bims-proteo 2024-11-24 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2024–11–24
thirty-six papers selected by
Eric Chevet, INSERM

PINK1 controls RTN3L-mediated ER autophagy by regulating peripheral tubule junctions.
Multiprotein bridging factor 1 is required for robust activation of the integrated stress response on collided ribosomes.
Excess dietary sodium restores electrolyte and water homeostasis caused by loss of the endoplasmic reticulum molecular chaperone, GRP170, in the mouse nephron.
Specific tRNAs promote mRNA decay by recruiting the CCR4-NOT complex to translating ribosomes.
Identification of phosphatases that dephosphorylate the co-chaperone BAG3.
CDR2 is a dynein adaptor recruited by kinectin to regulate ER sheet organization.
Structural landscape of AAA+ ATPase motor states in the substrate-degrading human 26S proteasome reveals conformation-specific binding of TXNL1.
Dominant-negative TP53 mutations potentiated by the HSF1-regulated proteostasis network.
STING induces HOIP-mediated synthesis of M1 ubiquitin chains to stimulate NF-κB signaling.
Autophagic stress activates distinct compensatory secretory pathways in neurons.
Human cartilage model of the precocious osteoarthritis-inducing COL2A1 p.Arg719Cys reveals pathology-driving matrix defects and a failure of the ER proteostasis network to recognize the defective procollagen-II.
p53 engagement is a hallmark of an unfolded protein response in the nucleus of mammalian cells.
Development of luciferase-based highly sensitive reporters that detect ER-associated protein biogenesis abnormalities.
Mutations in unfolded protein response regulator ATF6 cause hearing and vision loss syndrome.
Chronic stress antagonizes formation of Stress Granules.
A multi-subunit autophagic capture complex facilitates degradation of ER stalled MHC-I in pancreatic cancer.
Coordinated translational control of multiple immune checkpoints by the integrated stress response pathway in lung cancer.
Phagolysosomes break down the membrane of a non-apoptotic corpse independent of macroautophagy.
Protocol for detecting and quantifying hATF4-HA in non-stress versus stress conditions using automated and quantitative Jess western blotting.
m6A sites in the coding region trigger translation-dependent mRNA decay.
Dominant negative mutations in yeast Hsp90 indicate triage decision mechanism targeting client proteins for degradation.
Mechanism of NACHO-mediated assembly of pentameric ligand-gated ion channels.
Unremodeled GPI-anchored proteins at the plasma membrane trigger aberrant endocytosis.
Directed evolution of the multicopper oxidase laccase for cell surface proximity labeling and electron microscopy.
Modulation of stress granule dynamics by phosphorylation and ubiquitination in plants.
Interaction with the cysteine-free protein HAX1 expands the substrate specificity and function of MIA40 beyond protein oxidation.
An E2 ubiquitin-conjugating enzyme links diubiquitinated H2B to H3K27M oncohistone function.
Allostery can convert binding free energies into concerted domain motions in enzymes.
Proximity proteomics provides a new resource for exploring the function of Afadin and the complexity of cell-cell adherens junctions.
Visualizing ER-phagy and ER architecture in vivo.
Spc2 modulates substrate- and cleavage site-selection in the yeast signal peptidase complex.
O-GlcNAcylation modulates expression and abundance of N-glycosylation machinery in an inherited glycosylation disorder.
Autoinflammatory patients with Golgi-trapped CDC42 exhibit intracellular trafficking defects leading to STING hyperactivation and ER stress.
Chemical glycoproteomic profiling in rice seedlings reveals N-glycosylation in the ERAD-L machinery.
Spatiotemporal recruitment of the ubiquitin-specific protease USP8 directs endosome maturation.
Nutrient control of growth and metabolism through mTORC1 regulation of mRNA splicing.

J Cell Biol. 2024 Dec 02. pii: e202407193. [Epub ahead of print]223(12):

PINK1 controls RTN3L-mediated ER autophagy by regulating peripheral tubule junctions.

Ravi Chidambaram, Kamal Kumar, Smriti Parashar, Gowsalya Ramachandran, Shuliang Chen, Susan Ferro-Novick.

Here, we report that the RTN3L-SEC24C endoplasmic reticulum autophagy (ER-phagy) receptor complex, the CUL3KLHL12 E3 ligase that ubiquitinates RTN3L, and the FIP200 autophagy initiating protein, target mutant proinsulin (Akita) condensates for lysosomal delivery at ER tubule junctions. When delivery was blocked, Akita condensates accumulated in the ER. In exploring the role of tubulation in these events, we unexpectedly found that loss of the Parkinson's disease protein, PINK1, reduced peripheral tubule junctions and blocked ER-phagy. Overexpression of the PINK1 kinase substrate, DRP1, increased junctions, reduced Akita condensate accumulation, and restored lysosomal delivery in PINK1-depleted cells. DRP1 is a dual-functioning protein that promotes ER tubulation and severs mitochondria at ER-mitochondria contact sites. DRP1-dependent ER tubulating activity was sufficient for suppression. Supporting these findings, we observed PINK1 associating with ER tubules. Our findings show that PINK1 shapes the ER to target misfolded proinsulin for RTN3L-SEC24C-mediated macro-ER-phagy at defined ER sites called peripheral junctions. These observations may have important implications for understanding Parkinson's disease.

DOI: https://doi.org/10.1083/jcb.202407193
Mol Cell. 2024 Nov 13. pii: S1097-2765(24)00869-4. [Epub ahead of print]

Multiprotein bridging factor 1 is required for robust activation of the integrated stress response on collided ribosomes.

Kyusik Q Kim, Jeffrey J Li, Ankanahalli N Nanjaraj Urs, Miguel E Pacheco, Victor Lasehinde, Timo Denk, Petr Tesina, Shota Tomomatsu, Yoshitaka Matsuo, Elesa McDonald, Roland Beckmann, Toshifumi Inada, Rachel Green, Hani S Zaher.

  In yeast, multiprotein bridging factor 1 (Mbf1) has been proposed to function in the integrated stress response (ISR) as a transcriptional coactivator by mediating a direct interaction between general transcription machinery and the process's key effector, Gcn4. However, mounting evidence has demonstrated that Mbf1 (and its human homolog EDF1) is recruited to collided ribosomes, a known activator of the ISR. In this study, we connect these otherwise seemingly disparate functions of Mbf1. Our biochemical and structural analyses reveal that Mbf1 functions as a core ISR factor by interacting with collided ribosomes to mediate Gcn2 activation. We further show that Mbf1 serves no role as a transcriptional coactivator of Gcn4. Instead, Mbf1 is required for optimal stress-induced eukaryotic initiation factor 2α (eIF2α) phosphorylation and downstream de-repression of GCN4 translation. Collectively, our data establish that Mbf1 functions in ISR signaling by acting as a direct sensor of stress-induced ribosome collisions.

Keywords:  Gcn2; Gcn4; Mbf1; integrated stress response; ribosome; ribosome collisions; translation

DOI:  https://doi.org/10.1016/j.molcel.2024.10.029
Am J Physiol Renal Physiol. 2024 Nov 18.

Excess dietary sodium restores electrolyte and water homeostasis caused by loss of the endoplasmic reticulum molecular chaperone, GRP170, in the mouse nephron.

Aidan W Porter, Hannah E Vorndran, Allison Marciszyn, Stephanie M Mutchler, Arohan R Subramanya, Thomas R Kleyman, Linda M Hendershot, Jeffrey L Brodsky, Teresa M Buck.

The maintenance of fluid and electrolyte homeostasis by the kidney requires proper folding and trafficking of ion channels and transporters in kidney epithelia. Each of these processes requires a specific subset of a diverse class of proteins termed molecular chaperones. One such chaperone is GRP170, which is an Hsp70-like, endoplasmic reticulum (ER)-localized chaperone that plays roles in protein quality control and protein folding in the ER. We previously determined that loss of GRP170 in the mouse nephron leads to hypovolemia, electrolyte imbalance, and rapid weight loss. In addition, GRP170-deficient mice develop an AKI-like phenotype, typified by tubular injury, elevation of kidney injury markers, and induction of the unfolded protein response (UPR). By using an inducible GRP170 knockout cellular model, we confirmed that GRP170 depletion induces the UPR, triggers apoptosis, and disrupts protein homeostasis. Based on these data, we hypothesized that UPR induction underlies hyponatremia and volume depletion in these rodents, and that these and other phenotypes might be rectified by sodium supplementation. To test this hypothesis, control and GRP170 tubule-specific knockout mice were provided a diet containing 8% sodium chloride. We discovered that sodium supplementation improved electrolyte imbalance and kidney injury markers in a sex-specific manner but was unable to restore weight or tubule integrity. These results are consistent with UPR induction contributing to the kidney injury phenotype in the nephron-specific GR170 knockout model and indicate that GRP170 function in kidney epithelia is essential to both maintain electrolyte balance and ER homeostasis.

Keywords: AKI; Chaperone; GRP170; UPR; proteostasis
Science. 2024 Nov 22. 386(6724): eadq8587

Specific tRNAs promote mRNA decay by recruiting the CCR4-NOT complex to translating ribosomes.

Xiaoqiang Zhu, Victor Emmanuel Cruz, He Zhang, Jan P Erzberger, Joshua T Mendell.

The CCR4-NOT complex is a major regulator of eukaryotic messenger RNA (mRNA) stability. Slow decoding during translation promotes association of CCR4-NOT with ribosomes, accelerating mRNA degradation. We applied selective ribosome profiling to further investigate the determinants of CCR4-NOT recruitment to ribosomes in mammalian cells. This revealed that specific arginine codons in the P-site are strong signals for ribosomal recruitment of human CNOT3, a CCR4-NOT subunit. Cryo-electron microscopy and transfer RNA (tRNA) mutagenesis demonstrated that the D-arms of select arginine tRNAs interact with CNOT3 and promote its recruitment whereas other tRNA D-arms sterically clash with CNOT3. These effects link codon content to mRNA stability. Thus, in addition to their canonical decoding function, tRNAs directly engage regulatory complexes during translation, a mechanism we term P-site tRNA-mediated mRNA decay.

DOI: https://doi.org/10.1126/science.adq8587
Life Sci Alliance. 2025 Feb;pii: e202402734. [Epub ahead of print]8(2):

Identification of phosphatases that dephosphorylate the co-chaperone BAG3.

Thomas Kokot, Johannes P Zimmermann, Yamini Chand, Fabrice Krier, Lena Reimann, Laura Scheinost, Nico Höfflin, Alessandra Esch, Jörg Höhfeld, Bettina Warscheid, Maja Köhn.

The co-chaperone BAG3 plays critical roles in maintaining cellular proteostasis. It associates with 14-3-3 proteins during the trafficking of aggregation-prone proteins and facilitates their degradation through chaperone-assisted selective autophagy in cooperation with small heat shock proteins. Although reversible phosphorylation regulates BAG3 function, the involved phosphatases remain unknown. Here, we used affinity purification mass spectrometry to identify phosphatases that target BAG3. Of the hits, we evaluated the involvement of protein phosphatase-1 (PP1) using chemical inhibitors and activators in in vitro and cellular approaches. Our results demonstrate that PP1 can dephosphorylate BAG3-pS136 in cells and counteract 14-3-3γ association with BAG3 at this motif. Furthermore, protein phosphatase-5 (PP5) co-enriched with proteostasis-related proteins, and it has the capacity to dephosphorylate a BAG3 phosphorylation-site cluster regulating the interaction of BAG3 with small heat shock proteins and BAG3-mediated protein degradation. Our findings provide new insights into the regulation of BAG3 by phosphatases. This paves the way for future research focused on the precise control of BAG3 function through its regulatory proteins, potentially holding therapeutic promise for diseases characterized by disrupted proteostasis.

DOI: https://doi.org/10.26508/lsa.202402734
bioRxiv. 2024 Nov 06. pii: 2024.11.06.622207. [Epub ahead of print]

CDR2 is a dynein adaptor recruited by kinectin to regulate ER sheet organization.

Vanessa Teixeira, Kashish Singh, José B Gama, Ricardo Celestino, Ana Xavier Carvalho, Paulo Pereira, Carla M C Abreu, Tiago J Dantas, Andrew P Carter, Reto Gassmann.

The endoplasmic reticulum (ER) relies on the microtubule cytoskeleton for distribution and re-modelling of its extended membrane network, but how microtubule-based motors contribute to ER organization remains unclear. Using biochemical and cell-based assays, we identify cerebellar degeneration-related protein 2 (CDR2) and its paralog CDR2-like (CDR2L), onconeural antigens with poorly understood functions, as ER adaptors for cytoplasmic dynein-1 (dynein). We demonstrate that CDR2 is recruited by the integral ER membrane protein kinectin (KTN1) and that double knockout of CDR2 and CDR2L enhances KTN1-dependent ER sheet stacking, reversal of which by exogenous CDR2 requires its dynein-binding CC1 box motif. Exogenous CDR2 expression additionally promotes CC1 box-dependent clustering of ER sheets near centrosomes. CDR2 competes with the eEF1Bβ subunit of translation elongation factor 1 for binding to KTN1, and eEF1Bβ knockdown increases endogenous CDR2 levels on ER sheets, inducing their centrosome-proximal clustering. Our study describes a novel molecular pathway that implicates dynein in ER sheet organization and may be involved in the pathogenesis of paraneoplastic cerebellar degeneration.

DOI: https://doi.org/10.1101/2024.11.06.622207
bioRxiv. 2024 Nov 09. pii: 2024.11.08.622731. [Epub ahead of print]

Structural landscape of AAA+ ATPase motor states in the substrate-degrading human 26S proteasome reveals conformation-specific binding of TXNL1.

Connor Arkinson, Christine L Gee, Zeyuan Zhang, Ken C Dong, Andreas Martin.

The 26S proteasome targets many cellular proteins for degradation during general homeostasis, protein quality control, and the regulation of vital processes. A broad range of proteasome-interacting cofactors thereby modulates these functions and aids in substrate degradation. Here, we solved several high-resolution structures of the redox active cofactor TXNL1 bound to the human 26S proteasome at saturating and sub-stoichiometric concentrations by time resolved cryo-EM. We identified distinct binding modes of TXNL1 that depend on the proteasome conformational and ATPase motor states. Together with biophysical and biochemical experiments, our structural studies reveal that the resting-state proteasome prior to substrate engagement with the ATPase motor binds TXNL1 with low affinity and in variable positions on top of the Rpn11 deubiquitinase. In contrast, the actively degrading proteasome shows additional interactions leading to high-affinity TXNL1 binding, whereby TXNL1's C-terminal tail covers the catalytic groove of the Rpn11 deubiquitinase and coordinates the active-site Zn 2+ . Furthermore, these cryo-EM structures of the degrading proteasome capture the ATPase hexamer in all registers of spiral-staircase arrangements and thus visualize the complete ATP-hydrolysis cycle of the AAA+ motor, indicating temporally asymmetric hydrolysis and conformational changes in bursts during mechanical substrate unfolding and translocation. Remarkably, we catch the proteasome in the act of unfolding the beta-barrel mEos3.2 substrate while the ATPase hexamer is in a particular spiral staircase register. Our findings challenge current models for protein translocation through hexameric AAA+ motors and reveal how the proteasome uses its distinct but broad range of conformational states to coordinate cofactor binding and substrate processing.
Highlights: High resolution structures of the redox active cofactor TXNL1 in complex with the human 26S proteasome solved by time-resolved cryo-EM.TXNL1 binds the catalytic groove of the main proteasomal deubiquitinase Rpn11 and coordinates its active-site Zinc specifically in substrate-degrading states of the proteasome.Visualizing a partially unfolded intermediate of the mEos model substrate during processing.Structures of the actively degrading human proteasome reveal all spiral-staircase registers of the AAA+ ATPase hexamer with unexpected nucleotide occupancies that indicate asymmetric ATP hydrolysis mechanisms, conformational changes with burst phases, and thus new models for hand-over-hand substrate translocation.

DOI: https://doi.org/10.1101/2024.11.08.622731
bioRxiv. 2024 Nov 03. pii: 2024.11.01.621414. [Epub ahead of print]

Dominant-negative TP53 mutations potentiated by the HSF1-regulated proteostasis network.

Rebecca M Sebastian, Jessica E Patrick, Tiffani Hui, David R Amici, Andrew O Giacomelli, Vincent L Butty, William C Hahn, Marc L Mendillo, Yu-Shan Lin, Matthew D Shoulders.

Protein mutational landscapes are sculpted by the impacts of the resulting amino acid substitutions on the proteins stability and folding or aggregation kinetics. These properties can, in turn, be modulated by the composition and activities of the cellular proteostasis network. Heat shock factor 1 (HSF1) is the master regulator of the cytosolic and nuclear proteostasis networks, dynamically tuning the expression of cytosolic and nuclear chaperones and quality control factors to meet demand. Chronic increases in HSF1 levels and activity are prominent hallmarks of cancer cells. One plausible explanation for this observation is that the consequent upregulation of proteostasis factors could biophysically facilitate the acquisition of oncogenic mutations. Here, we experimentally evaluate the impacts of chronic HSF1 activation on the mutational landscape accessible to the quintessential oncoprotein p53. Specifically, we apply quantitative deep mutational scanning of p53 to assess how HSF1 activation shapes the mutational pathways by which p53 can escape cytotoxic pressure conferred by the small molecule nutlin-3, which is a potent antagonist of the p53 negative regulator MDM2. We find that activation of HSF1 broadly increases the fitness of dominant-negative substitutions within p53. This effect of HSF1 activation was particularly notable for non-conservative, biophysically unfavorable amino acid substitutions within buried regions of the p53 DNA-binding domain. These results indicate that chronic HSF1 activation profoundly shapes the oncogenic mutational landscape, preferentially supporting the acquisition of cancer-associated substitutions that are biophysically destabilizing. Along with providing the first experimental and quantitative insights into how HSF1 influences oncoprotein mutational spectra, these findings also implicate HSF1 inhibition as a strategy to reduce the accessibility of mutations that drive chemo-therapeutic resistance and metastasis.

DOI: https://doi.org/10.1101/2024.11.01.621414
EMBO J. 2024 Nov 22.

STING induces HOIP-mediated synthesis of M1 ubiquitin chains to stimulate NF-κB signaling.

Tara D Fischer, Eric N Bunker, Peng-Peng Zhu, François Le Guerroué, Mahan Hadjian, Eunice Dominguez-Martin, Francesco Scavone, Robert Cohen, Tingting Yao, Yan Wang, Achim Werner, Richard J Youle.

  STING activation by cyclic dinucleotides induces IRF3- and NF-κB-mediated gene expression in mammals, as well as lipidation of LC3B at Golgi-related membranes. While mechanisms of the IRF3 response are well understood, the mechanisms of NF-κB activation via STING remain unclear. We report here that STING activation induces linear/M1-linked ubiquitin chain (M1-Ub) formation and recruitment of the LUBAC E3 ligase, HOIP, to LC3B-associated Golgi membranes where ubiquitin is also localized. Loss of HOIP prevents formation of M1-Ub chains and reduces STING-induced NF-κB and IRF3 signaling in human THP1 monocytes and mouse bone marrow-derived macrophages, without affecting STING activation. STING-induced LC3B lipidation is not required for M1-Ub chain formation or for immune-related gene expression, but the recently reported STING function in neutralizing Golgi pH may be involved. Thus, LUBAC synthesis of M1-linked ubiquitin chains mediates STING-induced innate immune signaling.

Keywords:  Golgi; Innate Immunity; LC3B; LUBAC; NFkB

DOI:  https://doi.org/10.1038/s44318-024-00291-2
bioRxiv. 2024 Nov 07. pii: 2024.11.07.621551. [Epub ahead of print]

Autophagic stress activates distinct compensatory secretory pathways in neurons.

Sierra D Palumbos, Jacob Popolow, Juliet Goldsmith, Erika L F Holzbaur.

Autophagic dysfunction is a hallmark of neurodegenerative disease, leaving neurons vulnerable to the accumulation of damaged organelles and proteins. However, the late onset of diseases suggests that compensatory quality control mechanisms may be engaged to delay the deleterious effects induced by compromised autophagy. Neurons expressing common familial Parkinson's disease (PD)-associated mutations in LRRK2 kinase exhibit defective autophagy. Here, we demonstrate that both primary murine neurons and human iPSC-derived neurons harboring pathogenic LRRK2 upregulate the secretion of extracellular vesicles. We used unbiased proteomics to characterize the secretome of LRRK2 G2019S neurons and found that autophagic cargos including mitochondrial proteins were enriched. Based on these observations, we hypothesized that autophagosomes are rerouted toward secretion when cell-autonomous degradation is compromised, likely to mediate clearance of undegraded cellular waste. Immunoblotting confirmed the release of autophagic cargos and immunocytochemistry demonstrated that secretory autophagy was upregulated in LRRK2 G2019S neurons. We also found that LRRK2 G2019S neurons upregulate the release of exosomes containing miRNAs. Live-cell imaging confirmed that this upregulation of exosomal release was dependent on hyperactive LRRK2 activity, while pharmacological experiments indicate that this release staves off apoptosis. Finally, we show that markers of both vesicle populations are upregulated in plasma from mice expressing pathogenic LRRK2. In sum, we find that neurons expressing pathogenic LRRK2 upregulate the compensatory release of secreted autophagosomes and exosomes, to mediate waste disposal and transcellular communication, respectively. We propose that this increased secretion contributes to the maintenance of cellular homeostasis, delaying neurodegenerative disease progression over the short term while potentially contributing to increased neuroinflammation over the longer term.
SIGNIFICANCE: A hallmark feature of many neurodegenerative diseases is autophagy dysfunction, resulting in the accumulation of damaged proteins and organelles that is detrimental to neuronal health. The late onset of neurodegenerative diseases, however, suggests alternative quality control mechanisms may delay neuronal degeneration. Here, we demonstrate that neurons expressing a Parkinson's Disease-causing mutation upregulate the release of two extracellular vesicle populations. First, we observe the increased expulsion of secreted autophagosomes to mediate cellular waste disposal. Second, we observe the increased release of exosomes, likely to facilitate transcellular communication. Thus, we propose that increases in secretory autophagy and exosome release are a homeostatic response in neurons undergoing chronic stress.

DOI: https://doi.org/10.1101/2024.11.07.621551
bioRxiv. 2024 Nov 09. pii: 2024.11.07.622468. [Epub ahead of print]

Human cartilage model of the precocious osteoarthritis-inducing COL2A1 p.Arg719Cys reveals pathology-driving matrix defects and a failure of the ER proteostasis network to recognize the defective procollagen-II.

Kathryn M Yammine, Sophia Mirda Abularach, Michael Xiong, Seo-Yeon Kim, Agata A Bikovtseva, Vincent L Butty, Richard P Schiavoni, John F Bateman, Shireen R Lamandé, Matthew D Shoulders.

Objectives: Mutations in the procollagen-II gene ( COL2A1 ) often cause chondrodysplasias, including the precocious osteoarthritis-inducing p.Arg719Cys. Understanding the molecular basis of such diseases has long been challenging, owing to a lack of models accurately reflecting disease genotypes and phenotypes. To address this challenge, we develop and characterize in vitro human cartilage derived from wild-type and disease-causing Arg719Cys COL2A1 isogenic induced pluripotent stem cell (iPSC) lines.
Methods: Using directed differentiation of iPSCs to chondrocytes, we generated cartilage from wild-type and Arg719Cys COL2A1 lines. We compared the resulting protein, cell, and tissue properties using immunohistochemistry, electron microscopy, SDS-PAGE, RNA-sequencing, and quantitative interactomics.
Results: While both wild-type and disease lines deposited a cartilage matrix, the Arg719Cys matrix was deficient. Arg719Cys collagen-II was excessively post-translationally modified and modestly intracellularly retained, leading to endoplasmic reticulum (ER) distention suggestive of an ER storage defect. Interactomic studies indicated that Arg719Cys procollagen-II was not differentially engaged by the ER proteostasis network. RNA-sequencing showed that the ER storage defect engendered by Arg719Cys procollagen-II also did not activate cellular stress responses, including the unfolded protein response. These data suggest that cells fail to properly recognize Arg719Cys-associated procollagen-II defects.
Conclusions: A failure to identify and rectify defective procollagen-II folding in cells expressing Arg719Cys procollagen-II leads to the deposition of a sparse and defective collagen-II matrix, culminating in pathology. Combined with the highly expandable human cartilage disease model reported here, this work provides motivation and a platform to discover therapeutic strategies targeting procollagen folding, quality control, and secretion in this collagenopathy and others.
KEY MESSAGES: What is already known on this topic: The p.Arg719Cys substitution in procollagen-II ( COL2A1 ) causes precocious generalized osteoarthritis with mild chondrodysplasia, inherited in an autosomal dominant fashion. Previous studies of recombinant procollagen-II indicate that this pathologic substitution disrupts fibril formation in vitro . In homozygous transgenic mice expressing the human allele, this substitution disrupts growth plate organization and collagen-II fibril density. However, the molecular etiology of pathology remains unknown and has not been explored in relevant human models that properly represent the underlying genetics of this disorder. What this study adds: We compare human iPSC-derived cartilage from Arg719Cys procollagen-II expressing chondrocytes and an isogenic wild-type control to identify defects in procollagen-II, the chondrocytes expressing it, and the matrices they deposit.We observe phenotypes consistent with those described in patients and mouse models, including a sparse collagen-II network in the extracellular matrix and distended ER. We also observe that Arg719Cys procollagen-II is slow to fold and accumulates intracellularly, an ER storage defect that likely propagates to a pathologically insufficient matrix.RNA-sequencing and quantitative interactomic analyses reveal that this ER storage defect does not activate cellular stress responses, such as the unfolded protein response.The ER proteostasis network does not detectably recognize the intracellularly retained Arg719Cys procollagen-II.The cell's failure to identify the underlying folding defect likely leads to deposition of a defective matrix, and that defective matrix ultimately culminates in disease phenotypes.How this study might affect research, practice, or policy: This study uses human iPSC-derived cartilage to characterize the disease phenotype and test hypotheses regarding the molecular etiology of this disease. The results set the stage to explore collagen proteostasis-associated pathways as novel therapeutic targets. Moreover, the iPSC-derived system provides an ideal platform for pre-clinical testing and development of therapeutic strategies that benefit patients with this collagenopathy, and that could also be more broadly applied to other collagen-related diseases.

DOI: https://doi.org/10.1101/2024.11.07.622468
bioRxiv. 2024 Nov 08. pii: 2024.11.08.622663. [Epub ahead of print]

p53 engagement is a hallmark of an unfolded protein response in the nucleus of mammalian cells.

Joseph H Park, Thomas J Wandless.

Exposure to exogenous and endogenous stress is associated with the intracellular accumulation of aberrant unfolded and misfolded proteins. In eukaryotic cells, protein homeostasis within membrane-bound organelles is regulated by specialized signaling pathways, with the unfolded protein response in the endoplasmic reticulum serving as a foundational example. Yet, it is unclear if a similar surveillance mechanism exists in the nucleus. Here we leveraged engineered proteins called destabilizing domains to acutely expose mammalian cells to nuclear- or cytosolic- localized unfolded protein. We show that the appearance of unfolded protein in either compartment engages a common transcriptional response associated with the transcription factors Nrf1 and Nrf2. Uniquely, only in the nucleus does unfolded protein activate a robust p53-driven transcriptional response and a transient p53-independent cell cycle delay. These studies highlight the distinct effects of localized protein folding stress and the unique protein quality control environment of the nucleus.

DOI: https://doi.org/10.1101/2024.11.08.622663
iScience. 2024 Nov 15. 27(11): 111189

Development of luciferase-based highly sensitive reporters that detect ER-associated protein biogenesis abnormalities.

Hiroshi Kadokura, Nanshi Harada, Satoshi Yamaki, Naoya Hirai, Ryusuke Tsukuda, Kota Azuma, Yuta Amagai, Daisuke Nakamura, Kota Yanagitani, Hideki Taguchi, Kenji Kohno, Kenji Inaba.

  Localization to the endoplasmic reticulum (ER) and subsequent disulfide bond formation are crucial processes governing the biogenesis of secretory pathway proteins in eukaryotes. Hence, comprehending the mechanisms underlying these processes is important. Here, we have engineered firefly luciferase (FLuc) as a tool to detect deficiencies in these processes within mammalian cells. To achieve this, we introduced multiple cysteine substitutions into FLuc and targeted it to the ER. The reporter exhibited FLuc activity in response to defects in protein localization or disulfide bond formation within the ER. Notably, this system exhibited outstanding sensitivity, reproducibility, and convenience in detecting abnormalities in these processes. We applied this system to observe a protein translocation defect induced by an inhibitor of HIV receptor biogenesis. Moreover, utilizing the system, we showed that modulating LMF1 levels dramatically impacted the ER's redox environment, confirming that LMF1 plays some critical role in the redox control of the ER.

Keywords:  Bioengineering; Cell biology; Protein structure aspects

DOI:  https://doi.org/10.1016/j.isci.2024.111189
J Clin Invest. 2024 Nov 21. pii: e175562. [Epub ahead of print]

Mutations in unfolded protein response regulator ATF6 cause hearing and vision loss syndrome.

Eun-Jin Lee, Kyle Kim, Monica Sophia Diaz-Aguilar, Hyejung Min, Eduardo Chavez, Korina J Steinbergs, Lance A Safarta, Guirong Zhang, Allen F Ryan, Jonathan H Lin.

  Activating transcription factor 6 (Atf6) is a key regulator of the unfolded protein response (UPR) and is important for endoplasmic reticulum (ER) function and protein homeostasis in metazoan cells. Patients carrying loss-of-function ATF6 disease alleles develop the cone dysfunction disorder, achromatopsia. The impact of loss of ATF6 function on other cell types, organs, and diseases in people remains unclear. Here, we reported that progressive sensorineural hearing loss was a notable complaint in some patients carrying ATF6 disease alleles and that Atf6-/- mice also showed progressive auditory deficits affecting both genders. In mice with hearing deficits, we found disorganized stereocilia on hair cells and focal loss of outer hair cells. Transcriptomic analysis of Atf6-/- cochleae revealed marked induction of UPR, especially through the PERK arm. These findings identify ATF6 as an essential regulator of cochlear health and function. Furthermore, they supported that ATF6 inactivation in people causes progressive sensorineural hearing loss as part of a blindness-deafness genetic syndrome targeting hair cells and cone photoreceptors. Lastly, our genetic findings support ER stress as an important pathomechanism underlying cochlear damage and hearing loss with clinical implications for patient lifestyle modifications that minimize environmental/physiologic sources of ER stress to the ear.

Keywords:  Genetic diseases; Genetics; Molecular genetics; Ophthalmology; Retinopathy

DOI:  https://doi.org/10.1172/JCI175562
bioRxiv. 2024 Nov 03. pii: 2024.10.29.620814. [Epub ahead of print]

Chronic stress antagonizes formation of Stress Granules.

Yuichiro Adachi, Allison M Williams, Masashi Masuda, Yutaka Taketani, Paul J Anderson, Pavel Ivanov.

Chronic stress mediates cellular changes that can contribute to human disease. However, fluctuations in RNA metabolism caused by chronic stress have been largely neglected in the field. Stress granules (SGs) are cytoplasmic ribonucleoprotein condensates formed in response to stress-induced inhibition of mRNA translation and polysome disassembly. Despite the broad interest in SG assembly and disassembly in response to acute stress, SG assembly in response to chronic stress has not been extensively investigated. In this study, we show that cells pre-conditioned with low dose chronic (24-hour exposure) stresses such as oxidative stress, endoplasmic reticulum stress, mitochondrial stress, and starvation, fail to assemble SGs in response to acute stress. While translation is drastically decreased by acute stress in pre-conditioned cells, polysome profiling analysis reveals the partial preservation of polysomes resistant to puromycin-induced disassembly. We showed that chronic stress slows down the rate of mRNA translation at the elongation phase and triggers phosphorylation of translation elongation factor eEF2. Polysome profiling followed by RNase treatment confirmed that chronic stress induces ribosome stalling. Chronic stress-induced ribosome stalling is distinct from ribosome collisions that are known to trigger a specific stress response pathway. In summary, chronic stress triggers ribosome stalling, which blocks polysome disassembly and SG formation by subsequent acute stress.
Significant statements: Stress granules (SGs) are dynamic cytoplasmic biocondensates assembled in response to stress-induced inhibition of mRNA translation and polysome disassembly. SGs have been proposed to contribute to the survival of cells exposed to toxic conditions. Although the mechanisms of SG assembly and disassembly in the acute stress response are well understood, the role of SGs in modulating the response to chronic stress is unclear. Here, we show that human cells pre-conditioned with chronic stress fail to assemble SGs in response to acute stress despite inhibition of mRNA translation. Mechanistically, chronic stress induces ribosome stalling, which prevents polysome disassembly and subsequent SG formation. This finding suggests that chronically stressed or diseased human cells may have a dysfunctional SG response that could inhibit cell survival and promote disease.

DOI: https://doi.org/10.1101/2024.10.29.620814
bioRxiv. 2024 Oct 29. pii: 2024.10.27.620516. [Epub ahead of print]

A multi-subunit autophagic capture complex facilitates degradation of ER stalled MHC-I in pancreatic cancer.

Marine Berquez, Alexander L Li, Matthew A Luy, Anthony C Venida, Thomas O'Loughlin, Gilles Rademaker, Abhilash Barpanda, Jingjie Hu, Julian Yano, Arun Wiita, Luke A Gilbert, Peter M Bruno, Rushika M Perera.

Pancreatic ductal adenocarcinoma (PDA) evades immune detection partly via autophagic capture and lysosomal degradation of major histocompatibility complex class I (MHC-I). Why MHC-I is susceptible to capture via autophagy remains unclear. By synchronizing exit of proteins from the endoplasmic reticulum (ER), we show that PDAC cells display prolonged retention of MHC-I in the ER and fail to efficiently route it to the plasma membrane. A capture-complex composed of NBR1 and the ER-phagy receptor TEX264 facilitates targeting of MHC-I for autophagic degradation, and suppression of either receptor is sufficient to increase total levels and re-route MHC-I to the plasma membrane. Binding of MHC-I to the capture complex is linked to antigen presentation efficiency, as inhibiting antigen loading via knockdown of TAP1 or beta 2-Microglobulin led to increased binding between MHC-I and the TEX264-NBR1 capture complex. Conversely, expression of ER directed high affinity antigenic peptides led to increased MHC-I at the cell surface and reduced lysosomal degradation. A genome-wide CRISPRi screen identified NFXL1, as an ER-resident E3 ligase that binds to MHC-I and mediates its autophagic capture. High levels of NFXL1 are negatively correlated with MHC-I protein expression and predicts poor patient prognosis. These data highlight an ER resident capture complex tasked with sequestration and degradation of non-conformational MHC-I in PDAC cells, and targeting this complex has the potential to increase PDAC immunogenicity.

DOI: https://doi.org/10.1101/2024.10.27.620516
bioRxiv. 2024 Oct 28. pii: 2024.10.23.619897. [Epub ahead of print]

Coordinated translational control of multiple immune checkpoints by the integrated stress response pathway in lung cancer.

Shayna Thomas-Jardin, Shruthy Suresh, Ariana Arce, Nicole Novaresi, Emily Stein, Lisa Thomas, Cheryl Lewis, Chul Ahn, Bret M Evers, Maria E Salvatierra, Wei Lui, Khaja Khan, Luisa Maris Solis Soto, Ignacio Wistuba, John D Minna, Kathryn A O'Donnell.

The integrated stress response (ISR) is an adaptive pathway hijacked by cancer cells to survive cellular stresses in the tumor microenvironment. ISR activation potently induces Programmed Death Ligand 1 (PD-L1), leading to suppression of anti-tumor immunity. Here we sought to uncover additional immune checkpoint proteins regulated by the ISR to elucidate mechanisms of tumor immune escape. We show that CD155 and PD-L1 are coordinately induced by the ISR, enhancing translation of both immune checkpoint proteins through bypass of inhibitory upstream open reading frames (uORFs) in their 5' UTRs. Analysis of primary human lung tumors identifies a significant correlation between PD-L1 and CD155 expression. ISR activation accelerates tumorigenesis and inhibits T cell function, effects that can be overcome by combining PD-1 blockade with the ISR inhibitor ISRIB. These studies uncover a novel mechanism by which two immune checkpoint proteins are coordinately regulated and suggest a new therapeutic strategy for lung cancer patients.
Statement of Significance: This study uncovers a novel mechanism for the coordinated translational regulation of the PD-L1/PD1 and CD155/TIGIT immune checkpoint pathways and highlights the ISR as a therapeutic vulnerability for lung cancer. Inhibition of the ISR pathway bolsters PD-1 blockade, potentially unveiling a new therapeutic strategy for lung cancer patients.

DOI: https://doi.org/10.1101/2024.10.23.619897
PLoS One. 2024 ;19(11): e0306435

Phagolysosomes break down the membrane of a non-apoptotic corpse independent of macroautophagy.

Shruti Kolli, Cassidy J Kline, Kimya M Rad, Ann M Wehman.

Cell corpses must be cleared in an efficient manner to maintain tissue homeostasis and regulate immune responses. Ubiquitin-like Atg8/LC3 family proteins promote the degradation of membranes and internal cargo during both macroautophagy and corpse clearance, raising the question how macroautophagy contributes to corpse clearance. Studying the clearance of non-apoptotic dying polar bodies in Caenorhabditis elegans embryos, we show that the LC3 ortholog LGG-2 is enriched inside the polar body phagolysosome independent of autophagosome formation. We demonstrate that ATG-16.1 and ATG-16.2, which promote membrane association of lipidated Atg8/LC3 proteins, redundantly promote polar body membrane breakdown in phagolysosomes independent of their role in macroautophagy. We also show that the lipid scramblase ATG-9 is needed for autophagosome formation in early embryos but is dispensable for timely polar body membrane breakdown or protein cargo degradation. These findings demonstrate that macroautophagy is not required to promote polar body degradation, in contrast to recent findings with apoptotic corpse clearance in C. elegans embryos. Determining how factors regulating Atg8/LC3 promote the breakdown of different types of cell corpses in distinct cell types or metabolic states is likely to give insights into the mechanisms of immunoregulation during normal development, physiology, and disease.

DOI: https://doi.org/10.1371/journal.pone.0306435
STAR Protoc. 2024 Nov 20. pii: S2666-1667(24)00619-1. [Epub ahead of print]5(4): 103454

Protocol for detecting and quantifying hATF4-HA in non-stress versus stress conditions using automated and quantitative Jess western blotting.

Anna M Smirnova, Anna Herrmannová, Leoš Shivaya Valášek.

  Activating transcription factor 4 (ATF4) is a key player in the integrated stress response, whose expression is subject to tight translational control. Studying its stress-provoked induction, accompanied by the general translational shutdown, is intricate because the expression of reference genes declines rapidly, and finding appropriate normalization controls is challenging. We present a protocol for human hemagglutinin-tagged ATF4 (hATF4-HA) detection and high-throughput quantification in non-stress versus stress conditions using automated and quantitative western blotting. We describe steps for seeding cells, transfecting plasmids, thapsigargin treatment, sample preparation, and target protein detection. For complete details on the use and execution of this protocol, please refer to Smirnova et al.1.

Keywords:  cell culture; gene expression; protein expression and purification

DOI:  https://doi.org/10.1016/j.xpro.2024.103454
Mol Cell. 2024 Nov 19. pii: S1097-2765(24)00873-6. [Epub ahead of print]

m6A sites in the coding region trigger translation-dependent mRNA decay.

You Zhou, Miona Ćorović, Peter Hoch-Kraft, Nathalie Meiser, Mikhail Mesitov, Nadine Körtel, Hannah Back, Isabel S Naarmann-de Vries, Kritika Katti, Aleš Obrdlík, Anke Busch, Christoph Dieterich, Štěpánka Vaňáčová, Martin Hengesbach, Kathi Zarnack, Julian König.

  N6-Methyladenosine (m6A) is the predominant internal RNA modification in eukaryotic messenger RNAs (mRNAs) and plays a crucial role in mRNA stability. Here, using human cells, we reveal that m6A sites in the coding sequence (CDS) trigger CDS-m6A decay (CMD), a pathway that is distinct from previously reported m6A-dependent degradation mechanisms. Importantly, CDS m6A sites act considerably faster and more efficiently than those in the 3' untranslated region, which to date have been considered the main effectors. Mechanistically, CMD depends on translation, whereby m6A deposition in the CDS triggers ribosome pausing and transcript destabilization. The subsequent decay involves the translocation of the CMD target transcripts to processing bodies (P-bodies) and recruitment of the m6A reader protein YT521-B homology domain family protein 2 (YTHDF2). Our findings highlight CMD as a previously unknown pathway, which is particularly important for controlling the expression of developmental regulators and retrogenes.

Keywords:  P-bodies; RNA decay; RNA modification; YTHDF2; coding sequence; m6A; ribosomal A site; ribosome pausing; translation

DOI:  https://doi.org/10.1016/j.molcel.2024.10.033
Mol Biol Cell. 2024 Nov 20. mbcE24070309

Dominant negative mutations in yeast Hsp90 indicate triage decision mechanism targeting client proteins for degradation.

Julia M Flynn, Margot E Joyce, Daniel N A Bolon.

Dominant negative mutations provide valuable tools for investigating protein mechanisms but can be difficult to isolate because of their toxic effects. We used a mutational scanning approach to identify dominant negative (DN) mutations in yeast Hsp90. In a previous mutational scan of the ATPase domain of Hsp90, we noticed that many mutations were at very low frequency after outgrowth in cells co-expressing WT Hsp90. Most of these depleted variants were located at the hinge of a lid that closes over ATP. To quantify toxic effects in the hinge regions, we performed mutational scanning using an inducible promoter and identified 113 variants with strong toxic effects. We analyzed individual DN mutations in detail and found that addition of the E33A mutation that prevents ATP hydrolysis by Hsp90 abrogated the dominant negative phenotype. FRET assays performed on individual DN mutants indicate the linkage between ATPase activity and formation of the closed structure is disrupted. DN Hsp90 decreased the expression level of two model Hsp90 clients, glucocorticoid receptor (GR) and v-src kinase. Using MG132, we found that GR was rapidly destabilized in a proteasome-dependent fashion. Biochemical analyses indicate that ATP hydrolysis by Hsp90 from open conformations can lead to ubiquitin-dependent client degradation.

DOI: https://doi.org/10.1091/mbc.E24-07-0309
bioRxiv. 2024 Nov 15. pii: 2024.10.28.620708. [Epub ahead of print]

Mechanism of NACHO-mediated assembly of pentameric ligand-gated ion channels.

Yogesh Hooda, Andrija Sente, Ryan M Judy, Luka Smalinskaitė, Sew-Yeu Peak- Chew, Katerina Naydenova, Tomas Malinauskas, Steven W Hardwick, Dimitri Y Chirgadze, A Radu Aricescu, Ramanujan S Hegde.

Pentameric ligand-gated ion channels (pLGICs) are cell surface receptors of crucial importance for animal physiology 1-4 . This diverse protein family mediates the ionotropic signals triggered by major neurotransmitters and includes γ -aminobutyric acid receptors (GABA A Rs) and acetylcholine receptors (nAChRs). Receptor function is fine-tuned by a myriad of endogenous and pharmacological modulators 3 . A functional pLGIC is built from five homologous, sometimes identical, subunits, each containing a β-scaffold extracellular domain (ECD), a four-helix transmembrane domain (TMD) and intracellular loops of variable length. Although considerable progress has been made in understanding pLGICs in structural and functional terms, the molecular mechanisms that enable their assembly at the endoplasmic reticulum (ER) 5 in a vast range of potential subunit configurations 6 remain unknown. Here, we identified candidate pLGICs assembly factors selectively associated with an unassembled GABA A R subunit. Focusing on one of the candidates, we determined the cryo-EM structure of an assembly intermediate containing two α1 subunits of GABA A R each bound to an ER-resident membrane protein NACHO. The structure showed how NACHO shields the principal (+) transmembrane interface of α1 subunits containing an immature extracellular conformation. Crosslinking and structure-prediction revealed an adjacent surface on NACHO for β2 subunit interactions to guide stepwise oligimerisation. Mutations of either subunit-interacting surface on NACHO also impaired the formation of homopentameric α7 nAChRs, pointing to a generic framework for pLGIC assembly. Our work provides the foundation for understanding the regulatory principles underlying pLGIC structural diversity.

DOI: https://doi.org/10.1101/2024.10.28.620708
Life Sci Alliance. 2025 Feb;pii: e202402941. [Epub ahead of print]8(2):

Unremodeled GPI-anchored proteins at the plasma membrane trigger aberrant endocytosis.

Li Chen, David K Banfield.

The plasma membrane has a complex organization that includes the polarized distribution of membrane proteins and lipids. Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are ubiquitously expressed in eukaryotes and represent a functionally diverse, extensively remodeled, ER-derived group of proteins critical for the organization and function of the plasma membrane. Little is known about how the transport of incompletely remodeled GPI-APs to the plasma membrane affects cell function. Here, we investigated how failure to remodel mannose 2 (Man2) of the GPI moiety impacted endocytic activity on the plasma membrane. We find that Man2 unremodeled GPI-APs increased membrane disorder and generated a stress response that triggered abnormal ubiquitin- and clathrin-dependent endocytosis. The resulting stress-induced endocytosis disrupted the trafficking repertoire of a subset of plasma membrane proteins, which were redirected, via the multivesicular body, to numerous small vacuoles for degradation. Our findings highlight the critical importance of GPI-AP Man2 remodeling for maintaining the integrity and homeostasis of the plasma membrane.

DOI: https://doi.org/10.26508/lsa.202402941
bioRxiv. 2024 Oct 29. pii: 2024.10.29.620861. [Epub ahead of print]

Directed evolution of the multicopper oxidase laccase for cell surface proximity labeling and electron microscopy.

Song-Yi Lee, Heegwang Roh, David Gonzalez-Perez, Mason R Mackey, Keun-Young Kim, Daniel Hoces, Colleen N McLaughlin, Stephen R Adams, Khanh Nguyen, David J Luginbuhl, Liqun Luo, Namrata D Udeshi, Steven A Carr, Rogelio A Hernández-López, Mark H Ellisman, Miguel Alcalde, Alice Y Ting.

Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic H2O2. Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O2 instead of toxic H2O2, and exhibits activity selective to the surface plasma membrane of both living and fixed cells. We show that LaccID can be used with mass spectrometry-based proteomics to map the changing surface composition of T cells that engage with tumor cells via antigen-specific T cell receptors. In addition, we use LaccID as a genetically-encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID.

DOI: https://doi.org/10.1101/2024.10.29.620861
iScience. 2024 Nov 15. 27(11): 111162

Modulation of stress granule dynamics by phosphorylation and ubiquitination in plants.

Siou-Luan He, Xiling Wang, Sung-Il Kim, Liang Kong, Ailing Liu, Lei Wang, Ying Wang, Libo Shan, Ping He, Jyan-Chyun Jang.

  The Arabidopsis tandem CCCH zinc finger 1 (TZF1) is an RNA-binding protein that plays a pivotal role in plant growth and stress response. In this report, we show that TZF1 contains two intrinsically disordered regions necessary for its localization to stress granules (SGs). TZF1 recruits mitogen-activated protein kinase (MAPK) signaling components and an E3 ubiquitin ligase KEEP-ON-GOING (KEG) to SGs. TZF1 is phosphorylated by MPKs and ubiquitinated by KEG. Using a high throughput Arabidopsis protoplasts transient expression system, mutant studies reveal that the phosphorylation of specific residues plays differential roles in enhancing or reducing TZF1 SG assembly and protein-protein interaction with mitogen-activated kinase kinase 5 in SGs. Ubiquitination appears to play a positive role in TZF1 SG assembly, because mutations cause a reduction of typical SGs, while enhancing the assembly of large SGs encompassing the nucleus. Together, our results demonstrate that plant SG assembly is distinctively regulated by phosphorylation and ubiquitination.

Keywords:  Molecular biology; Plant Biology; Plant physiology

DOI:  https://doi.org/10.1016/j.isci.2024.111162
FEBS J. 2024 Nov 20.

Interaction with the cysteine-free protein HAX1 expands the substrate specificity and function of MIA40 beyond protein oxidation.

Robin Alexander Rothemann, Dylan Stobbe, Michaela Nicole Hoehne-Wiechmann, Lena Maria Murschall, Esra Peker, Lara Katharina Knaup, Julia Racho, Markus Habich, Sarah Gerlich, Kim Jasmin Lapacz, Kathrin Ulrich, Jan Riemer.

  The mitochondrial disulphide relay machinery is essential for the import and oxidative folding of many proteins in the mitochondrial intermembrane space. Its core component, the import receptor MIA40 (also CHCHD4), serves as an oxidoreductase but also as a chaperone holdase, which initially interacts with its substrates non-covalently before introducing disulphide bonds for folding and retaining proteins in the intermembrane space. Interactome studies have identified diverse substrates of MIA40, among them the intrinsically disordered HCLS1-associated protein X-1 (HAX1). Interestingly, this protein does not contain cysteines, raising the question of how and to what end HAX1 can interact with MIA40. Here, we demonstrate that MIA40 non-covalently interacts with HAX1 independent of its redox-active cysteines. While HAX1 import is driven by its weak mitochondrial targeting sequence, its subsequent transient interaction with MIA40 stabilizes the protein in the intermembrane space. HAX1 solely depends on the holdase activity of MIA40, and the absence of MIA40 results in the aggregation, degradation and loss of HAX1. Collectively, our study introduces HAX1 as the first endogenous MIA40 substrate without cysteines and demonstrates the diverse functions of this highly conserved oxidoreductase and import receptor.

Keywords:  HAX1; IMS; MIA40; mitochondria; mitochondrial disulphide relay

DOI:  https://doi.org/10.1111/febs.17328
Proc Natl Acad Sci U S A. 2024 Nov 26. 121(48): e2416614121

An E2 ubiquitin-conjugating enzyme links diubiquitinated H2B to H3K27M oncohistone function.

Alan L Jiao, Erdem Sendinc, Barry M Zee, Felice Wallner, Yang Shi.

  The H3K27M oncogenic histone (oncohistone) mutation drives ~80% of incurable childhood brain tumors known as diffuse midline gliomas (DMGs). The major molecular feature of H3K27M mutant DMGs is a global loss of H3K27 trimethylation (H3K27me3), a phenotype conserved in Caenorhabditis elegans (C. elegans). Here, we perform unbiased genome-wide suppressor screens in C. elegans expressing H3K27M and isolate 20 suppressors, all of which at least partially restore H3K27me3. 19/20 suppressor mutations map to the same histone H3.3 gene in which the K27M mutation was originally introduced. Most of these create single amino acid substitutions between residues R26-Y54, which do not disrupt oncohistone expression. Rather, they are predicted to impair interactions with the Polycomb Repressive Complex 2 (PRC2) and are functionally conserved in human cells. Further, we mapped a single extragenic H3K27M suppressor to ubc-20, an E2 ubiquitin-conjugating enzyme, whose loss rescued H3K27me3 to nearly 50% wild-type levels despite continued oncohistone expression and chromatin incorporation. We demonstrate that ubc-20 is the major enzyme responsible for generating diubiquitinated histone H2B. Our study provides in vivo support for existing models of PRC2 inhibition via direct oncohistone contact and suggests that the effects of H3K27M may be modulated by H2B ubiquitination.

Keywords:  C. elegans; H3K27M; Oncohistone; diffuse midline glioma; epigenetics

DOI:  https://doi.org/10.1073/pnas.2416614121
Nat Commun. 2024 Nov 22. 15(1): 10109

Allostery can convert binding free energies into concerted domain motions in enzymes.

Nicole Stéphanie Galenkamp, Sarah Zernia, Yulan B Van Oppen, Marco van den Noort, Andreas Milias Argeitis, Giovanni Maglia.

Enzymatic mechanisms are typically inferred from structural data. However, understanding enzymes require unravelling the intricate dynamic interplay between dynamics, conformational substates, and multiple protein structures. Here, we use single-molecule nanopore analysis to investigate the catalytic conformational changes of adenylate kinase (AK), an enzyme that catalyzes the interconversion of various adenosine phosphates (ATP, ADP, and AMP). Kinetic analysis validated by hidden Markov models unravels the details of domain motions during catalysis. Our findings reveal that allosteric interactions between ligands and cofactor enable converting binding energies into directional conformational changes of the two catalytic domains of AK. These coordinated motions emerged to control the exact sequence of ligand binding and the affinity for the three different substrates, thereby guiding the reactants along the reaction coordinates. Interestingly, we find that about 10% of enzymes show altered allosteric regulation and ligand affinities, indicating that a subset of enzymes folds in alternative catalytically active forms. Since molecules or proteins might be able to selectively stabilize one of the folds, this observation suggests an evolutionary path for allostery in enzymes. In AK, this complex catalytic framework has likely emerged to prevent futile ATP/ADP hydrolysis and to regulate the enzyme for different energy needs of the cell.

DOI: https://doi.org/10.1038/s41467-024-54421-9
bioRxiv. 2024 Nov 08. pii: 2024.11.07.622507. [Epub ahead of print]

Proximity proteomics provides a new resource for exploring the function of Afadin and the complexity of cell-cell adherens junctions.

Wangsun Choi, Dennis Goldfarb, Feng Yan, M Ben Major, Alan S Fanning, Mark Peifer.

The network of proteins at the interface between cell-cell adherens junctions and the actomyosin cytoskeleton provides robust yet dynamic connections that facilitate cell shape change and motility. While this was initially thought to be a simple linear connection via classic cadherins and their associated catenins, we now have come to appreciate that many more proteins are involved, providing robustness and mechanosensitivity. Defining the full network of proteins in this network remains a key objective in our field. Proximity proteomics provides a means to define these networks. Mammalian Afadin and its Drosophila homolog Canoe are key parts of this protein network, facilitating diverse cell shape changes during gastrulation and other events of embryonic morphogenesis. Here we report results of several proximity proteomics screens, defining proteins in the neighborhood of both the N- and C-termini of mammalian Afadin in the premier epithelial model, MDCK cells. We compare our results with previous screens done in other cell types, and with proximity proteomics efforts with other junctional proteins. These reveal the value of multiple screens in defining the full network of neighbors and offer interesting insights into the overlap in protein composition between different epithelial cell junctions.
Summary Statement: Afadin BioID reveals new adherens junction proteins.

DOI: https://doi.org/10.1101/2024.11.07.622507
J Cell Biol. 2024 Dec 02. pii: e202408061. [Epub ahead of print]223(12):

Visualizing ER-phagy and ER architecture in vivo.

Yongjuan Sang, Boran Li, Tinglin Su, Hanyu Zhan, Yue Xiong, Zhiming Huang, Changjing Wang, Xiaoxia Cong, Mengjie Du, Yang Wu, Hang Yu, Xi Yang, Kezhi Ding, Xuhua Wang, Xiaolong Miao, Weihua Gong, Liang Wang, Jingwei Zhao, Yiting Zhou, Wei Liu, Xinyang Hu, Qiming Sun.

ER-phagy is an evolutionarily conserved mechanism crucial for maintaining cellular homeostasis. However, significant gaps persist in our understanding of how ER-phagy and the ER network vary across cell subtypes, tissues, and organs. Furthermore, the pathophysiological relevance of ER-phagy remains poorly elucidated. Addressing these questions requires developing quantifiable methods to visualize ER-phagy and ER architecture in vivo. We generated two transgenic mouse lines expressing an ER lumen-targeting tandem RFP-GFP (ER-TRG) tag, either constitutively or conditionally. This approach enables precise spatiotemporal measurements of ER-phagy and ER structure at single-cell resolution in vivo. Systemic analysis across diverse organs, tissues, and primary cultures derived from these ER-phagy reporter mice unveiled significant variations in basal ER-phagy, both in vivo and ex vivo. Furthermore, our investigation uncovered substantial remodeling of ER-phagy and the ER network in different tissues under stressed conditions such as starvation, oncogenic transformation, and tissue injury. In summary, both reporter models represent valuable resources with broad applications in fundamental research and translational studies.

DOI: https://doi.org/10.1083/jcb.202408061
J Cell Biol. 2024 Dec 02. pii: e202211035. [Epub ahead of print]223(12):

Spc2 modulates substrate- and cleavage site-selection in the yeast signal peptidase complex.

Yeonji Chung, Chewon Yim, Gilberto P Pereira, Sungjoon Son, Lisbeth R Kjølbye, Lauren E Mazurkiewicz, Amy M Weeks, Friedrich Förster, Gunnar von Heijne, Paulo C T Souza, Hyun Kim.

Secretory proteins are critically dependent on the correct processing of their signal sequence by the signal peptidase complex (SPC). This step, which is essential for the proper folding and localization of proteins in eukaryotic cells, is still not fully understood. In eukaryotes, the SPC comprises four evolutionarily conserved membrane subunits (Spc1-3 and Sec11). Here, we investigated the role of Spc2, examining SPC cleavage efficiency on various models and natural signal sequences in yeast cells depleted of or with mutations in Spc2. Our data show that discrimination between substrates and identification of the cleavage site by SPC is compromised when Spc2 is absent or mutated. Molecular dynamics simulation of the yeast SPC AlphaFold2-Multimer model indicates that membrane thinning at the center of SPC is reduced without Spc2, suggesting a molecular explanation for the altered substrate recognition properties of SPC lacking Spc2. These results provide new insights into the molecular mechanisms by which SPC governs protein biogenesis.

DOI: https://doi.org/10.1083/jcb.202211035
Cell Rep. 2024 Nov 18. pii: S2211-1247(24)01327-5. [Epub ahead of print]43(11): 114976

O-GlcNAcylation modulates expression and abundance of N-glycosylation machinery in an inherited glycosylation disorder.

Courtney Matheny-Rabun, Sneha S Mokashi, Silvia Radenkovic, Kali Wiggins, Lynn Dukes-Rimsky, Peggi Angel, Bart Ghesquiere, Tamas Kozicz, Richard Steet, Eva Morava, Heather Flanagan-Steet.

  Core components of the N-glycosylation pathway are known, but the metabolic and post-translational mechanisms regulating this pathway in normal and disease states remain elusive. Using a multi-omic approach in zebrafish, we discovered a mechanism whereby O-GlcNAcylation directly impacts the expression and abundance of two rate-limiting proteins in the N-linked glycosylation pathway. We show in a model of an inherited glycosylation disorder PMM2-CDG, congenital disorders of glycosylation that phosphomannomutase deficiency is associated with increased levels of UDP-GlcNAc and protein O-GlcNAcylation. O-GlcNAc modification increases the transcript and protein abundance of both NgBR and Dpagt1 in pmm2m/m mutants. Modulating O-GlcNAc levels, NgBR abundance, or Dpagt1 activity exacerbated the cartilage phenotypes in pmm2 mutants, suggesting that O-GlcNAc-mediated increases in the N-glycosylation machinery are protective. These findings highlight nucleotide-sugar donors as metabolic sensors that regulate two spatially separated glycosylation pathways, demonstrating how their coordination is relevant to disease severity in the most common congenital disorder of glycosylation.

Keywords:  CDG; CP: Molecular biology; O-GlcNAc; disease modifiers; glycosylation; sugar metabolism; zebrafish

DOI:  https://doi.org/10.1016/j.celrep.2024.114976
Nat Commun. 2024 Nov 16. 15(1): 9940

Autoinflammatory patients with Golgi-trapped CDC42 exhibit intracellular trafficking defects leading to STING hyperactivation and ER stress.

Alberto Iannuzzo, Selket Delafontaine, Rana El Masri, Rachida Tacine, Giusi Prencipe, Masahiko Nishitani-Isa, Rogier T A van Wijck, Farzana Bhuyan, Adriana A de Jesus Rasheed, Simona Coppola, Paul L A van Daele, Antonella Insalaco, Raphaela Goldbach-Mansky, Takahiro Yasumi, Marco Tartaglia, Isabelle Meyts, Jérôme Delon.

Most autoinflammatory diseases are caused by mutations in innate immunity genes. Previously, four variants in the RHO GTPase CDC42 were discovered in patients affected by syndromes generally characterized by neonatal-onset of cytopenia and auto-inflammation, including hemophagocytic lymphohistiocytosis and rash in the most severe form (NOCARH syndrome). However, the mechanisms responsible for these phenotypes remain largely elusive. Here, we show that the recurrent p.R186C CDC42 variant, which is trapped in the Golgi apparatus, elicits a block in both anterograde and retrograde transports. Consequently, it favours STING accumulation in the Golgi in a COPI-dependent manner. This is also observed for the other Golgi-trapped p.*192 C*24 CDC42 variant, but not for the p.Y64C and p.C188Y variants that do not accumulate in the Golgi. We demonstrate that the two Golgi-trapped CDC42 variants are the only ones that exhibit overactivation of the STING pathway and the type I interferon response, and elicit endoplasmic reticulum stress. Consistent with these results, patients carrying Golgi-trapped CDC42 mutants present very high levels of circulating IFNα at the onset of their disease. In conclusion, we report further mechanistic insights on the impact of the Golgi-trapped CDC42 variants. This increase in STING activation provides a rationale for combination treatments for these severe cases.

DOI: https://doi.org/10.1038/s41467-024-54294-y
Mol Cell Proteomics. 2024 Nov 21. pii: S1535-9476(24)00173-7. [Epub ahead of print] 100883

Chemical glycoproteomic profiling in rice seedlings reveals N-glycosylation in the ERAD-L machinery.

Cong Lei, Xilong Li, Wenjia Li, Zihan Chen, Simiao Liu, Bo Cheng, Yili Hu, Qitao Song, Yahong Qiu, Yilan Zhou, Xiangbing Meng, Hong Yu, Wen Zhou, Xing Chen, Jiayang Li.

As a ubiquitous and essential posttranslational modification occurring in both plants and animals, protein N-linked glycosylation regulates various important biological processes. Unlike the well-studied animal N-glycoproteomes, the landscape of rice N-glycoproteome remains largely unexplored. Here, by developing a chemical glycoproteomic strategy based on metabolic glycan labeling (MGL), we report a comprehensive profiling of the N-glycoproteome in rice seedlings. The rice seedlings are incubated with N-azidoacetylgalactosamine (GalNAz) - a monosaccharide analog containing a bioorthogonal functional group - to metabolically label N-glycans, followed by conjugation with an affinity probe via click chemistry for enrichment of the N-glycoproteins. Subsequent mass spectrometry analyses identify a total of 403 N-glycosylation sites and 673 N-glycosylated proteins, which are involved in various important biological processes. In particular, the core components of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) machinery are N-glycosylated, and the N-glycosylation is important for the ERAD-L function. This work not only provides an invaluable resource for studying rice N-glycosylation, but also demonstrates the applicability of MGL in glycoproteomic profiling for crop species.

DOI: https://doi.org/10.1016/j.mcpro.2024.100883
Elife. 2024 Nov 22. pii: RP96353. [Epub ahead of print]13

Spatiotemporal recruitment of the ubiquitin-specific protease USP8 directs endosome maturation.

Yue Miao, Yongtao Du, Baolei Wang, Jingjing Liang, Yu Liang, Song Dang, Jiahao Liu, Dong Li, Kangmin He, Mei Ding.

  The spatiotemporal transition of small GTPase Rab5 to Rab7 is crucial for early-to-late endosome maturation, yet the precise mechanism governing Rab5-to-Rab7 switching remains elusive. USP8, a ubiquitin-specific protease, plays a prominent role in the endosomal sorting of a wide range of transmembrane receptors and is a promising target in cancer therapy. Here, we identified that USP8 is recruited to Rab5-positive carriers by Rabex5, a guanine nucleotide exchange factor (GEF) for Rab5. The recruitment of USP8 dissociates Rabex5 from early endosomes (EEs) and meanwhile promotes the recruitment of the Rab7 GEF SAND-1/Mon1. In USP8-deficient cells, the level of active Rab5 is increased, while the Rab7 signal is decreased. As a result, enlarged EEs with abundant intraluminal vesicles accumulate and digestive lysosomes are rudimentary. Together, our results reveal an important and unexpected role of a deubiquitinating enzyme in endosome maturation.

Keywords:  C. elegans; Rab5-to-Rab7 switch; Rabex5/RABX-5; USP8/USP-50; cell biology; endosome maturation

DOI:  https://doi.org/10.7554/eLife.96353
Mol Cell. 2024 Nov 15. pii: S1097-2765(24)00877-3. [Epub ahead of print]

Nutrient control of growth and metabolism through mTORC1 regulation of mRNA splicing.

Takafumi Ogawa, Meltem Isik, Ziyun Wu, Kiran Kurmi, Jin Meng, Sungyun Cho, Gina Lee, L Paulette Fernandez-Cardenas, Masaki Mizunuma, John Blenis, Marcia C Haigis, T Keith Blackwell.

  Cellular growth and organismal development are remarkably complex processes that require the nutrient-responsive kinase mechanistic target of rapamycin complex 1 (mTORC1). Anticipating that important mTORC1 functions remained to be identified, we employed genetic and bioinformatic screening in C. elegans to uncover mechanisms of mTORC1 action. Here, we show that during larval growth, nutrients induce an extensive reprogramming of gene expression and alternative mRNA splicing by acting through mTORC1. mTORC1 regulates mRNA splicing and the production of protein-coding mRNA isoforms largely independently of its target p70 S6 kinase (S6K) by increasing the activity of the serine/arginine-rich (SR) protein RSP-6 (SRSF3/7) and other splicing factors. mTORC1-mediated mRNA splicing regulation is critical for growth; mediates nutrient control of mechanisms that include energy, nucleotide, amino acid, and other metabolic pathways; and may be conserved in humans. Although mTORC1 inhibition delays aging, mTORC1-induced mRNA splicing promotes longevity, suggesting that when mTORC1 is inhibited, enhancement of this splicing might provide additional anti-aging benefits.

Keywords:  C. elegans; SR proteins; development; gene expression; growth; human cell growth; longevity; mRNA splicing; mTORC1; metabolism; nutrient response

DOI:  https://doi.org/10.1016/j.molcel.2024.10.037