidopsis, rice and Physcomitrium (moss) orthologs, and the termination-step stalling in the Selaginella (lycopod) ortholog, all of which occurred ∼50 amino acids downstream of a hydrophobic region. Transfection experiments showed that ribosome stalling contributes to cytoplasmic splicing in bZIP60u orthologs of Arabidopsis and Selaginella. In contrast, ribosome stalling was undetectable for liverwort, Klebsormidium (basal land plant), and green algae orthologs. This study highlights the evolutionary diversity of ribosome stalling and its contribution to ER targeting in plants.

DOI: https://doi.org/10.1093/nar/gkae101
Cell Metab. 2024 Feb 01. pii: S1550-4131(24)00011-1. [Epub ahead of print]

DGAT2 inhibition blocks SREBP-1 cleavage and improves hepatic steatosis by increasing phosphatidylethanolamine in the ER.

Shunxing Rong, Mingfeng Xia, Goncalo Vale, Simeng Wang, Chai-Wan Kim, Shili Li, Jeffrey G McDonald, Arun Radhakrishnan, Jay D Horton.

  Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step of triglyceride (TG) synthesis. DGAT2 deletion in mice lowers liver TGs, and DGAT2 inhibitors are under investigation for the treatment of fatty liver disease. Here, we show that DGAT2 inhibition also suppressed SREBP-1 cleavage, reduced fatty acid synthesis, and lowered TG accumulation and secretion from liver. DGAT2 inhibition increased phosphatidylethanolamine (PE) levels in the endoplasmic reticulum (ER) and inhibited SREBP-1 cleavage, while DGAT2 overexpression lowered ER PE concentrations and increased SREBP-1 cleavage in vivo. ER enrichment with PE blocked SREBP-1 cleavage independent of Insigs, which are ER proteins that normally retain SREBPs in the ER. Thus, inhibition of DGAT2 shunted diacylglycerol into phospholipid synthesis, increasing the PE content of the ER, resulting in reduced SREBP-1 cleavage and less hepatic steatosis. This study reveals a new mechanism that regulates SREBP-1 activation and lipogenesis that is independent of sterols and SREBP-2 in liver.

Keywords:  DGAT2; PE; SREBPs; diacylglycerol acyltransferase 2; hepatic steatosis; lipogenesis; phosphatidylethanolamine

DOI:  https://doi.org/10.1016/j.cmet.2024.01.011
J Biol Chem. 2024 Feb 12. pii: S0021-9258(24)00128-5. [Epub ahead of print] 105752

Modulation of Cullin-RING E3 ubiquitin ligase-dependent ubiquitination by small molecule compounds.

Kenneth Wu, Robert J DeVita, Zhen-Qiang Pan.

  Cullin (CUL)-RING (Really Interesting New Gene) E3 ubiquitin (Ub) ligases (CRLs) are the largest E3 family. The E3 CRL core ligase is a sub-complex formed by the CUL C-terminal domain bound with the ROC1/RBX1 RING finger protein, which acts as a hub that mediates and organizes multiple interactions with E2, Ub, Nedd8, and the ARIH family protein, thereby resulting in Ub transfer to the E3-bound substrate. This report describes modulation of CRL-dependent ubiquitination by small molecule compounds including KH-4-43, #33 and suramin, which target the CRL core ligases. We also show that both KH-4-43 and #33 inhibit the ubiquitination of CK1α by CRL4CRBN. However, either compound's inhibitory effect on this reaction is significantly reduced when a neddylated form of CRL4CRBN is used. On the other hand, both #33 and KH-4-43 inhibit the ubiquitination of β-catenin by CRL1β-TrCP and Nedd8-CRL1β-TrCP almost equally. Thus, neddylation of CRL1β-TrCP does not negatively impact the sensitivity to inhibition by #33 and KH-4-43. These findings suggest that the effects of neddylation to alter the sensitivity of CRL inhibition by KH-4-43/#33 is dependent upon the specific CRL type. Suramin, a compound that targets CUL's basic canyon, can effectively inhibit CRL1/4-dependent ubiquitination regardless of neddylation status, in contrast to the results observed with KH-4-43/#33. This observed differential drug sensitivity of KH-4-43/#33 appears to echo CUL-specific Nedd8 effects on CRLs as revealed by recent high resolution structural biology efforts. The highly diversified CRL core ligase structures may provide opportunities for specific targeting by small molecule modulators.

Keywords:  CRL core ligase; Cullin-RING E3 ubiquitin ligase; Nedd8 modification; Ubiquitination; small molecule modulators

DOI:  https://doi.org/10.1016/j.jbc.2024.105752
J Mol Biol. 2024 Feb 13. pii: S0022-2836(24)00064-0. [Epub ahead of print] 168492

Loss of Preproinsulin Interaction with Signal Recognition Particle Activates Protein Quality Control, Decreasing mRNA Stability.

Sarah C Miller, Elena B Tikhonova, Sarah M Hernandez, Jannette M Dufour, Andrey L Karamyshev.

  Many insulin gene variants alter the protein sequence and result in monogenic diabetes due to insulin insufficiency. However, the molecular mechanisms of various disease-causing mutations are unknown. Insulin is synthesized as preproinsulin containing a signal peptide (SP). SPs of secreted proteins are recognized by the signal recognition particle (SRP) or by another factor in a SRP-independent pathway. If preproinsulin uses SRP-dependent or independent pathways is still debatable. We demonstrate by the use of site-specific photocrosslinking that the SRP subunit, SRP54, interacts with the preproinsulin SP. Moreover, SRP54 depletion leads to the decrease of insulin mRNA and protein expression, supporting the involvement of the RAPP protein quality control in insulin biogenesis. RAPP regulates the quality of secretory proteins through degradation of their mRNA. We tested five disease-causing mutations in the preproinsulin SP on recognition by SRP and on their effects on mRNA and protein levels. We demonstrate that the effects of mutations are associated with their position in the SP and their severity. The data support diverse molecular mechanisms involved in the pathogenesis of these mutations. We show for the first time the involvement of the RAPP protein quality control pathway in insulin biogenesis that is implicated in the development of neonatal diabetes caused by the Leu13Arg mutation.

Keywords:  Protein secretion; Protein transport; Secretory proteins; Signal Recognition Particle (SRP); Signal peptide

DOI:  https://doi.org/10.1016/j.jmb.2024.168492
Nature. 2024 Feb 14.

A new family of bacterial ribosome hibernation factors.

Karla Helena-Bueno, Mariia Yu Rybak, Chinenye L Ekemezie, Rudi Sullivan, Charlotte R Brown, Charlotte Dingwall, Arnaud Baslé, Claudia Schneider, James P R Connolly, James N Blaza, Bálint Csörgő, Patrick J Moynihan, Matthieu G Gagnon, Chris H Hill, Sergey V Melnikov.

To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage1,2. In bacteria, two families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors have confounded their discovery3-6. Here, by combining cryogenic electron microscopy, genetics and biochemistry, we identify Balon, a new hibernation factor in the cold-adapted bacterium Psychrobacter urativorans. We show that Balon is a distant homologue of the archaeo-eukaryotic translation factor aeRF1 and is found in 20% of representative bacteria. During cold shock or stationary phase, Balon occupies the ribosomal A site in both vacant and actively translating ribosomes in complex with EF-Tu, highlighting an unexpected role for EF-Tu in the cellular stress response. Unlike typical A-site substrates, Balon binds to ribosomes in an mRNA-independent manner, initiating a new mode of ribosome hibernation that can commence while ribosomes are still engaged in protein synthesis. Our work suggests that Balon-EF-Tu-regulated ribosome hibernation is a ubiquitous bacterial stress-response mechanism, and we demonstrate that putative Balon homologues in Mycobacteria bind to ribosomes in a similar fashion. This finding calls for a revision of the current model of ribosome hibernation inferred from common model organisms and holds numerous implications for how we understand and study ribosome hibernation.

DOI: https://doi.org/10.1038/s41586-024-07041-8
J Mol Biol. 2024 Feb 08. pii: S0022-2836(24)00059-7. [Epub ahead of print] 168487

Synonymous mutations can alter protein dimerization through localized interface misfolding involving self-entanglements.

Pham Dang Lan, Daniel Allen Nissley, Ian Sitarik, Quyen V Vu, Yang Jiang, Philip To, Yingzi Xia, Stephen D Fried, Mai Suan Li, Edward P O'Brien.

  Synonymous mutations in messenger RNAs (mRNAs) can reduce protein-protein binding substantially without changing the protein's amino acid sequence. Here, we use coarse-grain simulations of protein synthesis, post-translational dynamics, and dimerization to understand how synonymous mutations can influence the dimerization of two E. coli homodimers, oligoribonuclease and ribonuclease T. We synthesize each protein from its wildtype, fastest- and slowest-translating synonymous mRNAs in silico and calculate the ensemble-averaged interaction energy between the resulting dimers. We find synonymous mutations alter oligoribonuclease's dimer properties. Relative to wildtype, the dimer interaction energy becomes 4% and 10% stronger, respectively, when translated from its fastest- and slowest-translating mRNAs. Ribonuclease T dimerization, however, is insensitive to synonymous mutations. The structural and kinetic origin of these changes are misfolded states containing non-covalent lasso-entanglements, many of which structurally perturb the dimer interface, and whose probability of occurrence depends on translation speed. These entangled states are kinetic traps that persist for long time scales. Entanglements cause altered dimerization energies for oligoribonuclease, as there is a large association (odds ratio: 52) between the co-occurrence of non-native self-entanglements and weak-binding dimer conformations. Simulated at all-atom resolution, these entangled structures persist for long timescales, indicating the conclusions are independent of model resolution. Finally, we show that regions of the protein we predict to have changes in entanglement are also structurally perturbed during refolding, as detected by limited-proteolysis mass spectrometry. Thus, non-native changes in entanglement at dimer interfaces is a mechanism through which oligomer structure and stability can be altered.

Keywords:  Function; Kinetics; mRNA; misfolding; non-equilibrium

DOI:  https://doi.org/10.1016/j.jmb.2024.168487
J Cell Sci. 2024 Feb 13. pii: jcs.261257. [Epub ahead of print]

ER stress impedes regulated secretion by governing key exocytotic and granulogenic molecular switches.

Mohima Mukherjee, Chandramouli Mukherjee, Vinayak Ghosh, Aamna Jain, Souren Sadhukhan, Sushma Dagar, Bhavani Shankar Sahu.

  Dense core vesicles (DCVs) and synaptic vesicles (SVs) are specialised secretory vesicles (SSVs) in neurons/neuroendocrine cells harbouring cargo whose abnormal release is associated with various pathophysiologies. Endoplasmic Reticulum (ER) stress and inter-organellar communication are also associated with disease biology. To investigate the functional status of regulated exocytosis arising from the crosstalk of a stressed ER and DCVs, ER stress was modelled in PC12 neuroendocrine cells using Thapsigargin (Tg). DCV exocytosis was severely compromised in ER-stressed PC12 cells, reversed to varied magnitudes by ER stress attenuators. Experiments with Tunicamycin (Tm), an independent ER stressor, yielded similar results. Concurrently, ER stress also caused impaired DCV exocytosis in INS-1 cells. Molecular analysis revealed blunted SNAP25 expression, potentially attributed to augmented levels of ATF4, an inhibitor of CREB that binds to the CREB binding site. The loss of function of ATF-4 experiments in ER-stressed cells substantiated this attribution. Our studies revealed severe defects in DCV exocytosis in ER-stressed cells for the first time, mediated by reduced levels of key 'exocytotic ' and 'granulogenic ' switches regulated via the eIF2α /ATF4 axis.

Keywords:  Chromogranin; Dense core vesicles; ER Stress; Regulated secretion; SNAREs; Synaptic vesicles

DOI:  https://doi.org/10.1242/jcs.261257
Nucleic Acids Res. 2024 Feb 16. pii: gkae119. [Epub ahead of print]

Nuclear RNA-related processes modulate the assembly of cytoplasmic RNA granules.

Mor Angel, Eden Fleshler, Mohammad Khaled Atrash, Noa Kinor, Jennifer I C Benichou, Yaron Shav-Tal.

Stress granules (SGs) are cytoplasmic assemblies formed under various stress conditions as a consequence of translation arrest. SGs contain RNA-binding proteins, ribosomal subunits and messenger RNAs (mRNAs). It is well known that mRNAs contribute to SG formation; however, the connection between SG assembly and nuclear processes that involve mRNAs is not well established. Here, we examine the effects of inhibiting mRNA transcription, splicing and export on the assembly of SGs and the related cytoplasmic P body (PB). We demonstrate that inhibition of mRNA transcription, splicing and export reduces the formation of canonical SGs in a eukaryotic initiation factor 2α phosphorylation-independent manner, and alters PB size and quantity. We find that the splicing inhibitor madrasin promotes the assembly of stress-like granules. We show that the addition of synthetic mRNAs directly to the cytoplasm is sufficient for SG assembly, and that the assembly of these SGs requires the activation of stress-associated protein synthesis pathways. Moreover, we show that adding an excess of mRNA to cells that do not have active splicing, and therefore have low levels of cytoplasmic mRNAs, promotes SG formation under stress conditions. These findings emphasize the importance of the cytoplasmic abundance of newly transcribed mRNAs in the assembly of SGs.

DOI: https://doi.org/10.1093/nar/gkae119
bioRxiv. 2024 Jan 30. pii: 2024.01.29.577729. [Epub ahead of print]

Understanding species-specific and conserved RNA-protein interactions in vivo and in vitro.

Sarah E Harris, Maria S Alexis, Gilbert Giri, Francisco F Cavazos, Jernej Murn, Maria M Aleman, Christopher B Burge, Daniel Dominguez.

While evolution is often considered from a DNA- and protein-centric view, RNA-based regulation can also impact gene expression and protein sequences. Here we examined interspecies differences in RNA-protein interactions using the conserved neuronal RNA binding protein, Unkempt (UNK) as model. We find that roughly half of mRNAs bound in human are also bound in mouse. Unexpectedly, even when transcript-level binding was conserved across species differential motif usage was prevalent. To understand the biochemical basis of UNK-RNA interactions, we reconstituted the human and mouse UNK-RNA interactomes using a high-throughput biochemical assay. We uncover detailed features driving binding, show that in vivo patterns are captured in vitro , find that highly conserved sites are the strongest bound, and associate binding strength with downstream regulation. Furthermore, subtle sequence differences surrounding motifs are key determinants of species-specific binding. We highlight the complex features driving protein-RNA interactions and how these evolve to confer species-specific regulation.

DOI: https://doi.org/10.1101/2024.01.29.577729
J Biol Chem. 2024 Feb 12. pii: S0021-9258(24)00122-4. [Epub ahead of print] 105746

Komagataella phaffii Erp41 is a protein disulfide isomerase with unprecedented disulfide bond catalyzing activity when coupled to glutathione.

Arianna Palma, Lukas A Rettenbacher, Antti Moilanen, Mirva Saaranen, Brigitte Gasser, Lloyd W Ruddock.

  In the methylotrophic yeast Komagataella phaffii, we identified an ER-resident protein disulfide isomerase (PDI) family member, Erp41, with a peculiar combination of active site motifs. Like fungal ERp38, it has two thioredoxin-like domains which contain active site motifs (a and a'), followed by an alpha-helical ERp29c C-terminal domain (c domain). However, while the a domain has a typical PDI-like active site motif (CGHC), the a' domain instead has CGYC, a glutaredoxin-like motif which confers to the protein an exceptional affinity for GSH/GSSG. This combination of active site motifs has so far been unreported in PDI-family members. Homology searches revealed ERp41 is present in the genome of some plants, fungal parasites and a few non-conventional yeasts, among which are Komagataella spp. and Yarrowia lipolytica. These yeasts are both used for the production of secreted recombinant proteins. Here, we analyzed the activity of K. phaffii Erp41. We report that it is non-essential in K. phaffii, and that it can catalyze disulfide bond formation in partnership with the sulfhydryl oxidase Ero1 in vitro with higher turnover rates than the canonical PDI from K. phaffii, Pdi1, but slower activation times. We show how Erp41 has unusually fast glutathione-coupled oxidation activity and relate it to its unusual combination of active sites in its thioredoxin-like domains. We further describe how this determines its unusually efficient catalysis of dithiol oxidation in peptide and protein substrates.

Keywords:  Disulfide; endoplasmic reticulum (ER); enzyme catalysis; glutathione; protein disulfide isomerase; protein folding; unfolded protein response (UPR); yeast

DOI:  https://doi.org/10.1016/j.jbc.2024.105746
Cell Rep. 2024 Feb 14. pii: S2211-1247(24)00092-5. [Epub ahead of print]43(2): 113764

Radiotherapy induces persistent innate immune reprogramming of microglia into a primed state.

Daniëlle C Voshart, Takuya Oshima, Yuting Jiang, Gideon P van der Linden, Anna P Ainslie, Luiza Reali Nazario, Fleur van Buuren-Broek, Ayla C Scholma, Hilmar R J van Weering, Nieske Brouwer, Jeffrey Sewdihal, Uilke Brouwer, Rob P Coppes, Inge R Holtman, Bart J L Eggen, Susanne M Kooistra, Lara Barazzuol.

  Over half of patients with brain tumors experience debilitating and often progressive cognitive decline after radiotherapy treatment. Microglia, the resident macrophages in the brain, have been implicated in this decline. In response to various insults, microglia can develop innate immune memory (IIM), which can either enhance (priming or training) or repress (tolerance) the response to subsequent inflammatory challenges. Here, we investigate whether radiation affects the IIM of microglia by irradiating the brains of rats and later exposing them to a secondary inflammatory stimulus. Comparative transcriptomic profiling and protein validation of microglia isolated from irradiated rats show a stronger immune response to a secondary inflammatory insult, demonstrating that radiation can lead to long-lasting molecular reprogramming of microglia. Transcriptomic analysis of postmortem normal-appearing non-tumor brain tissue of patients with glioblastoma indicates that radiation-induced microglial priming is likely conserved in humans. Targeting microglial priming or avoiding further inflammatory insults could decrease radiotherapy-induced neurotoxicity.

Keywords:  CP: Cancer; CP: Immunology; brain tumor; innate immune memory; microglia; microglial priming; neuroinflammation; radiotherapy

DOI:  https://doi.org/10.1016/j.celrep.2024.113764
Antioxid Redox Signal. 2024 Feb 17.

DAMPening tumor immune escape: the role of ER chaperones in immunogenic chemotherapy.

Selma Cifric, Marcello Turi, Pietro Folino, Cole Clericuzio, Francesca Barello, Tallya Maciel, Kenneth Carl Anderson, Annamaria Gulla.

Preclinical and clinical research in the past two decades has redefined the mechanism of action of some chemotherapeutics that are able to activate the immune system against cancer when cell death is perceived by the immune cells. This immunogenic cell death (ICD) activates antigen-presenting cells (APCs) and T cells to induce immune-mediated tumor clearance. One of the key requirements to achieve this effect is the externalization of the danger-associated molecular patterns (DAMPs), molecules released or exposed by cancer cells during ICD that increase the visibility of the cancer cells by the immune system. In this review, we focus on the role of calreticulin (CRT) and other ER chaperones, such as the heat-shock proteins (HSPs) and the protein disulfide isomerases (PDIs) as surface-exposed DAMPs. Once exposed on the cell membrane, these proteins shift their role from that of ER chaperone and regulator of Ca2+ and protein homeostasis to act as an immunogenic signal for APCs, driving DC-mediated phagocytosis and T-mediated anti-tumor response. However, cancer cells exploit several mechanisms of resistance to immune attack, including subverting the exposure of ER chaperones on their surface to avoid immune recognition. Overcoming these mechanisms of resistance represents a potential therapeutic opportunity to improve cancer treatment effectiveness and patient outcomes.

DOI: https://doi.org/10.1089/ars.2024.0558