bims-proteo 2022-11-13 papers

bims-proteo

Biomed News

on Proteostasis

Issue of 2022–11–13
thirty papers selected by
Eric Chevet, INSERM

Differential contributions of the proteasome, autophagy, and chaperones to the clearance of arsenite-induced protein aggregates in yeast.
How cells recognize and remove the perforated lysosome.
Integration of ER protein quality control mechanisms defines β-cell function and ER architecture.
An ERAD-independent role for rhomboid pseudoprotease Dfm1 in mediating sphingolipid homeostasis.
--Atg9 interactions via its transmembrane domains are required for phagophore expansion during autophagy.
A comprehensive set of ER protein disulfide isomerase family members supports the biogenesis of pro-inflammatory interleukin 12 family cytokines.
Dissociation of ERMES clusters plays a key role in attenuating the endoplasmic reticulum stress.
Novel protein complexes containing autophagy and UPS components regulate proteasome-dependent PARK2 recruitment onto mitochondria and PARK2-PARK6 activity during mitophagy.
Ovarian tumorB1-mediated heat shock transcription factor 1 deubiquitination is critical for glycolysis and development of endometriosis.
A conserved megaprotein-based molecular bridge critical for lipid trafficking and cold resilience.
Cancer cell-intrinsic XBP1 drives immunosuppressive reprogramming of intratumoral myeloid cells by promoting cholesterol production.
IRE1-mediated cytoplasmic splicing and regulated IRE1-dependent decay of mRNA in the liverwort Marchantia polymorpha.
ER-endosome contacts master the ins and outs of secretory endosomes.
Emerging role of protein modification by UFM1 in cancer.
Structure of Escherichia coli heat shock protein Hsp15 in complex with the ribosomal 50S subunit bearing peptidyl-tRNA.
ATG9A and ATG2A form a heteromeric complex essential for autophagosome formation.
Architecture of the outbred brown fat proteome defines regulators of metabolic physiology.
Selective PROTAC-mediated degradation of SMARCA2 is efficacious in SMARCA4 mutant cancers.
A nascent peptide code for translational control of mRNA stability in human cells.
pTINCR microprotein promotes epithelial differentiation and suppresses tumor growth through CDC42 SUMOylation and activation.
EXOSC8 promotes colorectal cancer tumorigenesis via regulating ribosome biogenesis-related processes.
Ydj1 interaction at nucleotide-binding-domain of yeast Ssa1 impacts Hsp90 collaboration and client maturation.
Processes in DNA damage response from a whole-cell multi-omics perspective.
Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation.
Genetic deletion and pharmacologic inhibition of E3 ubiquitin ligase HOIP impairs the propagation of myeloid leukemia.
COPA A-to-I RNA editing hijacks endoplasmic reticulum stress to promote metastasis in colorectal cancer.
Bortezomib inhibits ZIKV/DENV by interfering with viral polyprotein cleavage via the ERAD pathway.
Integrative omics indicate FMRP sequesters mRNA from translation and deadenylation in human neuronal cells.
Secreted Mutant Calreticulins As Rogue Cytokines in Myeloproliferative Neoplasms.
Inhibition of HSF1 Signaling Decreases Hepatoblastoma Growth Via Induction of Apoptosis.

J Biol Chem. 2022 Nov 07. pii: S0021-9258(22)01123-1. [Epub ahead of print] 102680

Differential contributions of the proteasome, autophagy, and chaperones to the clearance of arsenite-induced protein aggregates in yeast.

Sansan Hua, Agnieszka Kłosowska, Joana I Rodrigues, Gabriel Petelski, Lidia A Esquembre, Emma Lorentzon, Lars F Olsen, Krzysztof Liberek, Markus J Tamás.

  The poisonous metalloid arsenite induces widespread misfolding and aggregation of nascent proteins in vivo, and this mode of toxic action might underlie its suspected role in the pathology of certain protein misfolding diseases. Evolutionarily conserved protein quality-control systems protect cells against arsenite-mediated proteotoxicity and herein, we systematically assessed the contribution of the ubiquitin-proteasome system, the autophagy-vacuole pathway, and chaperone-mediated disaggregation to the clearance of arsenite-induced protein aggregates in Saccharomyces cerevisiae. We show that the ubiquitin-proteasome system is the main pathway that clears aggregates formed during arsenite stress and that cells depend on this pathway for optimal growth. The autophagy-vacuole pathway and chaperone-mediated disaggregation both contribute to clearance, but their roles appear less prominent than the ubiquitin-proteasome system. Our in vitro assays with purified components of the yeast disaggregating machinery demonstrated that chaperone binding to aggregates formed in the presence of arsenite is impaired. Hsp104 and Hsp70 chaperone activity was unaffected by arsenite, suggesting that this metalloid influences aggregate structure, making them less accessible for chaperone-mediated disaggregation. We further show that the defect in chaperone-mediated refolding of a model protein was abrogated in a cysteine-free version of the substrate, suggesting that arsenite directly modifies cysteines in non-native target proteins. In conclusion, our study sheds novel light on the differential contributions of protein quality-control systems to aggregate clearance and cell proliferation, and extends our understanding of how these systems operate during arsenite stress.

Keywords:  Hsp104; Hsp70; arsenite; protein aggregation; protein degradation; ubiquitin proteasome pathway

DOI:  https://doi.org/10.1016/j.jbc.2022.102680
Autophagy. 2022 Nov 11. 1-3

How cells recognize and remove the perforated lysosome.

Keisuke Tabata, Marika Saeki, Tamotsu Yoshimori, Maho Hamasaki.

  Macroautophagy (hereafter autophagy) is a highly conserved intracellular degradation system to maintain cellular homeostasis by degrading cellular components such as misfolded proteins, nonfunctional organelles, pathogens, and cytosol. Conversely, selective autophagy targets and degrades specific cargo, such as organelles, bacteria, etc. We previously reported that damaged lysosomes are autophagy targets, via a process called lysophagy. However, how cells target damaged lysosomes through autophagy is not known. We performed proteomics analysis followed by siRNA screening to identify genes involved in targeting damaged lysosomes and identified a new E3 ligase complex, involving CUL4A (cullin 4A), as a regulatory complex in lysophagy. We also found that this complex mediates K48-linked poly-ubiquitination on lysosome protein LAMP2 during lysosomal damage; particularly, the lumenal side of LAMP2 is important to recruit the complex to damaged lysosomes. This protein modification is thus critical to initiate the clearance of damaged lysosomes.

Keywords:  CUL4A; LAMP2; lysophagy; lysosomal membrane damage; selective autophagy; ubiquitination

DOI:  https://doi.org/10.1080/15548627.2022.2138686
J Clin Invest. 2022 Nov 08. pii: e163584. [Epub ahead of print]

Integration of ER protein quality control mechanisms defines β-cell function and ER architecture.

Neha Shrestha, Mauricio Torres, Jason Zhang, You Lu, Leena Haataja, Rachel B Reinert, Jeffrey Knupp, Yu-Jie Chen, Gunes Parlakgul, Ana Paula Arruda, Billy Tsai, Peter Arvan, Ling Qi.

  Three principal ER quality-control mechanisms, namely, unfolded protein response (UPR), ER-associated degradation (ERAD) and ER-phagy are each important for the maintenance of ER homeostasis, yet how they are integrated to regulate ER homeostasis and organellar architecture in vivo is largely unclear. Here we report intricate crosstalk among the three pathways, centered around the SEL1L-HRD1 protein complex of ERAD, in the regulation of organellar organization in β-cells. SEL1L-HRD1 ERAD deficiency in β-cells triggers activation of autophagy via IRE1α [an endogenous ERAD substrate]. In the absence of functional SEL1L-HRD1 ERAD, proinsulin is retained in the ER as high molecular weight conformers, which are subsequently cleared via ER-phagy. A combined loss of both SEL1L and autophagy in β-cells leads to diabetes in mice shortly after weaning, with premature death by ~11 weeks of age, associated with marked ER retention of proinsulin and β-cell loss. Using focus-ion beam scanning electron microscopy (FIB-SEM) powered by deep-learning automated image segmentation and 3D reconstruction, our data demonstrate a profound organellar restructuring with a massive expansion of ER volume and network in β-cells lacking both SEL1L and autophagy. These data reveal at an unprecedented detail the intimate crosstalk among the three ER quality-control mechanisms in the dynamic regulation of organellar architecture and β-cell function.

Keywords:  Autophagy; Cell Biology; Diabetes; Metabolism; Protein misfolding

DOI:  https://doi.org/10.1172/JCI163584
EMBO J. 2022 Nov 09. e112275

An ERAD-independent role for rhomboid pseudoprotease Dfm1 in mediating sphingolipid homeostasis.

Satarupa Bhaduri, Analine Aguayo, Yusuke Ohno, Marco Proietto, Jasmine Jung, Isabel Wang, Rachel Kandel, Narinderbir Singh, Ikran Ibrahim, Amit Fulzele, Eric J Bennett, Akio Kihara, Sonya E Neal.

  Nearly one-third of nascent proteins are initially targeted to the endoplasmic reticulum (ER), where they are correctly folded and assembled before being delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated degradation (ERAD) removes these client proteins from the ER membrane to the cytosol in a process known as retrotranslocation. Our previous work demonstrated that rhomboid pseudoprotease Dfm1 is involved in the retrotranslocation of ubiquitinated membrane integral ERAD substrates. Herein, we found that Dfm1 associates with the SPOTS complex, which is composed of serine palmitoyltransferase (SPT) enzymes and accessory components that are critical for catalyzing the first rate-limiting step of the sphingolipid biosynthesis pathway. Furthermore, Dfm1 employs an ERAD-independent role for facilitating the ER export and endosome- and Golgi-associated degradation (EGAD) of Orm2, which is a major antagonist of SPT activity. Given that the accumulation of human Orm2 homologs, ORMDLs, is associated with various pathologies, our study serves as a molecular foothold for understanding how dysregulation of sphingolipid metabolism leads to various diseases.

Keywords:  ERAD; Orm1/2; endoplasmic reticulum; rhomboid pseudoprotease; sphingolipid homeostasis

DOI:  https://doi.org/10.15252/embj.2022112275
Autophagy. 2022 Nov 10. 1-20

--Atg9 interactions via its transmembrane domains are required for phagophore expansion during autophagy.

Sabrina Chumpen Ramirez, Rubén Gómez-Sánchez, Pauline Verlhac, Ralph Hardenberg, Eleonora Margheritis, Katia Cosentino, Fulvio Reggiori, Christian Ungermann.

  During macroautophagy/autophagy, precursor cisterna known as phagophores expand and sequester portions of the cytoplasm and/or organelles, and subsequently close resulting in double-membrane transport vesicles called autophagosomes. Autophagosomes fuse with lysosomes/vacuoles to allow the degradation and recycling of their cargoes. We previously showed that sequential binding of yeast Atg2 and Atg18 to Atg9, the only conserved transmembrane protein in autophagy, at the extremities of the phagophore mediates the establishment of membrane contact sites between the phagophore and the endoplasmic reticulum. As the Atg2-Atg18 complex transfers lipids between adjacent membranes in vitro, it has been postulated that this activity and the scramblase activity of the trimers formed by Atg9 are required for the phagophore expansion. Here, we present evidence that Atg9 indeed promotes Atg2-Atg18 complex-mediated lipid transfer in vitro, although this is not the only requirement for its function in vivo. In particular, we show that Atg9 function is dramatically compromised by a F627A mutation within the conserved interface between the transmembrane domains of the Atg9 monomers. Although Atg9F627A self-interacts and binds to the Atg2-Atg18 complex, the F627A mutation blocks the phagophore expansion and thus autophagy progression. This phenotype is conserved because the corresponding human ATG9A mutant severely impairs autophagy as well. Importantly, Atg9F627A has identical scramblase activity in vitro like Atg9, and as with the wild-type protein enhances Atg2-Atg18-mediated lipid transfer. Collectively, our data reveal that interactions of Atg9 trimers via their transmembrane segments play a key role in phagophore expansion beyond Atg9's role as a lipid scramblase.Abbreviations: BafA1: bafilomycin A1; Cvt: cytoplasm-to-vacuole targeting; Cryo-EM: cryo-electron microscopy; ER: endoplasmic reticulum; GFP: green fluorescent protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCS: membrane contact site; NBD-PE: N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; PAS: phagophore assembly site; PE: phosphatidylethanolamine; prApe1: precursor Ape1; PtdIns3P: phosphatidylinositol-3-phosphate; SLB: supported lipid bilayer; SUV: small unilamellar vesicle; TMD: transmembrane domain; WT: wild type.

Keywords:  Autophagosome; lipid transfer; membrane contact site; phagophore; scramblase

DOI:  https://doi.org/10.1080/15548627.2022.2136340
J Biol Chem. 2022 Nov 03. pii: S0021-9258(22)01120-6. [Epub ahead of print] 102677

A comprehensive set of ER protein disulfide isomerase family members supports the biogenesis of pro-inflammatory interleukin 12 family cytokines.

Yonatan G Mideksa, Isabel Aschenbrenner, Anja Fux, Dinah Kaylani, Caroline A M Weiß, Tuan-Anh Nguyen, Nina C Bach, Kathrin Lang, Stephan A Sieber, Matthias J Feige.

  Cytokines of the interleukin 12 (IL-12) family are assembled combinatorially from shared α and β subunits. A common theme is that human IL-12 family α subunits remain incompletely structured in isolation until they pair with a designate β subunit. Accordingly, chaperones need to support and control specific assembly processes. It remains incompletely understood, which chaperones are involved in IL-12 family biogenesis. Here, we site-specifically introduce photo-crosslinking amino acids into the IL-12 and IL-23 α subunits (IL-12α and IL-23α) for stabilization of transient chaperone:client complexes for mass spectrometry. Our analysis reveals that a large set of ER chaperones interacts with IL-12α and IL-23α. Among these chaperones, we focus on protein disulfide isomerase (PDI) family members and reveal IL-12 family subunits to be clients of several incompletely characterized PDIs. We find that different PDIs show selectivity for different cysteines in IL-12α and IL-23α. Despite this, PDI binding generally stabilizes unassembled IL-12α and IL-23α against degradation. In contrast, α:β assembly appears robust, and only multiple simultaneous PDI depletions reduce IL-12 secretion. Our comprehensive analysis of the IL-12/IL-23 chaperone machinery reveals a hitherto uncharacterized role for several PDIs in this process. This extends our understanding of how cells accomplish the task of specific protein assembly reactions for signaling processes. Furthermore, our findings show that cytokine secretion can be modulated by targeting specific ER chaperones.

Keywords:  chaperone; genetic code expansion; interleukin; protein disulfide isomerase; protein folding

DOI:  https://doi.org/10.1016/j.jbc.2022.102677
iScience. 2022 Nov 18. 25(11): 105362

Dissociation of ERMES clusters plays a key role in attenuating the endoplasmic reticulum stress.

Yuriko Kakimoto-Takeda, Rieko Kojima, Hiroya Shiino, Manatsu Shinmyo, Kazuo Kurokawa, Akihiko Nakano, Toshiya Endo, Yasushi Tamura.

  In yeast, ERMES, which mediates phospholipid transport between the ER and mitochondria, forms a limited number of oligomeric clusters at ER-mitochondria contact sites in a cell. Although the number of the ERMES clusters appears to be regulated to maintain proper inter-organelle phospholipid trafficking, its underlying mechanism and physiological relevance remain poorly understood. Here, we show that mitochondrial dynamics control the number of ERMES clusters. Moreover, we find that ER stress causes dissociation of the ERMES clusters independently of Ire1 and Hac1, canonical ER-stress response pathway components, leading to a delay in the phospholipid transport from the ER to mitochondria. Our biochemical and genetic analyses strongly suggest that the impaired phospholipid transport contributes to phospholipid accumulation in the ER, expanding the ER for ER stress attenuation. We thus propose that the ERMES dissociation constitutes an overlooked pathway of the ER stress response that operates in addition to the canonical Ire1/Hac1-dependent pathway.

Keywords:  Biological sciences; Cell biology; Functional aspects of cell biology

DOI:  https://doi.org/10.1016/j.isci.2022.105362
Cell Death Dis. 2022 Nov 10. 13(11): 947

Novel protein complexes containing autophagy and UPS components regulate proteasome-dependent PARK2 recruitment onto mitochondria and PARK2-PARK6 activity during mitophagy.

Nur Mehpare Kocaturk, Nesibe Peker, Karin Eberhart, Yunus Akkoc, Gamze Deveci, Joern Dengjel, Devrim Gozuacik.

Autophagy is an evolutionarily conserved eukaryotic cellular mechanism through which cytosolic fragments, misfolded/aggregated proteins and organelles are degraded and recycled. Priming of mitochondria through ubiquitylation is required for the clearance the organelle by autophagy (mitophagy). Familial Parkinson's Disease-related proteins, including the E3-ligase PARK2 (PARKIN) and the serine/threonine kinase PARK6 (PINK1) control these ubiquitylation reactions and contribute to the regulation of mitophagy. Here we describe, novel protein complexes containing autophagy protein ATG5 and ubiquitin-proteasome system (UPS) components. We discovered that ATG5 interacts with PSMA7 and PARK2 upon mitochondrial stress. Results suggest that all three proteins translocate mitochondria and involve in protein complexes containing autophagy, UPS and mitophagy proteins. Interestingly, PARK2 and ATG5 recruitment onto mitochondria requires proteasome components PSMA7 and PSMB5. Strikingly, we discovered that subunit of 20 S proteasome, PSMA7, is required for the progression of PARK2-PARK6-mediated mitophagy and the proteasome activity following mitochondrial stress. Our results demonstrate direct, dynamic and functional interactions between autophagy and UPS components that contribute to the regulation of mitophagy.

DOI: https://doi.org/10.1038/s41419-022-05339-x
iScience. 2022 Nov 18. 25(11): 105363

Ovarian tumorB1-mediated heat shock transcription factor 1 deubiquitination is critical for glycolysis and development of endometriosis.

Xi Ling, Jiayi Lu, Xiaoyun Wang, Lan Liu, Lu Liu, Yadi Wang, Yujun Sun, Chune Ren, Chao Lu, Zhenhai Yu.

  Endometriosis is a common chronic condition characterized by abnormal growth of the endometrium outside the uterus. Heat shock transcription factor 1 (HSF1) is a significant regulator of the proteotoxic stress response and plays an essential role in developing endometriosis. However, the mechanisms regulating HSF1 protein stability in endometriosis remain unclear. Here, we demonstrate that OTUB1 interacts with HSF1 and promotes HSF1 protein stability through deubiquitination. In addition, OTUB1 enhances glycolysis and epithelial-mesenchymal transition of endometriosis cells, leading to promote proliferation, migration, and invasion of endometriosis cells. The progression of endometriosis is inhibited in an OTUB1-knockout mouse model. In summary, OTUB1 promotes the development of endometriosis by up-regulating HSF1. OTUB1/HSF1 axis may become a new therapeutic target for endometriosis.

Keywords:  Molecular physiology; molecular biology

DOI:  https://doi.org/10.1016/j.isci.2022.105363
Nat Commun. 2022 Nov 10. 13(1): 6805

A conserved megaprotein-based molecular bridge critical for lipid trafficking and cold resilience.

Changnan Wang, Bingying Wang, Taruna Pandey, Yong Long, Jianxiu Zhang, Fiona Oh, Jessica Sima, Ruyin Guo, Yun Liu, Chao Zhang, Shaeri Mukherjee, Michael Bassik, Weichun Lin, Huichao Deng, Goncalo Vale, Jeffrey G McDonald, Kang Shen, Dengke K Ma.

Cells adapt to cold by increasing levels of unsaturated phospholipids and membrane fluidity through conserved homeostatic mechanisms. Here we report an exceptionally large and evolutionarily conserved protein LPD-3 in C. elegans that mediates lipid trafficking to confer cold resilience. We identify lpd-3 mutants in a mutagenesis screen for genetic suppressors of the lipid desaturase FAT-7. LPD-3 bridges the endoplasmic reticulum (ER) and plasma membranes (PM), forming a structurally predicted hydrophobic tunnel for lipid trafficking. lpd-3 mutants exhibit abnormal phospholipid distribution, diminished FAT-7 abundance, organismic vulnerability to cold, and are rescued by Lecithin comprising unsaturated phospholipids. Deficient lpd-3 homologues in Zebrafish and mammalian cells cause defects similar to those observed in C. elegans. As mutations in BLTP1, the human orthologue of lpd-3, cause Alkuraya-Kucinskas syndrome, LPD-3 family proteins may serve as evolutionarily conserved highway bridges critical for ER-associated non-vesicular lipid trafficking and resilience to cold stress in eukaryotic cells.

DOI: https://doi.org/10.1038/s41467-022-34450-y
Cell Metab. 2022 Nov 02. pii: S1550-4131(22)00461-2. [Epub ahead of print]

Cancer cell-intrinsic XBP1 drives immunosuppressive reprogramming of intratumoral myeloid cells by promoting cholesterol production.

Zaili Yang, Yazhen Huo, Shixin Zhou, Jingya Guo, Xiaotu Ma, Tao Li, Congli Fan, Likun Wang.

  A hostile microenvironment in tumor tissues disrupts endoplasmic reticulum homeostasis and induces the unfolded protein response (UPR). A chronic UPR in both cancer cells and tumor-infiltrating leukocytes could facilitate the evasion of immune surveillance. However, how the UPR in cancer cells cripples the anti-tumor immune response is unclear. Here, we demonstrate that, in cancer cells, the UPR component X-box binding protein 1 (XBP1) favors the synthesis and secretion of cholesterol, which activates myeloid-derived suppressor cells (MDSCs) and causes immunosuppression. Cholesterol is delivered in the form of small extracellular vesicles and internalized by MDSCs through macropinocytosis. Genetic or pharmacological depletion of XBP1 or reducing the tumor cholesterol content remarkably decreases MDSC abundance and triggers robust anti-tumor responses. Thus, our data unravel the cell-non-autonomous role of XBP1/cholesterol signaling in the regulation of tumor growth and suggest its inhibition as a useful strategy for improving the efficacy of cancer immunotherapy.

Keywords:  ER stress; HMGCR; IRE1α; MDSC; XBP1; cancer immunosuppression; cholesterol; macropinocytosis; small extracellular vesicle; unfolded protein response

DOI:  https://doi.org/10.1016/j.cmet.2022.10.010
Plant Biotechnol (Tokyo). 2022 Sep 25. 39(3): 303-310

IRE1-mediated cytoplasmic splicing and regulated IRE1-dependent decay of mRNA in the liverwort Marchantia polymorpha.

Sho Takeda, Taisuke Togawa, Kei-Ichiro Mishiba, Katsuyuki T Yamato, Yuji Iwata, Nozomu Koizumi.

  The unfolded protein response (UPR) or the endoplasmic reticulum (ER) stress response is a homeostatic cellular response conserved in eukaryotes to alleviate the accumulation of unfolded proteins in the ER. In the present study, we characterized the UPR in the liverwort Marchantia polymorpha to obtain insights into the conservation and divergence of the UPR in the land plants. We demonstrate that the most conserved UPR transducer in eukaryotes, IRE1, is conserved in M. polymorpha, which harbors a single gene encoding IRE1. We showed that MpIRE1 mediates cytoplasmic splicing of mRNA encoding MpbZIP7, a M. polymorpha homolog of bZIP60 in flowering plants, and upregulation of ER chaperone genes in response to the ER stress inducer tunicamycin. We further showed that MpIRE1 also mediates downregulation of genes encoding secretory and membrane proteins in response to ER stress, indicating the conservation of regulated IRE1-dependent decay of mRNA. Consistent with their roles in the UPR, Mpire1 ge and Mpbzip7 ge mutants exhibited higher sensitivity to ER stress. Furthermore, an Mpire1 ge mutant also exhibited retarded growth even without ER stress inducers, indicating the importance of MpIRE1 for vegetative growth in addition to alleviation of ER stress. The present study provides insights into the evolution of the UPR in land plants.

Keywords:  Marchantia polymorpha; bZIP transcription factor; cytoplasmic splicing; endoplasmic reticulum stress; unfolded protein response

DOI:  https://doi.org/10.5511/plantbiotechnology.22.0704a
J Cell Biol. 2022 Dec 05. pii: e202210033. [Epub ahead of print]221(12):

ER-endosome contacts master the ins and outs of secretory endosomes.

Eva Maria Wenzel, Camilla Raiborg.

What defines whether an endosome follows the degradative pathway or fuses with the plasma membrane to release exosomes? In this issue of JCB, Fredrik Verweij and colleagues (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202112032) demonstrate how secretory endosomes are guided by ER-endosome contacts to take a cellular detour and several identity transitions for efficient exosome release.

DOI: https://doi.org/10.1083/jcb.202210033
Biochem Biophys Res Commun. 2022 Dec 10. pii: S0006-291X(22)01238-4. [Epub ahead of print]633 61-63

Emerging role of protein modification by UFM1 in cancer.

Chin Ha Chung, Hee Min Yoo.

Ubiquitin-fold modifier 1 (UFM1) is a newly identified ubiquitin-like protein. Like ubiquitin, UFM1 is conjugated to its target proteins through a three-step enzyme system: UBA5 (E1), UFC1 (E2), and UFL1 (E3), but with an additional essential component, UFBP1. This protein modification by UFM1 (ufmylation) can be reversed by UFM1-specific proteases (UFSPs). So far only a handful of target proteins for ufmylation have been identified, and they are mostly associated with either promotion or suppression of tumorigenesis. Here, we summarize the recent progress in the knowledge of tumor-suppressive and tumorigenic functions of ufmylation as well as in the development of therapeutic drugs against ufmylation-associated cancer.

DOI: https://doi.org/10.1016/j.bbrc.2022.08.093
Nucleic Acids Res. 2022 Nov 12. pii: gkac1035. [Epub ahead of print]

Structure of Escherichia coli heat shock protein Hsp15 in complex with the ribosomal 50S subunit bearing peptidyl-tRNA.

Haaris A Safdari, Sergo Kasvandik, Christine Polte, Zoya Ignatova, Tanel Tenson, Daniel N Wilson.

In Escherichia coli, the heat shock protein 15 (Hsp15) is part of the cellular response to elevated temperature. Hsp15 interacts with peptidyl-tRNA-50S complexes that arise upon dissociation of translating 70S ribosomes, and is proposed to facilitate their rescue and recycling. A previous structure of E. coli Hsp15 in complex with peptidyl-tRNA-50S complex reported a binding site located at the central protuberance of the 50S subunit. By contrast, recent structures of RqcP, the Hsp15 homolog in Bacillus subtilis, in complex with peptidyl-tRNA-50S complexes have revealed a distinct site positioned between the anticodon-stem-loop (ASL) of the P-site tRNA and H69 of the 23S rRNA. Here we demonstrate that exposure of E. coli cells to heat shock leads to a decrease in 70S ribosomes and accumulation of 50S subunits, thus identifying a natural substrate for Hsp15 binding. Additionally, we have determined a cryo-EM reconstruction of the Hsp15-50S-peptidyl-tRNA complex isolated from heat shocked E. coli cells, revealing that Hsp15 binds to the 50S-peptidyl-tRNA complex analogously to its B. subtilis homolog RqcP. Collectively, our findings support a model where Hsp15 stabilizes the peptidyl-tRNA in the P-site and thereby promotes access to the A-site for putative rescue factors to release the aberrant nascent polypeptide chain.

DOI: https://doi.org/10.1093/nar/gkac1035
Mol Cell. 2022 Nov 01. pii: S1097-2765(22)01017-6. [Epub ahead of print]

ATG9A and ATG2A form a heteromeric complex essential for autophagosome formation.

Alexander R van Vliet, George N Chiduza, Sarah L Maslen, Valerie E Pye, Dhira Joshi, Stefano De Tito, Harold B J Jefferies, Evangelos Christodoulou, Chloë Roustan, Emma Punch, Javier H Hervás, Nicola O'Reilly, J Mark Skehel, Peter Cherepanov, Sharon A Tooze.

  ATG9A and ATG2A are essential core members of the autophagy machinery. ATG9A is a lipid scramblase that allows equilibration of lipids across a membrane bilayer, whereas ATG2A facilitates lipid flow between tethered membranes. Although both have been functionally linked during the formation of autophagosomes, the molecular details and consequences of their interaction remain unclear. By combining data from peptide arrays, crosslinking, and hydrogen-deuterium exchange mass spectrometry together with cryoelectron microscopy, we propose a molecular model of the ATG9A-2A complex. Using this integrative structure modeling approach, we identify several interfaces mediating ATG9A-2A interaction that would allow a direct transfer of lipids from ATG2A into the lipid-binding perpendicular branch of ATG9A. Mutational analyses combined with functional activity assays demonstrate their importance for autophagy, thereby shedding light on this protein complex at the heart of autophagy.

Keywords:  AlphaFold; autophagosome; autophagy; integrative structure prediction; lipid scramblase; lipid transfer

DOI:  https://doi.org/10.1016/j.molcel.2022.10.017
Cell. 2022 Nov 02. pii: S0092-8674(22)01318-6. [Epub ahead of print]

Architecture of the outbred brown fat proteome defines regulators of metabolic physiology.

Haopeng Xiao, Luiz H M Bozi, Yizhi Sun, Christopher L Riley, Vivek M Philip, Mandy Chen, Jiaming Li, Tian Zhang, Evanna L Mills, Margo P Emont, Wenfei Sun, Anita Reddy, Ryan Garrity, Jiani Long, Tobias Becher, Laura Potano Vitas, Dina Laznik-Bogoslavski, Martha Ordonez, Xinyue Liu, Xiong Chen, Yun Wang, Weihai Liu, Nhien Tran, Yitong Liu, Yang Zhang, Aaron M Cypess, Andrew P White, Yuchen He, Rebecca Deng, Heiko Schöder, Joao A Paulo, Mark P Jedrychowski, Alexander S Banks, Yu-Hua Tseng, Paul Cohen, Linus T Tsai, Evan D Rosen, Samuel Klein, Maria Chondronikola, Fiona E McAllister, Nick Van Bruggen, Edward L Huttlin, Bruce M Spiegelman, Gary A Churchill, Steven P Gygi, Edward T Chouchani.

Brown adipose tissue (BAT) regulates metabolic physiology. However, nearly all mechanistic studies of BAT protein function occur in a single inbred mouse strain, which has limited the understanding of generalizable mechanisms of BAT regulation over physiology. Here, we perform deep quantitative proteomics of BAT across a cohort of 163 genetically defined diversity outbred mice, a model that parallels the genetic and phenotypic variation found in humans. We leverage this diversity to define the functional architecture of the outbred BAT proteome, comprising 10,479 proteins. We assign co-operative functions to 2,578 proteins, enabling systematic discovery of regulators of BAT. We also identify 638 proteins that correlate with protection from, or sensitivity to, at least one parameter of metabolic disease. We use these findings to uncover SFXN5, LETMD1, and ATP1A2 as modulators of BAT thermogenesis or adiposity, and provide OPABAT as a resource for understanding the conserved mechanisms of BAT regulation over metabolic physiology.

DOI: https://doi.org/10.1016/j.cell.2022.10.003
Nat Commun. 2022 Nov 10. 13(1): 6814

Selective PROTAC-mediated degradation of SMARCA2 is efficacious in SMARCA4 mutant cancers.

Jennifer Cantley, Xiaofen Ye, Emma Rousseau, Tom Januario, Brian D Hamman, Christopher M Rose, Tommy K Cheung, Trent Hinkle, Leofal Soto, Connor Quinn, Alicia Harbin, Elizabeth Bortolon, Xin Chen, Roy Haskell, Eva Lin, Shang-Fan Yu, Geoff Del Rosario, Emily Chan, Debra Dunlap, Hartmut Koeppen, Scott Martin, Mark Merchant, Matt Grimmer, Fabio Broccatelli, Jing Wang, Jennifer Pizzano, Peter S Dragovich, Michael Berlin, Robert L Yauch.

The mammalian SWItch/Sucrose Non-Fermentable (SWI/SNF) helicase SMARCA4 is frequently mutated in cancer and inactivation results in a cellular dependence on its paralog, SMARCA2, thus making SMARCA2 an attractive synthetic lethal target. However, published data indicates that achieving a high degree of selective SMARCA2 inhibition is likely essential to afford an acceptable therapeutic index, and realizing this objective is challenging due to the homology with the SMARCA4 paralog. Herein we report the discovery of a potent and selective SMARCA2 proteolysis-targeting chimera molecule (PROTAC), A947. Selective SMARCA2 degradation is achieved in the absence of selective SMARCA2/4 PROTAC binding and translates to potent in vitro growth inhibition and in vivo efficacy in SMARCA4 mutant models, compared to wild type models. Global ubiquitin mapping and proteome profiling reveal no unexpected off-target degradation related to A947 treatment. Our study thus highlights the ability to transform a non-selective SMARCA2/4-binding ligand into a selective and efficacious in vivo SMARCA2-targeting PROTAC, and thereby provides a potential new therapeutic opportunity for patients whose tumors contain SMARCA4 mutations.

DOI: https://doi.org/10.1038/s41467-022-34562-5
Nat Commun. 2022 Nov 11. 13(1): 6829

A nascent peptide code for translational control of mRNA stability in human cells.

Phillip C Burke, Heungwon Park, Arvind Rasi Subramaniam.

Stability of eukaryotic mRNAs is associated with their codon, amino acid, and GC content. Yet, coding sequence motifs that predictably alter mRNA stability in human cells remain poorly defined. Here, we develop a massively parallel assay to measure mRNA effects of thousands of synthetic and endogenous coding sequence motifs in human cells. We identify several families of simple dipeptide repeats whose translation triggers mRNA destabilization. Rather than individual amino acids, specific combinations of bulky and positively charged amino acids are critical for the destabilizing effects of dipeptide repeats. Remarkably, dipeptide sequences that form extended β strands in silico and in vitro slowdown ribosomes and reduce mRNA levels in vivo. The resulting nascent peptide code underlies the mRNA effects of hundreds of endogenous peptide sequences in the human proteome. Our work suggests an intrinsic role for the ribosome as a selectivity filter against the synthesis of bulky and aggregation-prone peptides.

DOI: https://doi.org/10.1038/s41467-022-34664-0
Nat Commun. 2022 Nov 11. 13(1): 6840

pTINCR microprotein promotes epithelial differentiation and suppresses tumor growth through CDC42 SUMOylation and activation.

Olga Boix, Marion Martinez, Santiago Vidal, Marta Giménez-Alejandre, Lluís Palenzuela, Laura Lorenzo-Sanz, Laura Quevedo, Olivier Moscoso, Jorge Ruiz-Orera, Pilar Ximénez-Embún, Nikaoly Ciriaco, Paolo Nuciforo, Camille Stephan-Otto Attolini, M Mar Albà, Javier Muñoz, Tian V Tian, Ignacio Varela, Ana Vivancos, Santiago Ramón Y Cajal, Purificación Muñoz, Carmen Rivas, María Abad.

The human transcriptome contains thousands of small open reading frames (sORFs) that encode microproteins whose functions remain largely unexplored. Here, we show that TINCR lncRNA encodes pTINCR, an evolutionary conserved ubiquitin-like protein (UBL) expressed in many epithelia and upregulated upon differentiation and under cellular stress. By gain- and loss-of-function studies, we demonstrate that pTINCR is a key inducer of epithelial differentiation in vitro and in vivo. Interestingly, low expression of TINCR associates with worse prognosis in several epithelial cancers, and pTINCR overexpression reduces malignancy in patient-derived xenografts. At the molecular level, pTINCR binds to SUMO through its SUMO interacting motif (SIM) and to CDC42, a Rho-GTPase critical for actin cytoskeleton remodeling and epithelial differentiation. Moreover, pTINCR increases CDC42 SUMOylation and promotes its activation, triggering a pro-differentiation cascade. Our findings suggest that the microproteome is a source of new regulators of cell identity relevant for cancer.

DOI: https://doi.org/10.1038/s41467-022-34529-6
Oncogene. 2022 Nov 08.

EXOSC8 promotes colorectal cancer tumorigenesis via regulating ribosome biogenesis-related processes.

Kaisa Cui, Liang Gong, Han Zhang, Ying Chen, Bingxin Liu, Zhicheng Gong, Jiuming Li, Yuanben Wang, Shengbai Sun, Yajun Li, Qiang Zhang, Yulin Cao, Qilin Li, Bojian Fei, Zhaohui Huang.

Extensive protein synthesis is necessary for uncontrolled cancer cell proliferation, requiring hyperactive ribosome biogenesis. Our previous Pan-cancer study has identified EXOSC8 as a potential copy number variation (CNV)-driven rRNA metabolism-related oncogene in colorectal cancer (CRC). Herein, we further investigated proliferation-prompting functions and mechanisms of EXOSC8 in CRC by performing in silico analyses and wet-lab experiments. We uncovered that increased EXOSC8 expression and CNV levels are strongly associated with ribosome biogenesis-related factor levels in CRC, including ribosome proteins (RPs), eukaryotic translation initiation factors and RNA polymerase I/III. EXOSC8 silence decreases nucleolar protein and proliferation marker levels, as well as rRNA/DNA and global protein syntheses. Clinically, EXOSC8 is upregulated across human cancers, particularly CNV-driven upregulation in CRC was markedly associated with poor clinical outcomes. Mechanistically, EXOSC8 knockdown increased p53 levels in CRC, and the oncogenic proliferation phenotypes of EXOSC8 depended on p53 in vitro and in vivo. We discovered that EXOSC8 knockdown in CRC cells triggers ribosomal stress, nucleolar RPL5/11 being released into the nucleoplasm and "hijacking" Mdm2 to block its E3 ubiquitin ligase function, thus releasing and activating p53. Furthermore, our therapeutic experiments provided initial evidence that EXOSC8 might serve as a potential therapeutic target in CRC. Our findings revealed, for the first time, that the RNA exosome gene (EXOSC8) promotes CRC tumorigenesis by regulating cancer-related ribosome biogenesis in CRC. This study further extends our previous Pan-cancer study of the rRNA metabolism-related genes. The inhibition of EXOSC8 is a novel therapeutic strategy for the RPs-Mdm2-p53 ribosome biogenesis surveillance pathway in CRC.

DOI: https://doi.org/10.1038/s41388-022-02530-4
PLoS Genet. 2022 11;18(11): e1010442

Ydj1 interaction at nucleotide-binding-domain of yeast Ssa1 impacts Hsp90 collaboration and client maturation.

Deepika Gaur, Navinder Kumar, Abhirupa Ghosh, Prashant Singh, Pradeep Kumar, Jyoti Guleria, Satinderdeep Kaur, Nikhil Malik, Sudipto Saha, Thomas Nystrom, Deepak Sharma.

Hsp90 constitutes one of the major chaperone machinery in the cell. The Hsp70 assists Hsp90 in its client maturation though the underlying basis of the Hsp70 role remains to be explored. In the present study, using S. cerevisiae strain expressing Ssa1 as sole Ssa Hsp70, we identified novel mutations in the nucleotide-binding domain of yeast Ssa1 Hsp70 (Ssa1-T175N and Ssa1-D158N) that adversely affect the maturation of Hsp90 clients v-Src and Ste11. The identified Ssa1 amino acids critical for Hsp90 function were also found to be conserved across species such as in E.coli DnaK and the constitutive Hsp70 isoform (HspA8) in humans. These mutations are distal to the C-terminus of Hsp70, that primarily mediates Hsp90 interaction through the bridge protein Sti1, and proximal to Ydj1 (Hsp40 co-chaperone of Hsp70 family) binding region. Intriguingly, we found that the bridge protein Sti1 is critical for cellular viability in cells expressing Ssa1-T175N (A1-T175N) or Ssa1-D158N (A1-D158N) as sole Ssa Hsp70. The growth defect was specific for sti1Δ, as deletion of none of the other Hsp90 co-chaperones showed lethality in A1-T175N or A1-D158N. Mass-spectrometry based whole proteome analysis of A1-T175N cells lacking Sti1 showed an altered abundance of various kinases and transcription factors suggesting compromised Hsp90 activity. Further proteomic analysis showed that pathways involved in signaling, signal transduction, and protein phosphorylation are markedly downregulated in the A1-T175N upon repressing Sti1 expression using doxycycline regulatable promoter. In contrast to Ssa1, the homologous mutations in Ssa4 (Ssa4-T175N/D158N), the stress inducible Hsp70 isoform, supported cell growth even in the absence of Sti1. Overall, our data suggest that Ydj1 competes with Hsp90 for binding to Hsp70, and thus regulates Hsp90 interaction with the nucleotide-binding domain of Hsp70. The study thus provides new insight into the Hsp70-mediated regulation of Hsp90 and broadens our understanding of the intricate complexities of the Hsp70-Hsp90 network.

DOI: https://doi.org/10.1371/journal.pgen.1010442
iScience. 2022 Nov 18. 25(11): 105341

Processes in DNA damage response from a whole-cell multi-omics perspective.

James C Pino, Alexander L R Lubbock, Leonard A Harris, Danielle B Gutierrez, Melissa A Farrow, Nicole Muszynski, Tina Tsui, Stacy D Sherrod, Jeremy L Norris, John A McLean, Richard M Caprioli, John P Wikswo, Carlos F Lopez.

  Technological advances have made it feasible to collect multi-condition multi-omic time courses of cellular response to perturbation, but the complexity of these datasets impedes discovery due to challenges in data management, analysis, visualization, and interpretation. Here, we report a whole-cell mechanistic analysis of HL-60 cellular response to bendamustine. We integrate both enrichment and network analysis to show the progression of DNA damage and programmed cell death over time in molecular, pathway, and process-level detail using an interactive analysis framework for multi-omics data. Our framework, Mechanism of Action Generator Involving Network analysis (MAGINE), automates network construction and enrichment analysis across multiple samples and platforms, which can be integrated into our annotated gene-set network to combine the strengths of networks and ontology-driven analysis. Taken together, our work demonstrates how multi-omics integration can be used to explore signaling processes at various resolutions and demonstrates multi-pathway involvement beyond the canonical bendamustine mechanism.

Keywords:  Bioinformatics; Complex system biology; Data processing in systems biology; Systems biology

DOI:  https://doi.org/10.1016/j.isci.2022.105341
Cell. 2022 Nov 02. pii: S0092-8674(22)01320-4. [Epub ahead of print]

Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation.

Jinfan Wang, Byung-Sik Shin, Carlos Alvarado, Joo-Ran Kim, Jonathan Bohlen, Thomas E Dever, Joseph D Puglisi.

  How the eukaryotic 43S preinitiation complex scans along the 5' untranslated region (5' UTR) of a capped mRNA to locate the correct start codon remains elusive. Here, we directly track yeast 43S-mRNA binding, scanning, and 60S subunit joining by real-time single-molecule fluorescence spectroscopy. 43S engagement with mRNA occurs through a slow, ATP-dependent process driven by multiple initiation factors including the helicase eIF4A. Once engaged, 43S scanning occurs rapidly and directionally at ∼100 nucleotides per second, independent of multiple cycles of ATP hydrolysis by RNA helicases post ribosomal loading. Scanning ribosomes can proceed through RNA secondary structures, but 5' UTR hairpin sequences near start codons drive scanning ribosomes at start codons backward in the 5' direction, requiring rescanning to arrive once more at a start codon. Direct observation of scanning ribosomes provides a mechanistic framework for translational regulation by 5' UTR structures and upstream near-cognate start codons.

Keywords:  5′ UTR; RNA helicases; RNA secondary structures; dynamics; eukaryotic ribosome; near-cognate start codons; scanning; single-molecule fluorescence spectroscopy; translation initiation; translation regulation

DOI:  https://doi.org/10.1016/j.cell.2022.10.005
Leukemia. 2022 Nov 09.

Genetic deletion and pharmacologic inhibition of E3 ubiquitin ligase HOIP impairs the propagation of myeloid leukemia.

Koji Jimbo, Ayuna Hattori, Shuhei Koide, Takahiro Ito, Katsuhiro Sasaki, Kazuhiro Iwai, Yasuhito Nannya, Atsushi Iwama, Arinobu Tojo, Takaaki Konuma.

We investigated the role of Hoip, a catalytic subunit of linear ubiquitin chain assembly complex (LUBAC), in adult hematopoiesis and myeloid leukemia by using both conditional deletion of Hoip and small-molecule chemical inhibitors of Hoip. Conditional deletion of Hoip led to significantly longer survival and marked depletion of leukemia burden in murine myeloid leukemia models. Nevertheless, a competitive transplantation assay showed the reduction of donor-derived cells in the bone marrow of recipient mice was relatively mild after conditional deletion of Hoip. Although both Hoip-deficient hematopoietic stem cells (HSCs) and leukemia stem cells (LSCs) impaired the maintenance of quiescence, conditional deletion of Hoipinduced apoptosis in LSCs but not HSCs in vivo. Structure-function analysis revealed that LUBAC ligase activity and the interaction of LUBAC subunits were critical for the propagation of leukemia. Hoip regulated oxidative phosphorylation pathway independently of nuclear factor kappa B pathway in leukemia, but not in normal hematopoietic cells. Finally, the administration of thiolutin, which inhibits the catalytic activity of Hoip, improved the survival of recipients in murine myeloid leukemia and suppressed propagation in the patient-derived xenograft model of myeloid leukemia. Collectively, these data indicate that inhibition of LUBAC activity may be a valid therapeutic target for myeloid leukemia.

DOI: https://doi.org/10.1038/s41375-022-01750-7
Cancer Lett. 2022 Nov 03. pii: S0304-3835(22)00482-7. [Epub ahead of print] 215995

COPA A-to-I RNA editing hijacks endoplasmic reticulum stress to promote metastasis in colorectal cancer.

Shu-Yang Wang, Ling-Jie Zhang, Guo-Jun Chen, Qi-Qi Ni, Yuan Huang, Dan Zhang, Fang-Yi Han, Wen-Feng He, Li-Ling He, Yan-Qing Ding, Hong-Li Jiao, Ya-Ping Ye.

  RNA editing is among the most common RNA level modifications for generating amino acid changes. We identified a COPA A-to-I RNA editing event in CRC metastasis. Our results showed that the COPA A-to-I RNA editing rate was significantly increased in metastatic CRC tissues and was closely associated with aggressive tumors in the T and N stages. The COPA I164V protein damaged the Golgi-ER reverse transport function, induced ER stress, promoted the translocation of the transcription factors ATF6, XBP1 and ATF4 into the nucleus, and activated the expression of MALAT1, MET, ZEB1, and lead to CRC cell invasion and metastasis. Moreover, the COPA A-to-I RNA editing rate was positively correlated with the immune infiltration score. Collectively, the COPA I164V protein hijacked ER stress to promote the metastasis of CRC, and the COPA A-to-I RNA editing rate may be a potential predictor for patient response to immune checkpoint inhibitor (ICIs) treatment.

Keywords:  COPA; ER stress; Immune checkpoint inhibitors (ICIs); RNA editing

DOI:  https://doi.org/10.1016/j.canlet.2022.215995
Cell Chem Biol. 2022 Oct 19. pii: S2451-9456(22)00357-9. [Epub ahead of print]

Bortezomib inhibits ZIKV/DENV by interfering with viral polyprotein cleavage via the ERAD pathway.

Yali Ci, Bin Yao, Kun Yue, Yang Yang, Caimin Xu, De-Feng Li, Cheng-Feng Qin, Lei Shi.

  Flaviviruses have posed a serious threat to human health in the past decades, and effective therapeutic drugs are lacking; thus, treatment of flavivirus infection is a great challenge. The flavivirus protease NS2B3 is an attractive target for antiviral drug screening. Here, we developed an intracellular Zika virus (ZIKV) NS2AB3 self-cleavage assay to identify inhibitors that interfere with viral polyprotein cleavage and block ZIKV/dengue virus (DENV) replication. Bortezomib was identified as the most potent inhibitor, with a half-maximal effective concentration (EC50) in the nanomolar range. We found that instead of directly inhibiting NS2B3 protease activity, bortezomib dramatically induced the ubiquitination and aggregation of NS3, leading to the attenuation of its protease activity in cells. Two E3 ligases, HRD1 and RNF126, were found to be responsible for NS3 ubiquitination. Our study identifies bortezomib as a potential drug for the treatment of ZIKV/DENV infection and reveals the central role of the ERAD pathway in the inhibition of flaviviruses by bortezomib.

Keywords:  E3 ligase; ERAD; NS2B3 protease; bortezomib; flavivirus; inhibitor; ubiquitination

DOI:  https://doi.org/10.1016/j.chembiol.2022.10.003
Mol Cell. 2022 Nov 01. pii: S1097-2765(22)01018-8. [Epub ahead of print]

Integrative omics indicate FMRP sequesters mRNA from translation and deadenylation in human neuronal cells.

Tatsuaki Kurosaki, Shuhei Mitsutomi, Alexander Hewko, Nobuyoshi Akimitsu, Lynne E Maquat.

  How fragile X syndrome protein (FMRP) binds mRNAs and regulates mRNA metabolism remains unclear. Our previous work using human neuronal cells focused on mRNAs targeted for nonsense-mediated mRNA decay (NMD), which we showed are generally bound by FMRP and destabilized upon FMRP loss. Here, we identify >400 high-confidence FMRP-bound mRNAs, only ∼35% of which are NMD targets. Integrative transcriptomics together with SILAC-LC-MS/MS reveal that FMRP loss generally results in mRNA destabilization and more protein produced per FMRP target. We use our established RIP-seq technology to show that FMRP footprints are independent of protein-coding potential, target GC-rich and structured sequences, and are densest in 5' UTRs. Regardless of where within an mRNA FMRP binds, we find that FMRP protects mRNAs from deadenylation and directly binds the cytoplasmic poly(A)-binding protein. Our results reveal how FMRP sequesters polyadenylated mRNAs into stabilized and translationally repressed complexes, whose regulation is critical for neurogenesis and synaptic plasticity.

Keywords:  FMRP RIP-seq footprinting; SILAC-LC-MS/MS; deadenylation; fragile X syndrome protein; human neuroblastoma cells; mRNA decay; mRNA translation; poly(A)-binding protein; translationally silenced and stabilized FMRP-mRNA complexes

DOI:  https://doi.org/10.1016/j.molcel.2022.10.018
Blood. 2022 Nov 10. pii: blood.2022016846. [Epub ahead of print]

Secreted Mutant Calreticulins As Rogue Cytokines in Myeloproliferative Neoplasms.

Christian Pecquet, Nicolas Papadopoulos, Thomas Balligand, Ilyas Chachoua, Amandine Tisserand, Gaëlle Vertenoeil, Audrey Nédélec, Didier Vertommen, Anita Roy, Caroline Marty, Harini Nivarthi, Jean-Philippe Defour, Mira El-Khoury, Eva Hug, Andrea Majoros, Erica Xu, Oleh Zagrijtschuk, Tudor Emanuel Fertig, Daciana S Marta, Heinz Gisslinger, Bettina Gisslinger, Martin Schalling, Ilaria Carola Casetti, Elisa Rumi, Daniela Pietra, Chiara Cavalloni, Luca Arcaini, Mario Cazzola, Norio Komatsu, Yoshihiko Kihara, Yoshitaka Sunami, Yoko Edahiro, Marito Araki, Roman Lesyk, Veronika Buxhofer-Ausch, Sonja Heibl, Florence Pasquier, Isabelle Plo, William Vainchenker, Robert Kralovics, Stefan N Constantinescu.

Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino-acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160ng/mL, with a mean of 25.64ng/mL. Plasma mutant CALR is found in complex with soluble Transferrin Receptor 1 (sTFRC) that acts as a carrier protein and increases CALR mutant half-life. Recombinant mutant CALR proteins bound and activated the TpoR on cell lines and primary megakaryocytic progenitors from CALR-mutated patients where they drive Tpo-independent colony formation. Importantly, the CALR-sTFRC complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in one cell can specifically interact in trans with the TpoR on a target cell. Cells that carry both TpoR and mutant CALR are hypersensitive to exogenous CALR mutant proteins in comparison to cells that only carry TpoR, and respond to levels of CALR mutant proteins similar to those in patient plasma. This is consistent with CALR mutated cells exposing at the cell-surface TpoR carrying immature N-linked sugars. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

DOI: https://doi.org/10.1182/blood.2022016846
Am J Pathol. 2022 Nov 03. pii: S0002-9440(22)00355-8. [Epub ahead of print]

Inhibition of HSF1 Signaling Decreases Hepatoblastoma Growth Via Induction of Apoptosis.

Edward H Hurley, Junyan Tao, Silvia Liu, Yekaterina Krutsenko, Sucha Singh, Satdarshan P Monga.

While rare compared to adult liver cancers, hepatoblastoma is the most common pediatric liver malignancy and its incidence is increasing. Treatment currently includes surgical resection with or without chemotherapy but in severe cases children require liver transplantation. The effort to develop more target, hepatoblastoma specific therapies have been stymied by the lack of fundamental knowledge about the hepatoblastoma biology. Heat shock factor 1 (HSF1) is a transcription factor that is a canonical inducer of heat shock proteins (HSPs), which act as chaperone proteins to prevent or undue protein misfolding. Recent work has shown a role for HSF1 in cancer beyond the canonical heat shock response. We found increased HSF1 signaling in hepatoblastoma versus normal liver. We also found that less differentiated, more embryonic tumors had higher levels of HSF1 than more differentiated, more fetal appearing tumors. Most strikingly, we found HSF1 expression levels correlated with mortality. Our lab has discovered a method to make hepatoblastoma-like disease develop in mice. We used this model to test how inhibiting HSF1 early in tumor development would impact cancer growth. We found less and smaller tumors when HSF1 was inhibited suggesting HSF1 is needed for aggressive tumor growth. Moreover, increased apoptosis in tumor foci was noted when HSF1 is inhibited. These data suggest HSF1 may be a viable pharmacologic target for hepatoblastoma treatment.

DOI: https://doi.org/10.1016/j.ajpath.2022.10.006