bims-proteo 2023-03-19 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2023–03–19
33 papers selected by
Eric Chevet, INSERM

Dysregulation of ribosome-associated quality control elicits cognitive disorders via overaccumulation of TTC3.
ER proteostasis regulators cell-non-autonomously control sleep.
Antitumor T-cell function requires CPEB4-mediated adaptation to chronic endoplasmic reticulum stress.
In situ snapshots along a mammalian selective autophagy pathway.
Effects of calreticulin mutations on cell transformation and immunity.
Insights into membrane association of the SMP domain of extended synaptotagmin.
Signal recognition particle receptor-β (SR-β) coordinates cotranslational N-glycosylation.
Integrated Stress Response signaling acts as a metabolic sensor in fat tissues to regulate oocyte maturation and ovulation.
The UFM1 Conjugation System in Mammalian Development.
FAM69C functions as a kinase for eIF2α and promotes stress granule assembly.
The ubiquitin-specific protease USP36 SUMOylates EXOSC10 and promotes the nucleolar RNA exosome function in rRNA processing.
Targeting unfolded protein response using albumin-encapsulated nanoparticles attenuates temozolomide resistance in glioblastoma.
STING signalling is terminated through ESCRT-dependent microautophagy of vesicles originating from recycling endosomes.
The exocyst complex is an essential component of the mammalian constitutive secretory pathway.
VCP/p97, a pleiotropic protein regulator of the DNA damage response and proteostasis, is a potential therapeutic target in KRAS-mutant pancreatic cancer.
SQSTM1/p62 and Hepatic Mallory Denk Body Formation in Alcohol-Associated Liver Disease.
Concurrent remodeling of nucleolar 60S subunit precursors by the Rea1 ATPase and Spb4 RNA helicase.
Genetically Encoded Boronolectin as a Specific Red Fluorescent UDP-GlcNAc Biosensor.
Bi-allelic mutation in SEC16B alters collagen trafficking and increases ER stress.
Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.
Structural basis of plp2-mediated cytoskeletal protein folding by TRiC/CCT.
CDNF rescues motor neurons in models of amyotrophic lateral sclerosis by targeting endoplasmic reticulum stress.
A Conserved Requirement for RME-8/DNAJC13 in Neuronal Autolysosome Reformation.
Chemoproteomics reveals arctigenin as a phagophore-closure blocker via targeting ESCRT-I subunit VPS28.
The P-body protein 4E-T represses translation to regulate the balance between cell genesis and establishment of the postnatal NSC pool.
A central chaperone-like role for 14-3-3 proteins in human cells.
Protein-protein interfaces in molecular glue-induced ternary complexes: classification, characterization, and prediction.
UBE2C-mediated autophagy inhibition via ubiquitination of SIRT1 contributes to endometrial cancer progression.
Platelet function studies in myeloproliferative neoplasms patients with Calreticulin or JAK2 V617F mutation.
UHRF1/UBE2L6/UBR4-mediated ubiquitination regulates EZH2 abundance and thereby melanocytic differentiation phenotypes in melanoma.
Cancer-Related Unconventional Protein Secretion: A New Role of the Endoplasmic Reticulum.
De novo lipogenesis fuels adipocyte autophagosome and lysosome membrane dynamics.
A general method for quantitative fractionation of mammalian cells.

Proc Natl Acad Sci U S A. 2023 Mar 21. 120(12): e2211522120

Dysregulation of ribosome-associated quality control elicits cognitive disorders via overaccumulation of TTC3.

Ryo Endo, Yi-Kai Chen, John Burke, Noriko Takashima, Nayan Suryawanshi, Kelvin K Hui, Tatsuhiko Miyazaki, Motomasa Tanaka.

  Ribosome-associated quality control (RQC) pathway is responsible for degradation of nascent polypeptides in aberrantly stalled ribosomes, and its defects may lead to neurological diseases. However, the underlying molecular mechanism of how RQC dysfunction elicits neurological disorders remains poorly understood. Here we revealed that neurons with knockout (KO) of ubiquitin ligase LTN1, a key gene in the RQC pathway, show developmental defects in neurons via upregulation of TTC3 and UFMylation signaling proteins. The abnormally enhanced TTC3 protein in Ltn1 KO neurons reduced further accumulation of translationally arrested products by preventing translation initiation of selective genes. However, the overaccumulated TTC3 protein in turn caused dendritic abnormalities and reduced surface-localized GABAA receptors during neuronal development. Ltn1 KO mice showed behavioral deficits associated with cognitive disorders, a subset of which were restored by TTC3 knockdown in medial prefrontal cortex. Together, the overactivated cellular compensatory mechanism against defective RQC through TTC3 overaccumulation induced synaptic and cognitive deficits. More broadly, these findings represent a novel cellular mechanism underlying neuronal dysfunctions triggered by exaggerated cellular stress response to accumulated abnormal translation products in neurons.

Keywords:  TTC3; UFMylation; cognitive disorders; ribosome stalling; ribosome-associated quality control

DOI:  https://doi.org/10.1073/pnas.2211522120
Cell Rep. 2023 Mar 15. pii: S2211-1247(23)00278-4. [Epub ahead of print]42(3): 112267

ER proteostasis regulators cell-non-autonomously control sleep.

Taizo Kawano, Mitsuaki Kashiwagi, Mika Kanuka, Chung-Kuan Chen, Shinnosuke Yasugaki, Sena Hatori, Shinichi Miyazaki, Kaeko Tanaka, Hidetoshi Fujita, Toshiro Nakajima, Masashi Yanagisawa, Yoshimi Nakagawa, Yu Hayashi.

  Sleep is regulated by peripheral tissues under fatigue. The molecular pathways in peripheral cells that trigger systemic sleep-related signals, however, are unclear. Here, a forward genetic screen in C. elegans identifies 3 genes that strongly affect sleep amount: sel-1, sel-11, and mars-1. sel-1 and sel-11 encode endoplasmic reticulum (ER)-associated degradation components, whereas mars-1 encodes methionyl-tRNA synthetase. We find that these machineries function in non-neuronal tissues and that the ER unfolded protein response components inositol-requiring enzyme 1 (IRE1)/XBP1 and protein kinase R-like ER kinase (PERK)/eukaryotic initiation factor-2α (eIF2α)/activating transcription factor-4 (ATF4) participate in non-neuronal sleep regulation, partly by reducing global translation. Neuronal epidermal growth factor receptor (EGFR) signaling is also required. Mouse studies suggest that this mechanism is conserved in mammals. Considering that prolonged wakefulness increases ER proteostasis stress in peripheral tissues, our results suggest that peripheral ER proteostasis factors control sleep homeostasis. Moreover, based on our results, peripheral tissues likely cope with ER stress not only by the well-established cell-autonomous mechanisms but also by promoting the individual's sleep.

Keywords:  C. elegans; CP: Cell biology; CP: Neuroscience; EGFR; cell non-autonomous; endoplasmic reticulum-associated degradation; proteostasis; sleep

DOI:  https://doi.org/10.1016/j.celrep.2023.112267
EMBO J. 2023 Mar 15. e111494

Antitumor T-cell function requires CPEB4-mediated adaptation to chronic endoplasmic reticulum stress.

Marcos Fernández-Alfara, Annarita Sibilio, Judit Martin, Elsa Tusquets Uxó, Marina Malumbres, Victor Alcalde, Verónica Chanes, Adrià Cañellas-Socias, Sergio Palomo-Ponce, Eduard Batlle, Raúl Méndez.

  Tumor growth is influenced by a complex network of interactions between multiple cell types in the tumor microenvironment (TME). These constrained conditions trigger the endoplasmic reticulum (ER) stress response, which extensively reprograms mRNA translation. When uncontrolled over time, chronic ER stress impairs the antitumor effector function of CD8 T lymphocytes. How cells promote adaptation to chronic stress in the TME without the detrimental effects of the terminal unfolded protein response (UPR) is unknown. Here, we find that, in effector CD8 T lymphocytes, RNA-binding protein CPEB4 constitutes a new branch of the UPR that allows cells to adapt to sustained ER stress, yet remains decoupled from the terminal UPR. ER stress, induced during CD8 T-cell activation and effector function, triggers CPEB4 expression. CPEB4 then mediates chronic stress adaptation to maintain cellular fitness, allowing effector molecule production and cytotoxic activity. Accordingly, this branch of the UPR is required for the antitumor effector function of T lymphocytes, and its disruption in these cells exacerbates tumor growth.

Keywords:  CPEB; T lymphocytes; endoplasmic reticulum stress; mRNA translation; tumor microenvironment

DOI:  https://doi.org/10.15252/embj.2022111494
Proc Natl Acad Sci U S A. 2023 Mar 21. 120(12): e2221712120

In situ snapshots along a mammalian selective autophagy pathway.

Meijing Li, Ishita Tripathi-Giesgen, Brenda A Schulman, Wolfgang Baumeister, Florian Wilfling.

  Selective macroautophagy (hereafter referred to as autophagy) describes a process in which cytosolic material is engulfed in a double membrane organelle called an autophagosome. Autophagosomes are carriers responsible for delivering their content to a lytic compartment for destruction. The cargo can be of diverse origin, ranging from macromolecular complexes to protein aggregates, organelles, and even invading pathogens. Each cargo is unique in composition and size, presenting different challenges to autophagosome biogenesis. Among the largest cargoes targeted by the autophagy machinery are intracellular bacteria, which can, in the case of Salmonella, range from 2 to 5 μm in length and 0.5 to 1.5 μm in width. How phagophores form and expand on such a large cargo remains mechanistically unclear. Here, we used HeLa cells infected with an auxotrophic Salmonella to study the process of phagophore biogenesis using in situ correlative cryo-ET. We show that host cells generate multiple phagophores at the site of damaged Salmonella-containing vacuoles (SCVs). The observed double membrane structures range from disk-shaped to expanded cup-shaped phagophores, which have a thin intermembrane lumen with a dilating rim region and expand using the SCV, the outer membrane of Salmonella, or existing phagophores as templates. Phagophore rims establish different forms of contact with the endoplasmic reticulum (ER) via structurally distinct molecular entities for membrane formation and expansion. Early omegasomes correlated with the marker Double-FYVE domain-Containing Protein 1 (DFCP1) are observed in close association with the ER without apparent membrane continuity. Our study provides insights into the formation of phagophores around one of the largest selective cargoes.

Keywords:  Salmonella; autophagosome; cryo-electron tomography; omegasome; xenophagy

DOI:  https://doi.org/10.1073/pnas.2221712120
J Cell Mol Med. 2023 Mar 13.

Effects of calreticulin mutations on cell transformation and immunity.

Harini Desikan, Amanpreet Kaur, Irina D Pogozheva, Malini Raghavan.

  Myeloproliferative neoplasms (MPNs) are cancers involving dysregulated production and function of myeloid lineage hematopoietic cells. Among MPNs, Essential thrombocythemia (ET), Polycythemia Vera (PV) and Myelofibrosis (MF), are driven by mutations that activate the JAK-STAT signalling pathway. Somatic mutations of calreticulin (CRT), an endoplasmic reticulum (ER)-localized lectin chaperone, are driver mutations in approximately 25% of ET and 35% of MF patients. The MPN-linked mutant CRT proteins have novel frameshifted carboxy-domain sequences and lack an ER retention motif, resulting in their secretion. Wild type CRT is a regulator of ER calcium homeostasis and plays a key role in the assembly of major histocompatibility complex (MHC) class I molecules, which are the ligands for antigen receptors of CD8+ T cells. Mutant CRT-linked oncogenesis results from the dysregulation of calcium signalling in cells and the formation of stable complexes of mutant CRT with myeloproliferative leukemia (MPL) protein, followed by downstream activation of the JAK-STAT signalling pathway. The intricate participation of CRT in ER protein folding, calcium homeostasis and immunity suggests the involvement of multiple mechanisms of mutant CRT-linked oncogenesis. In this review, we highlight recent findings related to the role of MPN-linked CRT mutations in the dysregulation of calcium homeostasis, MPL activation and immunity.

Keywords:  MHC Class I; MPL; calcium; calreticulin; endoplasmic reticulum; myeloproliferative neoplasms; thrombopoietin receptor

DOI:  https://doi.org/10.1111/jcmm.17713
Nat Commun. 2023 Mar 17. 14(1): 1504

Insights into membrane association of the SMP domain of extended synaptotagmin.

Yunyun Wang, Zhenni Li, Xinyu Wang, Ziyuan Zhao, Li Jiao, Ruming Liu, Keying Wang, Rui Ma, Yang Yang, Guo Chen, Yong Wang, Xin Bian.

The Synaptotagmin-like Mitochondrial-lipid-binding Protein (SMP) domain is a newly identified lipid transfer module present in proteins that regulate lipid homeostasis at membrane contact sites (MCSs). However, how the SMP domain associates with the membrane to extract and unload lipids is unclear. Here, we performed in vitro DNA brick-assisted lipid transfer assays and in silico molecular dynamics simulations to investigate the molecular basis of the membrane association by the SMP domain of extended synaptotagmin (E-Syt), which tethers the tubular endoplasmic reticulum (ER) to the plasma membrane (PM). We demonstrate that the SMP domain uses its tip region to recognize the extremely curved subdomain of tubular ER and the acidic-lipid-enriched PM for highly efficient lipid transfer. Supporting these findings, disruption of these mechanisms results in a defect in autophagosome biogenesis contributed by E-Syt. Our results suggest a model that provides a coherent picture of the action of the SMP domain at MCSs.

DOI: https://doi.org/10.1038/s41467-023-37202-8
Sci Adv. 2023 Mar 17. 9(11): eade8079

Signal recognition particle receptor-β (SR-β) coordinates cotranslational N-glycosylation.

Chatchai Phoomak, Natalie Rinis, Marta Baro, Shiteshu Shrimal, Daniel Bennett, Scott A Shaffer, Mark Lehrman, Reid Gilmore, Joseph N Contessa.

Proteins destined for the secretory compartment of the cell are cotranslationally translocated into the endoplasmic reticulum. The majority of these proteins are N-glycosylated, a co- and posttranslational modification that ensures proper protein folding, stability, solubility, and cellular localization. Here, we show that the [Formula: see text] subunit of the signal recognition particle receptor (SR) is required for assembly of the N-glycosylation-competent translocon. We report that guanine analog chemical probes identified by high-throughput screening or mutation of the SR-[Formula: see text] guanosine triphosphate binding site cause an N-glycosylation-deficient phenotype. Neither method alters the association of SR-[Formula: see text] with SR-[Formula: see text], but both approaches reduce the association of SR-[Formula: see text] with the oligosaccharyltransferase complex. These experiments demonstrate that SR-[Formula: see text] has a previously unrecognized function coordinating endoplasmic reticulum translation with N-glycosylation.

DOI: https://doi.org/10.1126/sciadv.ade8079
bioRxiv. 2023 Feb 28. pii: 2023.02.27.530289. [Epub ahead of print]

Integrated Stress Response signaling acts as a metabolic sensor in fat tissues to regulate oocyte maturation and ovulation.

Lydia Grmai, Manuel Michaca, Emily Lackner, Narayanan Nampoothiri V P, Deepika Vasudevan.

Reproduction is an energy-intensive process requiring systemic coordination. However, the inter-organ signaling mechanisms that relay nutrient status to modulate reproductive output are poorly understood. Here, we use Drosophila melanogaster as a model to establish the Integrated Stress response (ISR) transcription factor, Atf4, as a fat tissue metabolic sensor which instructs oogenesis. We demonstrate that Atf4 regulates the lipase Brummer to mediate yolk lipoprotein synthesis in the fat body. Depletion of Atf4 in the fat body also blunts oogenesis recovery after amino acid deprivation and re-feeding, suggestive of a nutrient sensing role for Atf4. We also discovered that Atf4 promotes secretion of a fat body-derived neuropeptide, CNMamide, which modulates neural circuits that promote egg-laying behavior (ovulation). Thus, we posit that ISR signaling in fat tissue acts as a "metabolic sensor" that instructs female reproduction: directly, by impacting yolk lipoprotein production and follicle maturation, and systemically, by regulating ovulation.

DOI: https://doi.org/10.1101/2023.02.27.530289
Dev Dyn. 2023 Mar 18.

The UFM1 Conjugation System in Mammalian Development.

Shuchun Yang, Nathan Moy, Rui Yang.

  Post-translational modifications by ubiquitin and ubiquitin-like proteins are important in regulating cellular protein functions. UFM1 (ubiquitin-fold modifier 1), first identified almost two decades ago, is a member of the ubiquitin-like protein family. UFM1 is covalently conjugated to the target proteins in an enzymatic cascade consisting of E1 (activating), E2 (conjugating), and E3 (ligating) enzymes. At the molecular level, modification by UFM1 (UFMylation) is an important mediator of protein function. Dysregulation of the UFM1 conjugation system, e.g., the knockout of UFMylation components, disturbs proteome homeostasis and triggers endoplasmic reticulum stress. Such changes are linked to developmental disorders, tumorigenesis, tissue injury, inflammation, and several hereditary neurological syndromes. This review will focus on the role of the UFMylation in animal development and associated congenital disorders. We will cover the hematopoietic system, liver, central nervous system, intestine, heart, kidney, immune and skeletal system to provide insight into disease pathogenesis and shed light on possible novel therapeutic methods. This article is protected by copyright. All rights reserved.

Keywords:  ER stress; UFM1; UFMylation; knockout; mammalian development

DOI:  https://doi.org/10.1002/dvdy.586
EMBO Rep. 2023 Mar 16. e55641

FAM69C functions as a kinase for eIF2α and promotes stress granule assembly.

Zhongyan Wu, Fan Mei, Yangyang Gan, Anhang Liu, Jiapan Hu, Yan Jin, Yuxin Yin.

  Stress granules are dynamic cytoplasmic ribonucleoprotein granules that assemble in response to cellular stress. Aberrant formation of stress granules has been linked to neurodegenerative diseases. However, the molecular mechanisms underlying the initiation of stress granules remain elusive. Here we report that the brain-enriched protein kinase FAM69C promotes stress granule assembly through phosphorylation of eukaryotic translation initiation factor 2 (eIF2α). FAM69C physically interacts with eIF2α and functions as a stress-specific kinase for eIF2α, leading to stress-induced protein translation arrest and stress granule assembly. Primary microglia derived from Fam69c knockout mice exhibit aberrant stress granule assembly in response to oxidative stress and ATP. Defective stress granule assembly in microglia correlates with the formation of ASC specks and NLRP3 inflammasome activation, whereas induction of stress granule precludes inflammasome formation. Consistently, increased NLRP3 levels, caspase-1 cleavage and Il18 expression corroborate microglia-associated neuroinflammation in aged Fam69c knockout mice. Our study demonstrates that FAM69C is critical for stress granule assembly and suggests its role in the regulation of microglia function.

Keywords:  FAM69C; eIF2α; inflammasome; microglia; stress granule

DOI:  https://doi.org/10.15252/embr.202255641
Nucleic Acids Res. 2023 Mar 13. pii: gkad140. [Epub ahead of print]

The ubiquitin-specific protease USP36 SUMOylates EXOSC10 and promotes the nucleolar RNA exosome function in rRNA processing.

Yingxiao Chen, Yanping Li, Roselyn S Dai, Jonathan C Savage, Ujwal Shinde, John Klimek, Larry L David, Emma A Young, Markus Hafner, Rosalie C Sears, Xiao-Xin Sun, Mu-Shui Dai.

The RNA exosome is an essential 3' to 5' exoribonuclease complex that mediates degradation, processing and quality control of virtually all eukaryotic RNAs. The nucleolar RNA exosome, consisting of a nine-subunit core and a distributive 3' to 5' exonuclease EXOSC10, plays a critical role in processing and degrading nucleolar RNAs, including pre-rRNA. However, how the RNA exosome is regulated in the nucleolus is poorly understood. Here, we report that the nucleolar ubiquitin-specific protease USP36 is a novel regulator of the nucleolar RNA exosome. USP36 binds to the RNA exosome through direct interaction with EXOSC10 in the nucleolus. Interestingly, USP36 does not significantly regulate the levels of EXOSC10 and other tested exosome subunits. Instead, it mediates EXOSC10 SUMOylation at lysine (K) 583. Mutating K583 impaired the binding of EXOSC10 to pre-rRNAs, and the K583R mutant failed to rescue the defects in rRNA processing and cell growth inhibition caused by knockdown of endogenous EXOSC10. Furthermore, EXOSC10 SUMOylation is markedly reduced in cells in response to perturbation of ribosomal biogenesis. Together, these results suggest that USP36 acts as a SUMO ligase to promote EXOSC10 SUMOylation critical for the RNA exosome function in ribosome biogenesis.

DOI: https://doi.org/10.1093/nar/gkad140
Br J Cancer. 2023 Mar 17.

Targeting unfolded protein response using albumin-encapsulated nanoparticles attenuates temozolomide resistance in glioblastoma.

Karrie Mei-Yee Kiang, Wanjun Tang, Qingchun Song, Jiaxin Liu, Ning Li, Tsz-Lung Lam, Ho Cheung Shum, Zhiyuan Zhu, Gilberto Ka-Kit Leung.

BACKGROUND: Chemoresistant cancer cells frequently exhibit a state of chronically activated endoplasmic reticulum (ER) stress. Engaged with ER stress, the unfolded protein response (UPR) is an adaptive reaction initiated by the accumulation of misfolded proteins. Protein disulfide isomerase (PDI) is a molecular chaperone known to be highly expressed in glioblastomas with acquired resistance to temozolomide (TMZ). We investigate whether therapeutic targeting of PDI provides a rationale to overcome chemoresistance.
METHODS: The activity of PDI was suppressed in glioblastoma cells using a small molecule inhibitor CCF642. Either single or combination treatment with TMZ was used. We prepared nanoformulation of CCF642 loaded in albumin as a drug carrier for orthotopic tumour model.
RESULTS: Inhibition of PDI significantly enhances the cytotoxic effect of TMZ on glioblastoma cells. More importantly, inhibition of PDI is able to sensitise glioblastoma cells that are initially resistant to TMZ treatment. Nanoformulation of CCF642 is well-tolerated and effective in suppressing tumour growth. It activates cell death-triggering UPR beyond repair and induces ER perturbations through the downregulation of PERK signalling. Combination treatment of TMZ with CCF642 significantly reduces tumour growth compared with either modality alone.
CONCLUSION: Our study demonstrates modulation of ER stress by targeting PDI as a promising therapeutic rationale to overcome chemoresistance.

DOI: https://doi.org/10.1038/s41416-023-02225-x
Nat Cell Biol. 2023 Mar;25(3): 453-466

STING signalling is terminated through ESCRT-dependent microautophagy of vesicles originating from recycling endosomes.

Yoshihiko Kuchitsu, Kojiro Mukai, Rei Uematsu, Yuki Takaada, Ayumi Shinojima, Ruri Shindo, Tsumugi Shoji, Shiori Hamano, Emari Ogawa, Ryota Sato, Kensuke Miyake, Akihisa Kato, Yasushi Kawaguchi, Masahiko Nishitani-Isa, Kazushi Izawa, Ryuta Nishikomori, Takahiro Yasumi, Takehiro Suzuki, Naoshi Dohmae, Takefumi Uemura, Glen N Barber, Hiroyuki Arai, Satoshi Waguri, Tomohiko Taguchi.

Stimulator of interferon genes (STING) is essential for the type I interferon response against a variety of DNA pathogens. Upon emergence of cytosolic DNA, STING translocates from the endoplasmic reticulum to the Golgi where STING activates the downstream kinase TBK1, then to lysosome through recycling endosomes (REs) for its degradation. Although the molecular machinery of STING activation is extensively studied and defined, the one underlying STING degradation and inactivation has not yet been fully elucidated. Here we show that STING is degraded by the endosomal sorting complexes required for transport (ESCRT)-driven microautophagy. Airyscan super-resolution microscopy and correlative light/electron microscopy suggest that STING-positive vesicles of an RE origin are directly encapsulated into Lamp1-positive compartments. Screening of mammalian Vps genes, the yeast homologues of which regulate Golgi-to-vacuole transport, shows that ESCRT proteins are essential for the STING encapsulation into Lamp1-positive compartments. Knockdown of Tsg101 and Vps4, components of ESCRT, results in the accumulation of STING vesicles in the cytosol, leading to the sustained type I interferon response. Knockdown of Tsg101 in human primary T cells leads to an increase the expression of interferon-stimulated genes. STING undergoes K63-linked ubiquitination at lysine 288 during its transit through the Golgi/REs, and this ubiquitination is required for STING degradation. Our results reveal a molecular mechanism that prevents hyperactivation of innate immune signalling, which operates at REs.

DOI: https://doi.org/10.1038/s41556-023-01098-9
J Cell Biol. 2023 May 01. pii: e202205137. [Epub ahead of print]222(5):

The exocyst complex is an essential component of the mammalian constitutive secretory pathway.

Conceição Pereira, Danièle Stalder, Georgina S F Anderson, Amber S Shun-Shion, Jack Houghton, Robin Antrobus, Michael A Chapman, Daniel J Fazakerley, David C Gershlick.

Secreted proteins fulfill a vast array of functions, including immunity, signaling, and extracellular matrix remodeling. In the trans-Golgi network, proteins destined for constitutive secretion are sorted into post-Golgi carriers which fuse with the plasma membrane. The molecular machinery involved is poorly understood. Here, we have used kinetic trafficking assays and transient CRISPR-KO to study biosynthetic sorting from the Golgi to the plasma membrane. Depletion of all canonical exocyst subunits causes cargo accumulation in post-Golgi carriers. Exocyst subunits are recruited to and co-localize with carriers. Exocyst abrogation followed by kinetic trafficking assays of soluble cargoes results in intracellular cargo accumulation. Unbiased secretomics reveals impairment of soluble protein secretion after exocyst subunit knockout. Importantly, in specialized cell types, the loss of exocyst prevents constitutive secretion of antibodies in lymphocytes and of leptin in adipocytes. These data identify exocyst as the functional tether of secretory post-Golgi carriers at the plasma membrane and an essential component of the mammalian constitutive secretory pathway.

DOI: https://doi.org/10.1083/jcb.202205137
Genes Cancer. 2023 ;14 30-49

VCP/p97, a pleiotropic protein regulator of the DNA damage response and proteostasis, is a potential therapeutic target in KRAS-mutant pancreatic cancer.

Ye S Lee, Jennifer E Klomp, Clint A Stalnecker, Craig M Goodwin, Yanzhe Gao, Gaith N Droby, Cyrus Vaziri, Kirsten L Bryant, Channing J Der, Adrienne D Cox.

  We and others have recently shown that proteins involved in the DNA damage response (DDR) are critical for KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cell growth in vitro. However, the CRISPR-Cas9 library that enabled us to identify these key proteins had limited representation of DDR-related genes. To further investigate the DDR in this context, we performed a comprehensive, DDR-focused CRISPR-Cas9 loss-of-function screen. This screen identified valosin-containing protein (VCP) as an essential gene in KRAS-mutant PDAC cell lines. We observed that genetic and pharmacologic inhibition of VCP limited cell growth and induced apoptotic death. Addressing the basis for VCP-dependent growth, we first evaluated the contribution of VCP to the DDR and found that loss of VCP resulted in accumulation of DNA double-strand breaks. We next addressed its role in proteostasis and found that loss of VCP caused accumulation of polyubiquitinated proteins. We also found that loss of VCP increased autophagy. Therefore, we reasoned that inhibiting both VCP and autophagy could be an effective combination. Accordingly, we found that VCP inhibition synergized with the autophagy inhibitor chloroquine. We conclude that concurrent targeting of autophagy can enhance the efficacy of VCP inhibitors in KRAS-mutant PDAC.

Keywords:  DNA damage response; KRAS; VCP; autophagy; pancreatic cancer

DOI:  https://doi.org/10.18632/genesandcancer.231
Am J Pathol. 2023 Mar 09. pii: S0002-9440(23)00084-6. [Epub ahead of print]

SQSTM1/p62 and Hepatic Mallory Denk Body Formation in Alcohol-Associated Liver Disease.

Hui Qian, Wen-Xing Ding.

  SQSTM1/p62 (hereafter referred to as p62) is an autophagy receptor protein for selective autophagy primarily due to its direct interaction with the microtubule light chain 3 (LC3) protein that specifically localizes on autophagosome membranes. As a result, impaired autophagy leads to the accumulation of p62. p62 is also a common component of many human liver disease-related cellular inclusion bodies, such as Mallory-Denk Bodies (MDB), intracytoplasmic hyaline bodies (IHBs), α1 antitrypsin aggregates, as well as p62 bodies/condensates. p62 also acts as an intracellular signaling hub, and it involves multiple signaling pathways including nuclear factor erythroid 2-related factor 2 (NRF2), nuclear factor kappa B (NFκB), and the mechanistic target of rapamycin (mTOR), which are critical for oxidative stress, inflammation, cell survival, metabolism, and liver tumorigenesis. In this review, we will discuss the recent insights of p62 in protein quality control, including the role of p62 in the formation and degradation of p62 stress granules and protein aggregates as well as regulation of multiple signaling pathways in the pathogenesis of alcohol-associated liver disease (ALD).

Keywords:  TFEB; autophagy; protein aggregates; proteostasis; stress granules

DOI:  https://doi.org/10.1016/j.ajpath.2023.02.015
Elife. 2023 Mar 17. pii: e84877. [Epub ahead of print]12

Concurrent remodeling of nucleolar 60S subunit precursors by the Rea1 ATPase and Spb4 RNA helicase.

Valentin Mitterer, Matthias Thoms, Robert Buschauer, Otto Berninghausen, Ed Hurt, Roland Beckmann.

  Biogenesis intermediates of nucleolar ribosomal 60S precursor particles undergo a number of structural maturation steps before they transit to the nucleoplasm and are finally exported into the cytoplasm. The AAA+-ATPase Rea1 participates in the nucleolar exit by releasing the Ytm1-Erb1 heterodimer from the evolving pre-60S particle. Here, we show that the DEAD-box RNA helicase Spb4 with its interacting partner Rrp17 is further integrated into this maturation event. Spb4 binds to a specific class of late nucleolar pre-60S intermediates, whose cryo-EM structure revealed how its helicase activity facilitates melting and restructuring of 25S rRNA helices H62 and H63/H63a prior to Ytm1-Erb1 release. In vitro maturation of such Spb4-enriched pre-60S particles, incubated with purified Rea1 and its associated pentameric Rix1-complex in the presence of ATP, combined with cryo-EM analysis depicted the details of the Rea1-dependent large-scale pre-ribosomal remodelling. Our structural insights unveil how the Rea1 ATPase and Spb4 helicase remodel late nucleolar pre-60S particles by rRNA restructuring and dismantling of a network of several ribosomal assembly factors.

Keywords:  S. cerevisiae; biochemistry; chemical biology; molecular biophysics; structural biology

DOI:  https://doi.org/10.7554/eLife.84877
bioRxiv. 2023 Mar 01. pii: 2023.03.01.530644. [Epub ahead of print]

Genetically Encoded Boronolectin as a Specific Red Fluorescent UDP-GlcNAc Biosensor.

Jing Zhang, Zefan Li, Yu Pang, Yichong Fan, Hui-Wang Ai.

There is great interest in developing boronolectins, which are synthetic lectin mimics containing a boronic acid functional group for reversible recognition of diol-containing molecules, such as glycans and ribonucleotides. However, it remains a significant challenge to gain specificity. Here, we present a genetically encoded boronolectin, which is a hybrid protein consisting of a noncanonical amino acid (ncAA) p-boronophenylalanine (pBoF), natural-lectin-derived peptide sequences, and a circularly permuted red fluorescent protein (cpRFP). The genetic encodability permitted a straightforward protein engineering process to derive a red fluorescent biosensor that can specifically bind uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), an important nucleotide sugar involved in metabolic sensing and cell signaling. We further characterized the resultant boronic acid-and peptide-assisted UDP-GlcNAc sensor (bapaUGAc) both in vitro and in live mammalian cells. Because UDP-GlcNAc in the endoplasmic reticulum (ER) and Golgi apparatus plays essential roles in glycosylating biomolecules in the secretory pathway, we genetically expressed bapaUGAc in the ER and Golgi and validated the sensor for its responses to metabolic disruption and pharmacological inhibition. In addition, we combined bapaUGAc with UGAcS, a recently reported green fluorescent UDP-GlcNAc sensor based on an alternative sensing mechanism, to monitor UDP-GlcNAc level changes in the ER and cytosol simultaneously. We expect our work to facilitate the future development of specific boronolectins for carbohydrates. In addition, this newly developed genetically encoded bapaUGAc sensor will be a valuable tool for studying UDP-GlcNAc and glycobiology.

DOI: https://doi.org/10.1101/2023.03.01.530644
EMBO Mol Med. 2023 Mar 14. e16834

Bi-allelic mutation in SEC16B alters collagen trafficking and increases ER stress.

Ahmed El-Gazzar, Barbara Voraberger, Frank Rauch, Mario Mairhofer, Katy Schmidt, Brecht Guillemyn, Goran Mitulović, Veronika Reiterer, Margot Haun, Michaela M Mayr, Johannes A Mayr, Susanne Kimeswenger, Oliver Drews, Vrinda Saraff, Nick Shaw, Nadja Fratzl-Zelman, Sofie Symoens, Hesso Farhan, Wolfgang Högler.

  Osteogenesis imperfecta (OI) is a genetically and clinically heterogeneous disorder characterized by bone fragility and reduced bone mass generally caused by defects in type I collagen structure or defects in proteins interacting with collagen processing. We identified a homozygous missense mutation in SEC16B in a child with vertebral fractures, leg bowing, short stature, muscular hypotonia, and bone densitometric and histomorphometric features in keeping with OI with distinct ultrastructural features. In line with the putative function of SEC16B as a regulator of trafficking between the ER and the Golgi complex, we showed that patient fibroblasts accumulated type I procollagen in the ER and exhibited a general trafficking defect at the level of the ER. Consequently, patient fibroblasts exhibited ER stress, enhanced autophagosome formation, and higher levels of apoptosis. Transfection of wild-type SEC16B into patient cells rescued the collagen trafficking. Mechanistically, we show that the defect is a consequence of reduced SEC16B expression, rather than due to alterations in protein function. These data suggest SEC16B as a recessive candidate gene for OI.

Keywords:  SEC16B; autophagy; endoplasmic reticulum; osteogenesis imperfecta; type I collagen

DOI:  https://doi.org/10.15252/emmm.202216834
Immunity. 2023 Mar 07. pii: S1074-7613(23)00092-4. [Epub ahead of print]

Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.

Jinzhi Duan, Juan D Matute, Lukas W Unger, Thomas Hanley, Alexandra Schnell, Xi Lin, Niklas Krupka, Paul Griebel, Conner Lambden, Brandon Sit, Joep Grootjans, Michal Pyzik, Felix Sommer, Sina Kaiser, Maren Falk-Paulsen, Helmut Grasberger, John Y Kao, Tobias Fuhrer, Hai Li, Donggi Paik, Yunjin Lee, Samuel Refetoff, Jonathan N Glickman, Adrienne W Paton, Lynn Bry, James C Paton, Uwe Sauer, Andrew J Macpherson, Philip Rosenstiel, Vijay K Kuchroo, Matthew K Waldor, Jun R Huh, Arthur Kaser, Richard S Blumberg.

  Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

Keywords:  Citrobacter rodentium; ROS signals; TH17 cells; commensal bacterial; epithelial endoplasmic reticulum stress; inflammatory bowel disease; purine metabolism

DOI:  https://doi.org/10.1016/j.immuni.2023.02.018
Sci Adv. 2023 Mar 17. 9(11): eade1207

Structural basis of plp2-mediated cytoskeletal protein folding by TRiC/CCT.

Wenyu Han, Mingliang Jin, Caixuan Liu, Qiaoyu Zhao, Shutian Wang, Yifan Wang, Yue Yin, Chao Peng, Yanxing Wang, Yao Cong.

The cytoskeletal proteins tubulin and actin are the obligate substrates of TCP-1 ring complex/Chaperonin containing TCP-1 (TRiC/CCT), and their folding involves co-chaperone. Through cryo-electron microscopy analysis, we present a more complete picture of TRiC-assisted tubulin/actin folding along TRiC adenosine triphosphatase cycle, under the coordination of co-chaperone plp2. In the open S1/S2 states, plp2 and tubulin/actin engaged within opposite TRiC chambers. Notably, we captured an unprecedented TRiC-plp2-tubulin complex in the closed S3 state, engaged with a folded full-length β-tubulin and loaded with a guanosine triphosphate, and a plp2 occupying opposite rings. Another closed S4 state revealed an actin in the intermediate folding state and a plp2. Accompanying TRiC ring closure, plp2 translocation could coordinate substrate translocation on the CCT6 hemisphere, facilitating substrate stabilization and folding. Our findings reveal the folding mechanism of the major cytoskeletal proteins tubulin/actin under the coordination of the biogenesis machinery TRiC and plp2 and extend our understanding of the links between cytoskeletal proteostasis and related human diseases.

DOI: https://doi.org/10.1126/sciadv.ade1207
Brain. 2023 Mar 16. pii: awad087. [Epub ahead of print]

CDNF rescues motor neurons in models of amyotrophic lateral sclerosis by targeting endoplasmic reticulum stress.

Francesca De Lorenzo, Patrick Lüningschrör, Jinhan Nam, Liam Beckett, Federica Pilotto, Emilia Galli, Päivi Lindholm, Cora Rüdt von Collenberg, Simon Tii Mungwa, Sibylle Jablonka, Julia Kauder, Nadine Thau-Habermann, Susanne Petri, Dan Lindholm, Smita Saxena, Michael Sendtner, Mart Saarma, Merja H Voutilainen.

  Amyotrophic lateral sclerosis is a progressive neurodegenerative disease that affects motor neurons (MNs) in the spinal cord, brainstem, and motor cortex, leading to paralysis and eventually to death within 3 to 5 years of symptom onset. To date, no cure or effective therapy is available. The role of chronic endoplasmic reticulum (ER) stress in the pathophysiology of amyotrophic lateral sclerosis, as well as a potential drug target, has received increasing attention. Here, we investigated the mode of action and therapeutic effect of the ER-resident protein cerebral dopamine neurotrophic factor (CDNF) in three preclinical models of amyotrophic lateral sclerosis, exhibiting different disease development and etiology: (i) the conditional choline acetyltransferase (ChAT)-tTA/TRE-hTDP43-M337V rat model previously described, (ii) the widely used SOD1-G93A mouse model, and (iii) a novel slow-progressive TDP43-M337V mouse model. To specifically analyse the ER stress response in MNs, we used three main methods: (i) primary culture of MNs derived from E13 days embryos, (ii) immunohistochemical analyses of spinal cord sections with ChAT as spinal MNs marker, and (iii) qPCR analyses of lumbar MNs isolated via laser microdissection. We show that intracerebroventricular administration of CDNF significantly halts the progression of the disease and improves motor behavior in TDP43-M337V and SOD1-G93A rodent models of amyotrophic lateral sclerosis. CDNF rescues motor neurons in vitro and in vivo from ER stress-associated cell death and its beneficial effect is independent of genetic disease etiology. Notably, CDNF regulates the unfolded protein response (UPR) initiated by transducers IRE1α, PERK, and ATF6, thereby enhancing MN survival. Thus, CDNF holds great promise for the design of new rational treatments for amyotrophic lateral sclerosis.

Keywords:  CDNF; ER stress; amyotrophic lateral sclerosis; motor neurons; unfolded protein response

DOI:  https://doi.org/10.1093/brain/awad087
bioRxiv. 2023 Feb 28. pii: 2023.02.27.530319. [Epub ahead of print]

A Conserved Requirement for RME-8/DNAJC13 in Neuronal Autolysosome Reformation.

Sierra Swords, Nuo Jia, Anne Norris, Jil Modi, Qian Cai, Barth D Grant.

Autophagosomes fuse with lysosomes, forming autolysosomes that degrade engulfed cargo. To maintain lysosomal capacity, autolysosome reformation (ALR) must regenerate lysosomes from autolysosomes using a membrane tubule-based process. Maintaining lysosomal capacity is required to maintain proteostasis and cellular health, especially in neurons where lysosomal dysfunction has been repeatedly implicated in neurodegenerative disease. Cell biological studies have linked the DNA-J domain Hsc70 co-chaperone RME-8/DNAJC13 to endosomal coat protein regulation, while human genetics studies have linked RME-8/DNAJC13 to neurological disease, including Parkinsonism and Essential Tremor. We report new analysis of the requirements for the RME-8/DNAJC13 protein in neurons, focusing on C. elegans mechanosensory neurons in the intact animal, and in primary mouse cortical neurons in culture. We find that loss of RME-8/DNAJC13 in both systems results in accumulation of grossly elongated autolysosomal tubules. Further C. elegans analysis revealed a similar autolysosome tubule accumulation defect in mutants known to be required for ALR in mammals, including bec-1/beclin and vps-15/PIK3R4/p150 that regulate type-III PI3-kinase VPS-34, and dyn-1 /dynamin that severs ALR tubules. Clathrin is also an important ALR regulator implicated in autolysosome tubule formation and release. In C. elegans we found that loss of RME-8 causes severe depletion of clathrin from neuronal autolysosomes, a phenotype shared with bec-1 and vps-15 mutants. We conclude that RME-8/DNAJC13 plays a conserved but previously unrecognized role in autolysosome reformation, likely affecting ALR tubule initiation and/or severing. Additionally, in both systems, we found that loss of RME-8/DNAJC13 appeared to reduce autophagic flux, suggesting feedback regulation from ALR to autophagy. Our results connecting RME-8/DNAJC13 to ALR and autophagy provide a potential mechanism by which RME-8/DNAJC13 could influence neuronal health and the progression of neurodegenerative disease.

DOI: https://doi.org/10.1101/2023.02.27.530319
Bioorg Chem. 2023 Mar 07. pii: S0045-2068(23)00117-7. [Epub ahead of print]134 106457

Chemoproteomics reveals arctigenin as a phagophore-closure blocker via targeting ESCRT-I subunit VPS28.

Jinyan Han, Yifang Yu, Shasha Li, Ruoyang Miao, Weiyan Cheng, Han Wei.

  Arctigenin is the active ingredient of the traditional medicines Arctium lappa and Fructus Arctii and has been extensively investigated for its diverse pharmacological functions, including its novel anti-austerity activity. Although several mechanisms have been proposed, the direct target of arctigenin to induce anti-austerity activity remains unclear. In this study, we designed and synthesized photo-crosslinkable arctigenin probes and utilized them in the chemoproteomic profiling of potential target proteins directly in living cells. Vacuolar protein sorting-associated protein 28 (VPS28), a key subunit of the ESCRT-I complex implicated in phagophore closure, was successfully identified. Unexpectedly, we found that arctigenin degraded VPS28 via the ubiquitin-proteasome pathway. We also demonstrated that arctigenin induces a prominent phagophore closure-blockade phenotype in PANC-1 cells. To the best of our knowledge, this is the first report of a small molecule acting as a phagophore-closure blocker and a VPS28 degrader. The arctigenin-modulating phagophore closure provides a new druggable target for cancers that rely heavily on autophagy activation and may also be used for other diseases associated with the ESCRT system.

Keywords:  Anti-austerity; Arctigenin; Chemical proteomics; Phagophore closure; VPS28

DOI:  https://doi.org/10.1016/j.bioorg.2023.106457
Cell Rep. 2023 Mar 14. pii: S2211-1247(23)00253-X. [Epub ahead of print]42(3): 112242

The P-body protein 4E-T represses translation to regulate the balance between cell genesis and establishment of the postnatal NSC pool.

Adelaida Kolaj, Siraj K Zahr, Beatrix S Wang, Taylor Krawec, Hilal Kazan, Guang Yang, David R Kaplan, Freda D Miller.

  Here, we ask how developing precursors maintain the balance between cell genesis for tissue growth and establishment of adult stem cell pools, focusing on postnatal forebrain neural precursor cells (NPCs). We show that these NPCs are transcriptionally primed to differentiate and that the primed mRNAs are associated with the translational repressor 4E-T. 4E-T also broadly associates with other NPC mRNAs encoding transcriptional regulators, and these are preferentially depleted from ribosomes, consistent with repression. By contrast, a second translational regulator, Cpeb4, associates with diverse target mRNAs that are largely ribosome associated. The 4E-T-dependent mRNA association is functionally important because 4E-T knockdown or conditional knockout derepresses proneurogenic mRNA translation and perturbs maintenance versus differentiation of early postnatal NPCs in culture and in vivo. Thus, early postnatal NPCs are primed to differentiate, and 4E-T regulates the balance between cell genesis and stem cell expansion by sequestering and repressing mRNAs encoding transcriptional regulators.

Keywords:  4E-T; CP: Neuroscience; CP: Stem cell research; Cpeb4; brain development; neural stem cell; neurogenesis; translational repression

DOI:  https://doi.org/10.1016/j.celrep.2023.112242
Mol Cell. 2023 Mar 16. pii: S1097-2765(23)00121-1. [Epub ahead of print]83(6): 974-993.e15

A central chaperone-like role for 14-3-3 proteins in human cells.

Dmitri Segal, Stefan Maier, Giovanni J Mastromarco, Wesley Wei Qian, Syed Nabeel-Shah, Hyunmin Lee, Gaelen Moore, Jessica Lacoste, Brett Larsen, Zhen-Yuan Lin, Abeeshan Selvabaskaran, Karen Liu, Craig Smibert, Zhaolei Zhang, Jack Greenblatt, Jian Peng, Hyun O Lee, Anne-Claude Gingras, Mikko Taipale.

  14-3-3 proteins are highly conserved regulatory proteins that interact with hundreds of structurally diverse clients and act as central hubs of signaling networks. However, how 14-3-3 paralogs differ in specificity and how they regulate client protein function are not known for most clients. Here, we map the interactomes of all human 14-3-3 paralogs and systematically characterize the effect of disrupting these interactions on client localization. The loss of 14-3-3 binding leads to the coalescence of a large fraction of clients into discrete foci in a client-specific manner, suggesting a central chaperone-like function for 14-3-3 proteins. Congruently, the engraftment of 14-3-3 binding motifs to nonclients can suppress their aggregation or phase separation. Finally, we show that 14-3-3s negatively regulate the localization of the RNA-binding protein SAMD4A to cytoplasmic granules and inhibit its activity as a translational repressor. Our work suggests that 14-3-3s have a more prominent role as chaperone-like molecules than previously thought.

Keywords:  14-3-3 proteins; affinity purification; aggregation; chaperones; functional proteomics; mass spectrometry; phase transitions; protein quality control; proximity-dependent biotinylation

DOI:  https://doi.org/10.1016/j.molcel.2023.02.018
RSC Chem Biol. 2023 Mar 08. 4(3): 192-215

Protein-protein interfaces in molecular glue-induced ternary complexes: classification, characterization, and prediction.

Huan Rui, Kate S Ashton, Jaeki Min, Connie Wang, Patrick Ryan Potts.

Molecular glues are a class of small molecules that stabilize the interactions between proteins. Naturally occurring molecular glues are present in many areas of biology where they serve as central regulators of signaling pathways. Importantly, several clinical compounds act as molecular glue degraders that stabilize interactions between E3 ubiquitin ligases and target proteins, leading to their degradation. Molecular glues hold promise as a new generation of therapeutic agents, including those molecular glue degraders that can redirect the protein degradation machinery in a precise way. However, rational discovery of molecular glues is difficult in part due to the lack of understanding of the protein-protein interactions they stabilize. In this review, we summarize the structures of known molecular glue-induced ternary complexes and the interface properties. Detailed analysis shows different mechanisms of ternary structure formation. Additionally, we also review computational approaches for predicting protein-protein interfaces and highlight the promises and challenges. This information will ultimately help inform future approaches for rational molecular glue discovery.

DOI: https://doi.org/10.1039/d2cb00207h
Mol Cancer Res. 2023 Mar 17. pii: MCR-22-0825. [Epub ahead of print]

UBE2C-mediated autophagy inhibition via ubiquitination of SIRT1 contributes to endometrial cancer progression.

Rong Zhao, Yan Liu, Ziwei Wang, Jun Zhang, Wei Zhang, Xing Zhou, Sevjid Tsedendorj, Amarsanaa Enkhtur, Dilu Feng, Hongbo Wang.

Recent studies have shown that autophagy plays an important role in gynecological tumours, and ubiquitin modification of autophagy regulatory components is essential to regulate autophagic. In this study, we found that UBE2C affects endometrial cancer cell apoptosis and proliferation by inhibiting autophagy. Electron microscopy observation of cell ultrastructure and experimental biochemical analysis showed that EC cells with UBE2C expression knocked down display typical autophagic characteristics. Cells were cotreated with the autophagy pharmacological inhibitors chloroquine and/or bafilomycin A1, and mRFP-GFP-LC3 assays were performed to monitor autophagic flux and determine whether UBE2C suppresses the autophagy program. Investigation of the corresponding mechanism by which UBE2C inhibits autophagy revealed that UBE2C induces K48-linked SIRT1 ubiquitination and promotes ubiquitination-dependent degradation of SIRT1, subsequently reducing H4K16 deacetylation levels and epigenetically inhibiting the expression of autophagy-related genes. The results of CCK-8, Hoechst staining, and immunofluorescence assays further indicated that deletion of the autophagy-related gene BECN1 significantly attenuates UBE2C knockdown-induced cell apoptosis. Moreover, overexpression of UBE2C promoted tumor growth in the xenograft mice model. While, the introduction of rapamycin, an agonist of autophagy, successfully reversed tumor growth and apoptosis inhibition mediated by UBE2C overexpression in vitro and in vivo. Taken together, our results suggested that UBE2C-mediated ubiquitination and degradation of SIRT1 contribute to the malignant progression of endometrial cancer through epigenetic inhibition of autophagy. Implications: Our study highlights the tumorigenic role and regulatory mechanism of UBE2C in EC; UBE2C inhibits EC cell apoptosis through autophagy-related mechanisms and our findings provide new insights into the treatment of endometrial cancer.

DOI: https://doi.org/10.1158/1541-7786.MCR-22-0825
Res Pract Thromb Haemost. 2023 Feb;7(2): 100060

Platelet function studies in myeloproliferative neoplasms patients with Calreticulin or JAK2 V617F mutation.

Alexandre Guy, Khalil Helzy, Olivier Mansier, Jean-Claude Bordet, Etienne Rivière, Mathieu Fiore, Chloe James.

   Background: JAK2V617F and Calreticulin (CALR) mutations are the most frequent molecular causes of Phi-negative myeloproliferative neoplasms (MPN). Patients with CALR mutations are at lower risk of thrombosis than patients with JAK2 V617F. We hypothesized that CALR-mutated blood platelets would have platelet function defects that might explain why these patients are at lower risk of thrombosis.
Objectives: Our main objective was to explore and compare platelet function depending on the MPN molecular marker.
Methods: We analyzed platelet function in 16 patients with MPN with CALR mutations and 17 patients with JAK2 V617F mutation and compared them with healthy controls. None of these patients was taking antiplatelet therapy. We performed an extensive analysis of platelet function and measured plasmatic soluble P-selectin and CD40L levels.
Results: We observed significant defects in platelet aggregation, surface glycoprotein expression, fibrinogen binding, and granule content in platelets from patients with MPN compared with that in controls. Moreover, soluble CD40L and P-selectin levels were elevated in patients with MPN compared with that in controls, suggesting an in vivo platelet preactivation. Comparison of platelet function between patients with CALR and JAK2 V617F MPN revealed only minor differences in platelets from patients with CALR. However, these results need to be interpreted within the context of absence of an inflammatory environment that could impact platelet function during MPN.
Conclusions: These results do not support the hypothesis that calreticulin-mutated platelets have platelet function defects that could explain the lower thrombotic risk of patients with CALR.

Keywords:  JAK2V617F mutation; blood platelets; calreticulin; myeloproliferative neoplasms; platelet aggregation; thrombosis

DOI:  https://doi.org/10.1016/j.rpth.2023.100060
Oncogene. 2023 Mar 11.

UHRF1/UBE2L6/UBR4-mediated ubiquitination regulates EZH2 abundance and thereby melanocytic differentiation phenotypes in melanoma.

Gamze Kuser-Abali, Youfang Zhang, Pacman Szeto, Peinan Zhao, Samar Masoumi-Moghaddam, Clare G Fedele, Isobel Leece, Cheng Huang, Jen G Cheung, Malaka Ameratunga, Fumihito Noguchi, Miles C Andrews, Nicholas C Wong, Ralf B Schittenhelm, Mark Shackleton.

Cellular heterogeneity in cancer is linked to disease progression and therapy response, although mechanisms regulating distinct cellular states within tumors are not well understood. We identified melanin pigment content as a major source of cellular heterogeneity in melanoma and compared RNAseq data from high-pigmented (HPCs) and low-pigmented melanoma cells (LPCs), suggesting EZH2 as a master regulator of these states. EZH2 protein was found to be upregulated in LPCs and inversely correlated with melanin deposition in pigmented patient melanomas. Surprisingly, conventional EZH2 methyltransferase inhibitors, GSK126 and EPZ6438, had no effect on LPC survival, clonogenicity and pigmentation, despite fully inhibiting methyltransferase activity. In contrast, EZH2 silencing by siRNA or degradation by DZNep or MS1943 inhibited growth of LPCs and induced HPCs. As the proteasomal inhibitor MG132 induced EZH2 protein in HPCs, we evaluated ubiquitin pathway proteins in HPC vs LPCs. Biochemical assays and animal studies demonstrated that in LPCs, the E2-conjugating enzyme UBE2L6 depletes EZH2 protein in cooperation with UBR4, an E3 ligase, via ubiquitination at EZH2's K381 residue, and is downregulated in LPCs by UHRF1-mediated CpG methylation. Targeting UHRF1/UBE2L6/UBR4-mediated regulation of EZH2 offers potential for modulating the activity of this oncoprotein in contexts in which conventional EZH2 methyltransferase inhibitors are ineffective.

DOI: https://doi.org/10.1038/s41388-023-02631-8
DNA Cell Biol. 2023 Mar 17.

Cancer-Related Unconventional Protein Secretion: A New Role of the Endoplasmic Reticulum.

Kohji Yamada, Kiyotsugu Yoshida.

  Unconventional protein secretion (UPS) is a crucial mechanism controlling the localization of cytosolic proteins lacking signal peptides and is implicated in inflammation, neurodegenerative diseases, and cancer. Several previous studies on immune cells have demonstrated the mechanisms of UPS. In cancer, the active secretion of several cytosolic proteins, including PKCδ and nucleolin, has been described. Moreover, we have recently demonstrated that extended synaptotagmin 1, one of the membrane proteins of the endoplasmic reticulum, plays a critical role in UPS in liver cancer cells. Importantly, UPS in cancer cells shows characteristics that are markedly different from those of the previously known UPS, and therefore, we categorize them as cancer-related UPS (CUPS). In this article, we provide an overview of UPS mechanisms and discuss the process that leads to the naming of cancer-specific UPS as CUPS.

Keywords:  cancer; cytosolic proteins; endoplasmic reticulum; extended-synaptotagmin 1; unconventional protein secretion

DOI:  https://doi.org/10.1089/dna.2023.0044
Nat Commun. 2023 Mar 13. 14(1): 1362

De novo lipogenesis fuels adipocyte autophagosome and lysosome membrane dynamics.

Leslie A Rowland, Adilson Guilherme, Felipe Henriques, Chloe DiMarzio, Sean Munroe, Nicole Wetoska, Mark Kelly, Keith Reddig, Gregory Hendricks, Meixia Pan, Xianlin Han, Olga R Ilkayeva, Christopher B Newgard, Michael P Czech.

Adipocytes robustly synthesize fatty acids (FA) from carbohydrate through the de novo lipogenesis (DNL) pathway, yet surprisingly DNL contributes little to their abundant triglyceride stored in lipid droplets. This conundrum raises the hypothesis that adipocyte DNL instead enables membrane expansions to occur in processes like autophagy, which requires an abundant supply of phospholipids. We report here that adipocyte Fasn deficiency in vitro and in vivo markedly impairs autophagy, evident by autophagosome accumulation and severely compromised degradation of the autophagic substrate p62. Our data indicate the impairment occurs at the level of autophagosome-lysosome fusion, and indeed, loss of Fasn decreases certain membrane phosphoinositides necessary for autophagosome and lysosome maturation and fusion. Autophagy dependence on FA produced by Fasn is not fully alleviated by exogenous FA in cultured adipocytes, and interestingly, imaging studies reveal that Fasn colocalizes with nascent autophagosomes. Together, our studies identify DNL as a critical source of FAs to fuel autophagosome and lysosome maturation and fusion in adipocytes.

DOI: https://doi.org/10.1038/s41467-023-37016-8
J Cell Biol. 2023 Jun 05. pii: e202209062. [Epub ahead of print]222(6):

A general method for quantitative fractionation of mammalian cells.

Yael Udi, Wenzhu Zhang, Milana E Stein, Inna Ricardo-Lax, Hilda A Pasolli, Brian T Chait, Michael P Rout.

Subcellular fractionation in combination with mass spectrometry-based proteomics is a powerful tool to study localization of key proteins in health and disease. Here we offered a reliable and rapid method for mammalian cell fractionation, tuned for such proteomic analyses. This method proves readily applicable to different cell lines in which all the cellular contents are accounted for, while maintaining nuclear and nuclear envelope integrity. We demonstrated the method's utility by quantifying the effects of a nuclear export inhibitor on nucleoplasmic and cytoplasmic proteomes.

DOI: https://doi.org/10.1083/jcb.202209062