bims-proteo Biomed News
on Proteostasis
Issue of 2024–09–01
thirty-one papers selected by
Eric Chevet, INSERM
  1. Function of CSNK2/CK2 selectively affects the endoplasmic reticulum and the Golgi apparatus in mtor-mediated autophagy induction.
  2. USP8 PROMOTES INTRACELLULAR INFECTION by ENHANCING ESCRT-MEDIATED MEMBRANE REPAIR, LIMITING XENOPHAGY, and REDUCING OXIDATIVE STRESS.
  3. INF2-mediated actin polymerization at ER-organelle contacts regulates organelle size and movement.
  4. Calibrated ribosome profiling assesses the dynamics of ribosomal flux on transcripts.
  5. Ubiquitin gets sweet: Sugar-mediated ubiquitination regulates Nrf1 function.
  6. Development of Ligands and Degraders Targeting MAGE-A3.
  7. HSP110 is a modulator of amyloid beta (Aβ) aggregation and proteotoxicity.
  8. The ubiquitin-proteasome system regulates the formation of specialized ribosomes during high salt stress in yeast.
  9. Multifaceted role of GCN2 in tumor adaptation and therapeutic targeting.
  10. The RNA Demethylases ALKBH5 and FTO Regulate the Translation of ATF4 mRNA in Sorafenib-Treated Hepatocarcinoma Cells.
  11. Histone chaperone HIRA, promyelocytic leukemia protein, and p62/SQSTM1 coordinate to regulate inflammation during cell senescence.
  12. GCN2 drives diurnal patterns in the hepatic integrated stress response and maintains circadian rhythms in whole body metabolism during amino acid insufficiency.
  13. Lactylation stabilizes TFEB to elevate autophagy and lysosomal activity.
  14. Phosphorylation of the selective autophagy receptor TAX1BP1 by TBK1 and IKBKE/IKKi promotes Atg8-family protein-dependent clearance of MAVS aggregates.
  15. hnRNP R promotes O-GlcNAcylation of eIF4G and facilitates axonal protein synthesis.
  16. Engineering acyclovir-induced RNA nanodevices for reversible and tunable control of aptamer function.
  17. Force-induced dephosphorylation activates the cochaperone BAG3 to coordinate protein homeostasis and membrane traffic.
  18. Massively parallel measurement of protein-protein interactions by sequencing using MP3-seq.
  19. Inhibition of TOPORS ubiquitin ligase augments the efficacy of DNA hypomethylating agents through DNMT1 stabilization.
  20. Botulinum toxin intoxication requires retrograde transport and membrane translocation at the ER in RenVM neurons.
  21. Opto-CLIP reveals dynamic FMRP regulation of mRNAs upon CA1 neuronal activation.
  22. Design of intrinsically disordered protein variants with diverse structural properties.
  23. Secreted PTEN binds PLXDC2 on macrophages to drive antitumor immunity and tumor suppression.
  24. Ubiquitin-mediated endosomal stress: A novel organelle stress of early endosomes that initiates cellular signaling pathways: USP8 serves as a gatekeeper of ubiquitin-mediated endosomal stress to counteract the activation of cellular signaling pathways.
  25. Structural Analyses of a GABARAP~ATG3 Conjugate Uncover a Novel Non-covalent Ubl-E2 Backside Interaction.
  26. Functional analysis of a common BAG3 allele associated with protection from heart failure.
  27. Synthetic modification of protein surfaces to mediate induced-proximity pharmacology.
  28. TOPORS E3 ligase mediates resistance to hypomethylating agent cytotoxicity in acute myeloid leukemia cells.
  29. Chemoproteomics reveals immunogenic and tumor-associated cell surface substrates of ectokinase CK2α.
  30. The RNA-binding protein EIF4A3 promotes axon development by direct control of the cytoskeleton.
  31. Release of mitochondrial dsRNA into the cytosol is a key driver of the inflammatory phenotype of senescent cells.
  1. Autophagy. 2024 Aug 23.
    Function of CSNK2/CK2 selectively affects the endoplasmic reticulum and the Golgi apparatus in mtor-mediated autophagy induction.
    Pablo Sanz-Martinez, Rayene Berkane, Alexandra Stolz.
      Selective macroautophagy/autophagy of the endoplasmic reticulum, known as reticulophagy/ER-phagy, is essential to maintain ER homeostasis. We recently showed that members of the autophagy receptor family RETREG/FAM134 are regulated by phosphorylation-dependent ubiquitination. In an unbiased screen we had identified several kinases downstream of MTOR with profound impact on reticulophagy flux, including ATR and CSNK2/CK2. Inhibition of CSNK2 by SGC-CK2-1 prevented regulatory ubiquitination of RETREG1/FAM134B and RETREG3/FAM134C upon autophagy activation as well as the formation of high-density RETREG1- and RETREG3-clusters. Here we report on additional resource data of global proteomics upon CSNK2 and ATR inhibition, respectively. Our data suggests that the function of CSNK2 is mainly limited to the ER/reticulophagy and Golgi/Golgiphagy, while ATR inhibition by VE-822 affects the vast majority of organelles/selective autophagy pathways.
    Keywords:  Kinase signaling; LAMP1; RETREG; methuosis; selective autophagy; vacuolization
    DOI:  https://doi.org/10.1080/15548627.2024.2395725
  2. Autophagy. 2024 Aug 23.
    USP8 PROMOTES INTRACELLULAR INFECTION by ENHANCING ESCRT-MEDIATED MEMBRANE REPAIR, LIMITING XENOPHAGY, and REDUCING OXIDATIVE STRESS.
    Pallavi Chandra, Jennifer A Philips.
      The host ESCRT-machinery repairs damaged endolysosomal membranes. If damage persists, selective macroautophagy/autophagy clears the damaged compartment. Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that damages the phagosomal membrane and targets ESCRT-mediated repair as part of its virulence program. The E3 ubiquitin ligases PRKN and SMURF1 promote autophagic capture of damaged, Mtb-containing phagosomes. Because ubiquitination is a reversible process, we anticipated that host deubiquitinases (DUBs) would also be involved. Here, we screened all predicted mouse DUBs for their role in ubiquitin targeting and control of intracellular Mtb. We show that USP8 (ubiquitin specific peptidase 8) colocalizes with intracellular Mtb, recognizes phagosomal membrane damage, and is required for ESCRT-dependent membrane repair. Furthermore, we show that USP8 regulates the NFE2L2/NRF2-dependent antioxidant signature. Taken together, our study demonstrates a central role of USP8 in promoting Mtb intracellular growth by promoting phagosomal membrane repair, limiting ubiquitin-driven selective autophagy, and reducing oxidative stress.
    Keywords:  Autophagy; deubiquitinase; endomembrane damage; mycobacterium tuberculosis
    DOI:  https://doi.org/10.1080/15548627.2024.2395134
  3. Res Sq. 2024 Aug 16. pii: rs.3.rs-4720604. [Epub ahead of print]
    INF2-mediated actin polymerization at ER-organelle contacts regulates organelle size and movement.
    Cara R Schiavon, Yuning Wang, Jasmine W Feng, Stephanie Garrett, Tsung-Chang Sung, Yelena Dayn, Chunxin Wang, Richard J Youle, Omar A Quintero-Carmona, Gerald S Shadel, Uri Manor.
      Proper regulation of organelle dynamics and inter-organelle contacts is critical for cellular health and function. Both the endoplasmic reticulum (ER) and actin cytoskeleton are known to regulate organelle dynamics, but how, when, and where these two subcellular components are coordinated to control organelle dynamics remains unclear. Here, we show that ER-associated actin consistently marks mitochondrial, endosomal, and lysosomal fission sites. We also show that actin polymerization by the ER-anchored isoform of the formin protein INF2 is a key regulator of the morphology and mobility of these organelles. Together, our findings establish a mechanism by which INF2-mediated polymerization of ER-associated actin at ER-organelle contacts regulates organelle dynamics.
    DOI:  https://doi.org/10.21203/rs.3.rs-4720604/v1
  4. Nat Commun. 2024 Aug 26. 15(1): 7061
    Calibrated ribosome profiling assesses the dynamics of ribosomal flux on transcripts.
    Kotaro Tomuro, Mari Mito, Hirotaka Toh, Naohiro Kawamoto, Takahito Miyake, Siu Yu A Chow, Masao Doi, Yoshiho Ikeuchi, Yuichi Shichino, Shintaro Iwasaki.
      Ribosome profiling, which is based on deep sequencing of ribosome footprints, has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However, the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to measure ribosome numbers in transcripts. Here, we overcome these hurdles through the development of "Ribo-FilterOut", which is based on the separation of footprints from ribosome subunits by ultrafiltration, and "Ribo-Calibration", which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches estimates the number of ribosomes on a transcript, the translation initiation rate, and the overall number of translation events before its decay, all in a genome-wide manner. Moreover, our method reveals the allocation of ribosomes under heat shock stress, during aging, and across cell types. Our strategy of modified ribosome profiling measures kinetic and stoichiometric parameters of cellular translation across the transcriptome.
    DOI:  https://doi.org/10.1038/s41467-024-51258-0
  5. Mol Cell. 2024 Aug 22. pii: S1097-2765(24)00621-X. [Epub ahead of print]84(16): 3003-3005
    Ubiquitin gets sweet: Sugar-mediated ubiquitination regulates Nrf1 function.
    Alison C Mody, Christina M Woo.
      In this issue of Molecular Cell, Yoshida et al.1 report an unconventional sugar-dependent ubiquitination event on Nrf1 that disrupts Nrf1 transcriptional activation.
    DOI:  https://doi.org/10.1016/j.molcel.2024.07.023
  6. J Am Chem Soc. 2024 Aug 27.
    Development of Ligands and Degraders Targeting MAGE-A3.
    Ke Li, Mackenzie W Krone, Arseniy Butrin, Michael J Bond, Brian M Linhares, Craig M Crews.
      Type I melanoma antigen (MAGE) family members are detected in numerous tumor types, and expression is correlated with poor prognosis, high tumor grade, and increased metastasis. Type I MAGE proteins are typically restricted to reproductive tissues, but expression can recur during tumorigenesis. Several biochemical functions have been elucidated for them, and notably, MAGEs regulate proteostasis by serving as substrate recognition modules for E3 ligase complexes. The repertoire of E3 ligase complexes that can be hijacked for targeted protein degradation continues to expand, and MAGE-E3 complexes are an especially attractive platform given their cancer-selective expression. Additionally, type I MAGE-derived peptides are presented on cancer cell surfaces, so targeted MAGE degradation may increase antigen presentation and improve immunotherapy outcomes. Motivated by these applications, we developed novel, small-molecule ligands for MAGE-A3, a type I MAGE that is widely expressed in tumors and associates with TRIM28, a RING E3 ligase. Chemical matter was identified through DNA-encoded library (DEL) screening, and hit compounds were validated for in vitro binding to MAGE-A3. We obtained a cocrystal structure with a DEL analog and hypothesize that the small molecule binds at a dimer interface. We utilized this ligand to develop PROTAC molecules that induce MAGE-A3 degradation through VHL recruitment and inhibit the proliferation of MAGE-A3 positive cell lines. These ligands and degraders may serve as valuable probes for investigating MAGE-A3 biology and as foundations for the ongoing development of tumor-specific PROTACs.
    DOI:  https://doi.org/10.1021/jacs.4c05393
  7. J Neurochem. 2024 Aug 23.
    HSP110 is a modulator of amyloid beta (Aβ) aggregation and proteotoxicity.
    Sabrina Montresor, Maria Lucia Pigazzini, Sudarson Baskaran, Mira Sleiman, Govinda Adhikari, Lukas Basilicata, Luca Secker, Natascha Jacob, Yara Ehlert, Anushree Kelkar, Gurleen Kaur Kalsi, Niraj Kulkarni, Paul Spellerberg, Janine Kirstein.
      Chaperones safeguard protein homeostasis by promoting folding and preventing aggregation. HSP110 is a cytosolic chaperone that functions as a nucleotide exchange factor for the HSP70 cycle. Together with HSP70 and a J-domain protein (JDP), HSP110 maintains protein folding and resolubilizes aggregates. Interestingly, HSP110 is vital for the HSP70/110/JDP-mediated disaggregation of amyloidogenic proteins implicated in neurodegenerative diseases (i.e., α-synuclein, HTT, and tau). However, despite its abundance, HSP110 remains still an enigmatic chaperone, and its functional spectrum is not very well understood. Of note, the disaggregation activity of neurodegenerative disease-associated amyloid fibrils showed both beneficial and detrimental outcomes in vivo. To gain a more comprehensive understanding of the chaperone HSP110 in vivo, we analyzed its role in neuronal proteostasis and neurodegeneration in C. elegans. Specifically, we investigated the role of HSP110 in the regulation of amyloid beta peptide (Aβ) aggregation using an established Aβ-C. elegans model that mimics Alzheimer's disease pathology. We generated a novel C. elegans model that over-expresses hsp-110 pan-neuronally, and we also depleted hsp-110 by RNAi-mediated knockdown. We assessed Aβ aggregation in vivo and in situ by fluorescence lifetime imaging. We found that hsp-110 over-expression exacerbated Aβ aggregation and appeared to reduce the conformational variability of the Aβ aggregates, whereas hsp-110 depletion reduced aggregation more significantly in the IL2 neurons, which marked the onset of Aβ aggregation. HSP-110 also plays a central role in growth and fertility as its over-expression compromises nematode physiology. In addition, we found that HSP-110 modulation affects the autophagy pathway. While hsp-110 over-expression impairs the autophagic flux, a depletion enhances it. Thus, HSP-110 regulates multiple nodes of the proteostasis network to control amyloid protein aggregation, disaggregation, and autophagic clearance.
    Keywords:  Abeta; C. elegans; aggregation; chaperones; proteostasis
    DOI:  https://doi.org/10.1111/jnc.16214
  8. bioRxiv. 2024 Aug 15. pii: 2024.08.15.608112. [Epub ahead of print]
    The ubiquitin-proteasome system regulates the formation of specialized ribosomes during high salt stress in yeast.
    Yoon-Mo Yang, Katrin Karbstein.
      Rps26-deficient ribosomes are a physiologically relevant ribosome population which arises during osmotic stress to support the translation of mRNAs involved in the response to high salt in yeast. They are formed by binding of the chaperone Tsr2 to fully assembled ribosomes to release Rps26 when intracellular Na+ concentrations rise. Tsr2-mediated Rps26 release is reversible, enabling a rapid response that conserves ribosomes. However, because the concentration of Tsr2 relative to ribosomes is low, how the released Rps26•Tsr2 complex is managed to allow for accumulation of Rps26-deficient ribosomes to nearly 50% of all ribosomes remains unclear. Here we show that released Rps26 is degraded via the Pro/N-degron pathway, enabling the accumulation of Rps26-deficient ribosomes. Substitution of the N-terminal proline of Rps26 to serine increases the stability of free Rps26, limits the accumulation of Rps26-deficient ribosomes and renders yeast sensitive to high salt. The GID-complex, an E3 ubiquitin ligase, and its adaptor Gid4, mediate polyubiquitination of Rps26 at Lys66 and Lys70. Moreover, this ubiquitination event is required for Rps26 degradation, the accumulation of Rps26-deficient ribosomes and the high salt stress resistance. Together, the data show that targeted degradation of released Rps26 from the Rps26•Tsr2 complex allows Tsr2 to be recycled, thus facilitating multiple rounds of Rps26 release.
    Keywords:  GID complex; N-degron pathway; Rps26; specialized ribosomes
    DOI:  https://doi.org/10.1101/2024.08.15.608112
  9. Transl Oncol. 2024 Aug 22. pii: S1936-5233(24)00223-7. [Epub ahead of print]49 102096
    Multifaceted role of GCN2 in tumor adaptation and therapeutic targeting.
    Can Chen, Yaping Xie, Shenxian Qian.
      Tumor cells voraciously consume nutrients from their environment to facilitate rapid proliferation, necessitating effective strategies to manage nutrient scarcity during tumor growth and progression. A pivotal regulatory mechanism in this context is the Integrated Stress Response (ISR), which ensures cellular homeostasis under conditions such as endoplasmic reticulum stress, the unfolded protein response, and nutrient deprivation. Within the ISR framework, the kinase GCN2 is critical, orchestrating a myriad of cellular processes including the inhibition of protein synthesis, the enhancement of amino acid transport, autophagy initiation, and angiogenesis. These processes collectively enable tumor survival and adaptation under nutrient-limited conditions. Furthermore, GCN2-mediated pathways may induce apoptosis, a property exploited by specific therapeutic agents. Leveraging extensive datasets from TCGA, GEO, and GTEx projects, we conducted a pan-cancer analysis to investigate the prognostic significance of GCN2 expression across diverse cancer types. Our analysis indicates that GCN2 expression significantly varies and correlates with both adverse and favorable prognoses depending on the type of cancer, illustrating its complex role in tumorigenesis. Importantly, GCN2 also modulates the tumor immune microenvironment, influencing immune checkpoint expression and the functionality of immune cells, thereby affecting immunotherapy outcomes. This study highlights the potential of targeting GCN2 with specific inhibitors, as evidenced by their efficacy in preclinical models to augment treatment responses and combat resistance in oncology. These findings advocate for a deeper exploration of GCN2's multifaceted roles, which could pave the way for novel targeted therapies in cancer treatment, aiming to improve clinical outcomes.
    Keywords:  Drug resistance; GCN2; Inhibitor; Pan-cancer; Tumor adaptation
    DOI:  https://doi.org/10.1016/j.tranon.2024.102096
  10. Biomolecules. 2024 Aug 01. pii: 932. [Epub ahead of print]14(8):
    The RNA Demethylases ALKBH5 and FTO Regulate the Translation of ATF4 mRNA in Sorafenib-Treated Hepatocarcinoma Cells.
    Pauline Adjibade, Sergio Di-Marco, Imed-Eddine Gallouzi, Rachid Mazroui.
      Translation is one of the main gene expression steps targeted by cellular stress, commonly referred to as translational stress, which includes treatment with anticancer drugs. While translational stress blocks the translation initiation of bulk mRNAs, it nonetheless activates the translation of specific mRNAs known as short upstream open reading frames (uORFs)-mRNAs. Among these, the ATF4 mRNA encodes a transcription factor that reprograms gene expression in cells responding to various stresses. Although the stress-induced translation of the ATF4 mRNA relies on the presence of uORFs (upstream to the main ATF4 ORF), the mechanisms mediating this effect, particularly during chemoresistance, remain elusive. Here, we report that ALKBH5 (AlkB Homolog 5) and FTO (FTO: Fat mass and obesity-associated protein), the two RNA demethylating enzymes, promote the translation of ATF4 mRNA in a transformed liver cell line (Hep3B) treated with the chemotherapeutic drug sorafenib. Using the in vitro luciferase reporter translational assay, we found that depletion of both enzymes reduced the translation of the reporter ATF4 mRNA upon drug treatment. Consistently, depletion of either protein abrogates the loading of the ATF3 mRNA into translating ribosomes as assessed by polyribosome assays coupled to RT-qPCR. Collectively, these results indicate that the ALKBH5 and FTO-mediated translation of the ATF4 mRNA is regulated at its initiation step. Using in vitro methylation assays, we found that ALKBH5 is required for the inhibition of the methylation of a reporter ATF4 mRNA at a conserved adenosine (A235) site located at its uORF2, suggesting that ALKBH5-mediated translation of ATF4 mRNA involves demethylation of its A235. Preventing methylation of A235 by introducing an A/G mutation into an ATF4 mRNA reporter renders its translation insensitive to ALKBH5 depletion, supporting the role of ALKBH5 demethylation activity in translation. Finally, targeting either ALKBH5 or FTO sensitizes Hep3B to sorafenib-induced cell death, contributing to their resistance. In summary, our data show that ALKBH5 and FTO are novel factors that promote resistance to sorafenib treatment, in part by mediating the translation of ATF4 mRNA.
    Keywords:  ALKBH5; FTO; RNA methylation; stress response; translation regulation
    DOI:  https://doi.org/10.3390/biom14080932
  11. Mol Cell. 2024 Aug 16. pii: S1097-2765(24)00660-9. [Epub ahead of print]
    Histone chaperone HIRA, promyelocytic leukemia protein, and p62/SQSTM1 coordinate to regulate inflammation during cell senescence.
    Nirmalya Dasgupta, Xue Lei, Christina Huan Shi, Rouven Arnold, Marcos G Teneche, Karl N Miller, Adarsh Rajesh, Andrew Davis, Valesca Anschau, Alexandre R Campos, Rebecca Gilson, Aaron Havas, Shanshan Yin, Zong Ming Chua, Tianhui Liu, Jessica Proulx, Michael Alcaraz, Mohammed Iqbal Rather, Josue Baeza, David C Schultz, Kevin Y Yip, Shelley L Berger, Peter D Adams.
      Cellular senescence, a stress-induced stable proliferation arrest associated with an inflammatory senescence-associated secretory phenotype (SASP), is a cause of aging. In senescent cells, cytoplasmic chromatin fragments (CCFs) activate SASP via the anti-viral cGAS/STING pathway. Promyelocytic leukemia (PML) protein organizes PML nuclear bodies (NBs), which are also involved in senescence and anti-viral immunity. The HIRA histone H3.3 chaperone localizes to PML NBs in senescent cells. Here, we show that HIRA and PML are essential for SASP expression, tightly linked to HIRA's localization to PML NBs. Inactivation of HIRA does not directly block expression of nuclear factor κB (NF-κB) target genes. Instead, an H3.3-independent HIRA function activates SASP through a CCF-cGAS-STING-TBK1-NF-κB pathway. HIRA physically interacts with p62/SQSTM1, an autophagy regulator and negative SASP regulator. HIRA and p62 co-localize in PML NBs, linked to their antagonistic regulation of SASP, with PML NBs controlling their spatial configuration. These results outline a role for HIRA and PML in the regulation of SASP.
    Keywords:  CCF; HIRA; NF-κB pathway; PML; PML NBs; SASP; cGAS-STING signaling; p62/SQSTM; senescence
    DOI:  https://doi.org/10.1016/j.molcel.2024.08.006
  12. Am J Physiol Endocrinol Metab. 2024 Aug 28.
    GCN2 drives diurnal patterns in the hepatic integrated stress response and maintains circadian rhythms in whole body metabolism during amino acid insufficiency.
    Jordan L Levy, Emily T Mirek, Esther M Rodriguez, Maria J Tolentino, Brian Zalma, Troy A Roepke, Ronald C Wek, Ruifeng Cao, Tracy G Anthony.
      Disruptions in circadian rhythms are associated with increased risk of developing metabolic diseases. General control nonderepressible 2 (GCN2), a primary sensor of amino acid insufficiency and activator of the integrated stress response (ISR), has emerged as a conserved regulator of the circadian clock in multiple organisms. The objective of this study was to examine diurnal patterns in hepatic ISR activation in the liver and whole-body rhythms in metabolism. We hypothesized that GCN2 activation cues hepatic ISR signaling over a natural 24 h feeding fasting cycle. To address our objective, wild type (WT) and whole body Gcn2 knockout (GCN2 KO) mice were housed in metabolic cages and provided free access to either a Control or leucine-devoid diet (LeuD) for 8-days in total darkness. On the last day, blood and livers were collected at circadian time (CT) 3 and CT15. In livers of WT mice, GCN2 phosphorylation followed a diurnal pattern that was guided by intracellular branched chain amino acid concentrations (r2=0.93). Feeding LeuD to WT mice increased hepatic ISR activation at CT15 only. Diurnal oscillation in hepatic ISR signaling, the hepatic transcriptome including lipid metabolic genes, and triglyceride concentrations were substantially reduced or absent in GCN2 KO mice. Further, mice lacking GCN2 were unable to maintain circadian rhythms in whole body energy expenditure, respiratory exchange ratio and physical activity when fed LeuD. In conclusion, GCN2 activation functions to maintain diurnal ISR activation in the liver and has a vital role in the mechanisms by which nutrient stress affects whole-body metabolism.
    Keywords:  RNA sequencing; dietary protein quality; eukaryotic initiation factor 2 (eIF2); peripheral circadian clock
    DOI:  https://doi.org/10.1152/ajpendo.00129.2024
  13. J Cell Biol. 2024 Nov 04. pii: e202308099. [Epub ahead of print]223(11):
    Lactylation stabilizes TFEB to elevate autophagy and lysosomal activity.
    Yewei Huang, Gan Luo, Kesong Peng, Yue Song, Yusha Wang, Hongtao Zhang, Jin Li, Xiangmin Qiu, Maomao Pu, Xinchang Liu, Chao Peng, Dante Neculai, Qiming Sun, Tianhua Zhou, Pintong Huang, Wei Liu.
      The transcription factor TFEB is a major regulator of lysosomal biogenesis and autophagy. There is growing evidence that posttranslational modifications play a crucial role in regulating TFEB activity. Here, we show that lactate molecules can covalently modify TFEB, leading to its lactylation and stabilization. Mechanically, lactylation at K91 prevents TFEB from interacting with E3 ubiquitin ligase WWP2, thereby inhibiting TFEB ubiquitination and proteasome degradation, resulting in increased TFEB activity and autophagy flux. Using a specific antibody against lactylated K91, enhanced TFEB lactylation was observed in clinical human pancreatic cancer samples. Our results suggest that lactylation is a novel mode of TFEB regulation and that lactylation of TFEB may be associated with high levels of autophagy in rapidly proliferating cells, such as cancer cells.
    DOI:  https://doi.org/10.1083/jcb.202308099
  14. Autophagy. 2024 Aug 28.
    Phosphorylation of the selective autophagy receptor TAX1BP1 by TBK1 and IKBKE/IKKi promotes Atg8-family protein-dependent clearance of MAVS aggregates.
    Jesse White, Young Bong Choi, Jiawen Zhang, Mai Tram Vo, Chaoxia He, Kashif Shaikh, Edward W Harhaj.
      TAX1BP1 is a selective macroautophagy/autophagy receptor that inhibits NFKB and RIGI-like receptor (RLR) signaling to prevent excessive inflammation and maintain homeostasis. Selective autophagy receptors such as SQSTM1/p62 and OPTN are phosphorylated by the kinase TBK1 to stimulate their selective autophagy function. However, it is unknown if TAX1BP1 is regulated by TBK1 or other kinases under basal conditions or during RNA virus infection. Here, we found that TBK1 and IKBKE/IKKi function redundantly to phosphorylate TAX1BP1 and regulate its autophagic turnover through canonical macroautophagy. TAX1BP1 phosphorylation promotes its localization to lysosomes resulting in its degradation. Additionally, we found that during vesicular stomatitis virus infection, TAX1BP1 is targeted to lysosomes in an Atg8-family protein-independent manner. Furthermore, TAX1BP1 plays a critical role in the clearance of MAVS aggregates, and phosphorylation of TAX1BP1 controls its MAVS aggrephagy function. Together, our data support a model whereby TBK1 and IKBKE license TAX1BP1-selective autophagy function to inhibit MAVS and RLR signaling.
    Keywords:  Aggrephagy; IKBKE/Ikki; MAVS; TAX1BP1; TBK1; autophagy
    DOI:  https://doi.org/10.1080/15548627.2024.2394306
  15. Nat Commun. 2024 Aug 28. 15(1): 7430
    hnRNP R promotes O-GlcNAcylation of eIF4G and facilitates axonal protein synthesis.
    Abdolhossein Zare, Saeede Salehi, Jakob Bader, Cornelius Schneider, Utz Fischer, Alexander Veh, Panagiota Arampatzi, Matthias Mann, Michael Briese, Michael Sendtner.
      Motoneurons critically depend on precise spatial and temporal control of translation for axon growth and the establishment and maintenance of neuromuscular connections. While defects in local translation have been implicated in the pathogenesis of motoneuron disorders, little is known about the mechanisms regulating axonal protein synthesis. Here, we report that motoneurons derived from Hnrnpr knockout mice show reduced axon growth accompanied by lowered synthesis of cytoskeletal and synaptic components in axons. Mutant mice display denervated neuromuscular junctions and impaired motor behavior. In axons, hnRNP R is a component of translation initiation complexes and, through interaction with O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (Ogt), modulates O-GlcNAcylation of eIF4G. Restoring axonal O-GlcNAc levels rescued local protein synthesis and axon growth defects of hnRNP R knockout motoneurons. Together, these findings demonstrate a function of hnRNP R in controlling the local production of key factors required for axon growth and formation of neuromuscular innervations.
    DOI:  https://doi.org/10.1038/s41467-024-51678-y
  16. Cell Chem Biol. 2024 Aug 26. pii: S2451-9456(24)00319-2. [Epub ahead of print]
    Engineering acyclovir-induced RNA nanodevices for reversible and tunable control of aptamer function.
    Timo Hagen, Jacob L Litke, Nahian Nasir, Qian Hou, Samie R Jaffrey.
      Small molecule-regulated RNA devices have the potential to modulate diverse aspects of cellular function, but the small molecules used to date have potential toxicities limiting their use in cells. Here we describe a method for creating drug-regulated RNA nanodevices (RNs) using acyclovir, a biologically compatible small molecule with minimal toxicity. Our modular approach involves a scaffold comprising a central F30 three-way junction, an integrated acyclovir aptamer on the input arm, and a variable effector-binding aptamer on the output arm. This design allows for the rapid engineering of acyclovir-regulated RNs, facilitating temporal, tunable, and reversible control of intracellular aptamers. We demonstrate the control of the Broccoli aptamer and the iron-responsive element (IRE) by acyclovir. Regulating the IRE with acyclovir enables precise control over iron-regulatory protein (IRP) sequestration, consequently promoting the inhibition of ferroptosis. Overall, the method described here provides a platform for transforming aptamers into acyclovir-controllable antagonists against physiologic target proteins.
    Keywords:  RNA nanodevices; RNA three-way junction; acyclovir; circular RNA; drug-regulated aptamers; ferroptosis; iron-regulatory protein; iron-responisve element
    DOI:  https://doi.org/10.1016/j.chembiol.2024.07.017
  17. Curr Biol. 2024 Aug 16. pii: S0960-9822(24)01027-3. [Epub ahead of print]
    Force-induced dephosphorylation activates the cochaperone BAG3 to coordinate protein homeostasis and membrane traffic.
    Judith Ottensmeyer, Alessandra Esch, Henrique Baeta, Sandro Sieger, Yamini Gupta, Maximilian F Rathmann, Andreas Jeschke, Daniel Jacko, Kirill Schaaf, Thorsten Schiffer, Bahareh Rahimi, Lukas Lövenich, Angela Sisto, Peter F M van der Ven, Dieter O Fürst, Albert Haas, Wilhelm Bloch, Sebastian Gehlert, Bernd Hoffmann, Vincent Timmerman, Pitter F Huesgen, Jörg Höhfeld.
      Proteome maintenance in contracting skeletal and cardiac muscles depends on the chaperone-regulating protein BAG3. Reduced BAG3 activity leads to muscle weakness and heart failure in animal models and patients. BAG3 and its chaperone partners recognize mechanically damaged muscle proteins and initiate their disposal through chaperone-assisted selective autophagy (CASA). However, molecular details of the force-dependent regulation of BAG3 have remained elusive so far. Here, we demonstrate that mechanical stress triggers the dephosphorylation of BAG3 in human muscle and in isolated cells. We identify force-regulated phospho-switches in BAG3 that control CASA complex assembly and CASA activity. Differential proteomics reveal RAB GTPases, which organize membrane traffic and fusion, as dephosphorylation-dependent interactors of BAG3. In fact, RAB7A and RAB11B are shown here to be essential for CASA in skeletal muscle cells. Moreover, BAG3 dephosphorylation is also observed upon induction of mitophagy, suggesting an involvement of the cochaperone in the RAB7A-dependent autophagic engulfment of damaged mitochondria in exercised muscle. Cooperation of BAG3 with RAB7A relies on a direct interaction of both proteins, which is regulated by the nucleotide state of the GTPase and by association with the autophagosome membrane protein LC3B. Finally, we provide evidence that BAG3 and RAB7A also cooperate in non-muscle cells and propose that overactivation of CASA in RAB7A-L129F patients contributes to the loss of peripheral neurons in Charcot-Marie-Tooth neuropathy.
    Keywords:  autophagy; exercise; membrane trafficking; molecular chaperones; myopathy; neurodegeneration; protein degradation
    DOI:  https://doi.org/10.1016/j.cub.2024.07.088
  18. Nat Chem Biol. 2024 Aug 27.
    Massively parallel measurement of protein-protein interactions by sequencing using MP3-seq.
    Alexandr Baryshev, Alyssa La Fleur, Benjamin Groves, Cirstyn Michel, David Baker, Ajasja Ljubetič, Georg Seelig.
      Protein-protein interactions (PPIs) regulate many cellular processes and engineered PPIs have cell and gene therapy applications. Here, we introduce massively parallel PPI measurement by sequencing (MP3-seq), an easy-to-use and highly scalable yeast two-hybrid approach for measuring PPIs. In MP3-seq, DNA barcodes are associated with specific protein pairs and barcode enrichment can be read by sequencing to provide a direct measure of interaction strength. We show that MP3-seq is highly quantitative and scales to over 100,000 interactions. We apply MP3-seq to characterize interactions between families of rationally designed heterodimers and to investigate elements conferring specificity to coiled-coil interactions. Lastly, we predict coiled heterodimer structures using AlphaFold-Multimer (AF-M) and train linear models on physics-based energy terms to predict MP3-seq values. We find that AF-M-based models could be valuable for prescreening interactions but experimentally measuring interactions remains necessary to rank their strengths quantitatively.
    DOI:  https://doi.org/10.1038/s41589-024-01718-x
  19. Nat Commun. 2024 Aug 28. 15(1): 7359
    Inhibition of TOPORS ubiquitin ligase augments the efficacy of DNA hypomethylating agents through DNMT1 stabilization.
    Satoshi Kaito, Kazumasa Aoyama, Motohiko Oshima, Akiho Tsuchiya, Makiko Miyota, Masayuki Yamashita, Shuhei Koide, Yaeko Nakajima-Takagi, Hiroko Kozuka-Hata, Masaaki Oyama, Takao Yogo, Tomohiro Yabushita, Ryoji Ito, Masaya Ueno, Atsushi Hirao, Kaoru Tohyama, Chao Li, Kimihito Cojin Kawabata, Kiyoshi Yamaguchi, Yoichi Furukawa, Hidetaka Kosako, Akihide Yoshimi, Susumu Goyama, Yasuhito Nannya, Seishi Ogawa, Karl Agger, Kristian Helin, Satoshi Yamazaki, Haruhiko Koseki, Noriko Doki, Yuka Harada, Hironori Harada, Atsuya Nishiyama, Makoto Nakanishi, Atsushi Iwama.
      DNA hypomethylating agents (HMAs) are used for the treatment of myeloid malignancies, although their therapeutic effects have been unsatisfactory. Here we show that CRISPR-Cas9 screening reveals that knockout of topoisomerase 1-binding arginine/serine-rich protein (TOPORS), which encodes a ubiquitin/SUMO E3 ligase, augments the efficacy of HMAs on myeloid leukemic cells with little effect on normal hematopoiesis, suggesting that TOPORS is involved in resistance to HMAs. HMAs are incorporated into the DNA and trap DNA methyltransferase-1 (DNMT1) to form DNA-DNMT1 crosslinks, which undergo SUMOylation, followed by proteasomal degradation. Persistent crosslinking is cytotoxic. The TOPORS RING finger domain, which mediates ubiquitination, is responsible for HMA resistance. In TOPORS knockout cells, DNMT1 is stabilized by HMA treatment due to inefficient ubiquitination, resulting in the accumulation of unresolved SUMOylated DNMT1. This indicates that TOPORS ubiquitinates SUMOylated DNMT1, thereby promoting the resolution of DNA-DNMT1 crosslinks. Consistently, the ubiquitination inhibitor, TAK-243, and the SUMOylation inhibitor, TAK-981, show synergistic effects with HMAs through DNMT1 stabilization. Our study provides a novel HMA-based therapeutic strategy that interferes with the resolution of DNA-DNMT1 crosslinks.
    DOI:  https://doi.org/10.1038/s41467-024-50498-4
  20. Elife. 2024 Aug 28. pii: RP92806. [Epub ahead of print]12
    Botulinum toxin intoxication requires retrograde transport and membrane translocation at the ER in RenVM neurons.
    Jeremy C Yeo, Felicia P Tay, Rebecca Bennion, Omar Loss, Jacquie Maignel, Laurent Pons, Keith Foster, Matthew Beard, Frederic Bard.
      Botulinum neurotoxin A (BoNT/A) is a highly potent proteolytic toxin specific for neurons with numerous clinical and cosmetic uses. After uptake at the synapse, the protein is proposed to translocate from synaptic vesicles to the cytosol through a self-formed channel. Surprisingly, we found that after intoxication proteolysis of a fluorescent reporter occurs in the neuron soma first and then centrifugally in neurites. To investigate the molecular mechanisms at play, we use a genome-wide siRNA screen in genetically engineered neurons and identify over three hundred genes. An organelle-specific split-mNG complementation indicates BoNT/A traffic from the synapse to the soma-localized Golgi in a retromer-dependent fashion. The toxin then moves to the ER and appears to require the Sec61 complex for retro-translocation to the cytosol. Our study identifies genes and trafficking processes hijacked by the toxin, revealing a new pathway mediating BoNT/A cellular toxicity.
    Keywords:  botulinum toxin; cell biology; endoplasmic reticulum; human; intoxication; neuron; translocation
    DOI:  https://doi.org/10.7554/eLife.92806
  21. bioRxiv. 2024 Aug 14. pii: 2024.08.13.607210. [Epub ahead of print]
    Opto-CLIP reveals dynamic FMRP regulation of mRNAs upon CA1 neuronal activation.
    Ruth A Singer, Veronika Rajchin, Kwanghoon Park, Nathaniel Heintz, Robert B Darnell.
      Neuronal diversity and function are intricately linked to the dynamic regulation of RNA metabolism, including splicing, localization, and translation. Electrophysiologic studies of synaptic plasticity, models for learning and memory, are disrupted in Fragile X Syndrome (FXS). FXS is characterized by the loss of FMRP, an RNA-binding protein (RBP) known to bind and suppress translation of specific neuronal RNAs. Since molecular studies have demonstrated that synaptic plasticity in CA1 excitatory hippocampal neurons is protein-synthesis dependent, together these observations have suggested a potential role for FMRP in synaptic plasticity in FXS. To explore this model, we developed a new experimental platform, Opto-CLIP, to integrate optogenetics with cell-type specific FMRP CLIP and RiboTag in CA1 hippocampal neurons, allowing investigation of FMRP-regulated dynamics after neuronal activation. We tracked changes in FMRP binding and ribosome-associated RNA profiles 30 minutes after neuronal activation. Our findings reveal a significant reduction in FMRP-RNA binding to transcripts encoding nuclear proteins, suggesting FMRP translational inhibition may be de-repressed to allow rapid translational responses required for neuronal homeostasis. In contrast, FMRP binding to transcripts encoding synaptic targets were generally stable after activation, but all categories of targets demonstrated variability in FMRP translational control. Opto-CLIP revealed differential regulation of subsets of transcripts within CA1 neurons rapidly after depolarization, and offers promise as a generally useful platform to uncover mechanisms of RBP-mediated RNA regulation in the context of synaptic plasticity.
    DOI:  https://doi.org/10.1101/2024.08.13.607210
  22. Sci Adv. 2024 Aug 30. 10(35): eadm9926
    Design of intrinsically disordered protein variants with diverse structural properties.
    Francesco Pesce, Anne Bremer, Giulio Tesei, Jesse B Hopkins, Christy R Grace, Tanja Mittag, Kresten Lindorff-Larsen.
      Intrinsically disordered proteins (IDPs) perform a broad range of functions in biology, suggesting that the ability to design IDPs could help expand the repertoire of proteins with novel functions. Computational design of IDPs with specific conformational properties has, however, been difficult because of their substantial dynamics and structural complexity. We describe a general algorithm for designing IDPs with specific structural properties. We demonstrate the power of the algorithm by generating variants of naturally occurring IDPs that differ in compaction, long-range contacts, and propensity to phase separate. We experimentally tested and validated our designs and analyzed the sequence features that determine conformations. We show how our results are captured by a machine learning model, enabling us to speed up the algorithm. Our work expands the toolbox for computational protein design and will facilitate the design of proteins whose functions exploit the many properties afforded by protein disorder.
    DOI:  https://doi.org/10.1126/sciadv.adm9926
  23. Dev Cell. 2024 Aug 26. pii: S1534-5807(24)00486-6. [Epub ahead of print]
    Secreted PTEN binds PLXDC2 on macrophages to drive antitumor immunity and tumor suppression.
    Cheng Zhang, Hong-Ming Ma, Shuai Wu, Jia-Ming Shen, Na Zhang, Yi-Lu Xu, Cheng-Xiao Li, Ping He, Meng-Kai Ge, Xi-Li Chu, Yu-Xue Zhang, Jun-Ke Zheng, Guo-Qiang Chen, Shao-Ming Shen.
      Loss of phosphatase and tensin homolog (PTEN) has been linked to an immunosuppressive tumor microenvironment, but its underlying mechanisms remain largely enigmatic. Here, we report that PTEN can be secreted by the transmembrane emp24 domain-containing protein 10 (TMED10)-channeled protein secretion pathway. Inhibiting PTEN secretion from tumor cells contributes to immunosuppression and impairs the tumor-suppressive role of PTEN, while intratumoral injection of PTEN protein promotes antitumor immunity and suppresses tumor growth in mice. Mechanistically, extracellular PTEN binds to the plexin domain-containing protein 2 (PLXDC2) on macrophages, triggering subsequent activation of JAK2-STAT1 signaling, which switches tumor-associated macrophages (TAMs) from the immunosuppressive to inflammatory phenotype, leading to enhanced activation of CD8+ T and natural killer cells. Importantly, PTEN treatment also enhances the therapeutic efficacy of anti-PD-1 treatment in mice and reverses the immune-suppressive phenotype of patient-derived primary TAMs. These data identify a cytokine-like role of PTEN in immune activation and tumor suppression and demonstrate the therapeutic potential for extracellular administration of PTEN in cancer immunotherapy.
    Keywords:  JAK2; PLXDC2; PTEN; TMED10; antitumor immunity; macrophages; unconventional protein secretion
    DOI:  https://doi.org/10.1016/j.devcel.2024.08.003
  24. Bioessays. 2024 Aug 28. e2400127
    Ubiquitin-mediated endosomal stress: A novel organelle stress of early endosomes that initiates cellular signaling pathways: USP8 serves as a gatekeeper of ubiquitin-mediated endosomal stress to counteract the activation of cellular signaling pathways.
    Akinori Endo, Masayuki Komada, Yukiko Yoshida.
      Cells utilize diverse organelles to maintain homeostasis and to respond to extracellular stimuli. Recently, multifaceted aspects of organelle stress caused by various factors have been emerging. The endosome is an essential organelle, functioning as the central hub for membrane trafficking in cooperation with the ubiquitin system. However, knowledge regarding endosomal stress, which refers to organelle stress of the endosome, is currently limited. We recently revealed ubiquitin-mediated endosomal stress of early endosomes (EEs) and its responsive signaling pathways. These findings shed light on the relevance of ubiquitin-mediated endosomal stress to physiological and pathological processes. Here, we present a hypothesis that ubiquitin-mediated endosomal stress may have significant roles in biological contexts and that ubiquitin-specific protease 8 is a key regulator of ubiquitin clearance from EEs.
    DOI:  https://doi.org/10.1002/bies.202400127
  25. bioRxiv. 2024 Aug 15. pii: 2024.08.14.607425. [Epub ahead of print]
    Structural Analyses of a GABARAP~ATG3 Conjugate Uncover a Novel Non-covalent Ubl-E2 Backside Interaction.
    Kazuto Ohashi, Takanori Otomo.
      Members of the ATG8 family of ubiquitin-like proteins (Ubls) are conjugated to phosphatidylethanolamine (PE) in the autophagosomal membrane, where they recruit degradation substrates and facilitate membrane biogenesis. Despite this well-characterized function, the mechanisms underlying the lipidation process, including the action of the E2 enzyme ATG3, remain incompletely understood. Here, we report the crystal structure of human ATG3 conjugated to the mammalian ATG8 protein GABARAP via an isopeptide bond, mimicking the Ubl~E2 thioester intermediate. In this structure, the GABARAP~ATG3 conjugate adopts an open configuration with minimal contacts between the two proteins. Notably, the crystal lattice reveals non-covalent contacts between GABARAP and the backside of ATG3's E2 catalytic center, resulting in the formation of a helical filament of the GABARAP~ATG3 conjugate. While similar filament formations have been observed with canonical Ub~E2 conjugates, the E2 backside-binding interface of GABARAP is distinct from those of Ub/Ubl proteins and overlaps with the binding site for LC3 interacting region (LIR) peptides. NMR analysis confirms the presence of this non-covalent interaction in solution, and mutagenesis experiments demonstrate the involvement of the E2 backside in PE conjugation. These findings highlight the critical role of the E2 backside in the lipidation process and suggest evolutionary adaptations in the unique E2 enzyme ATG3.
    DOI:  https://doi.org/10.1101/2024.08.14.607425
  26. Nat Cardiovasc Res. 2023 Jul;2(7): 615-628
    Functional analysis of a common BAG3 allele associated with protection from heart failure.
    Juan A Perez-Bermejo, Luke M Judge, Christina L Jensen, Kenneth Wu, Hannah L Watry, Annie Truong, Jaclyn J Ho, Matthew Carter, Wendy V Runyon, Robyn M Kaake, Ernst H Pulido, Mohammad A Mandegar, Danielle L Swaney, Po-Lin So, Nevan J Krogan, Bruce R Conklin.
      Multiple genetic association studies have correlated a common allelic block linked to the BAG3 gene with a decreased incidence of heart failure, but the molecular mechanism remains elusive. In this study, we used induced pluripotent stem cells to test if the only coding variant in this allele block, BAG3C151R, alters protein and cellular function in human cardiomyocytes. Quantitative protein interaction analysis identified changes in BAG3C151R protein partners specific to cardiomyocytes. Knockdown of genes encoding for BAG3-interacting factors in cardiomyocytes followed by myofibrillar analysis revealed that BAG3C151R associates more strongly with proteins involved in the maintenance of myofibrillar integrity. Finally, we demonstrate that cardiomyocytes expressing the BAG3C151R variant have improved response to proteotoxic stress in a dose-dependent manner. This study suggests that BAG3C151R could be responsible for the cardioprotective effect of the haplotype block, by increasing cardiomyocyte protection from stress. Preferential binding partners of BAG3C151R may reveal potential targets for cardioprotective therapies.
    DOI:  https://doi.org/10.1038/s44161-023-00288-w
  27. RSC Med Chem. 2024 Aug 09.
    Synthetic modification of protein surfaces to mediate induced-proximity pharmacology.
    Lyn H Jones.
      Molecular glues and bifunctional small molecules, such as targeted protein degraders, induce protein proximity to mediate gain-of-function pharmacology. Emerging technologies that synthetically manipulate protein surfaces to create neoproteins, and the development of covalent chemical probes for intra- and inter-protein surface labeling are described. Ligand-directed protein surface modification strategies have the potential to enhance the induced-proximity pharmacology toolkit and expand the druggable proteome, and this Opinion considers the opportunities and challenges that lie ahead.
    DOI:  https://doi.org/10.1039/d4md00388h
  28. Nat Commun. 2024 Aug 28. 15(1): 7360
    TOPORS E3 ligase mediates resistance to hypomethylating agent cytotoxicity in acute myeloid leukemia cells.
    Peter Truong, Sylvie Shen, Swapna Joshi, Md Imtiazul Islam, Ling Zhong, Mark J Raftery, Ali Afrasiabi, Hamid Alinejad-Rokny, Mary Nguyen, Xiaoheng Zou, Golam Sarower Bhuyan, Chowdhury H Sarowar, Elaheh S Ghodousi, Olivia Stonehouse, Sara Mohamed, Cara E Toscan, Patrick Connerty, Purvi M Kakadia, Stefan K Bohlander, Katharine A Michie, Jonas Larsson, Richard B Lock, Carl R Walkley, Julie A I Thoms, Christopher J Jolly, John E Pimanda.
      Hypomethylating agents (HMAs) are frontline therapies for Myelodysplastic Neoplasms (MDS) and Acute Myeloid Leukemia (AML). However, acquired resistance and treatment failure are commonplace. To address this, we perform a genome-wide CRISPR-Cas9 screen in a human MDS-derived cell line, MDS-L, and identify TOPORS as a loss-of-function target that synergizes with HMAs, reducing leukemic burden and improving survival in xenograft models. We demonstrate that depletion of TOPORS mediates sensitivity to HMAs by predisposing leukemic blasts to an impaired DNA damage response (DDR) accompanied by an accumulation of SUMOylated DNMT1 in HMA-treated TOPORS-depleted cells. The combination of HMAs with targeting of TOPORS does not impair healthy hematopoiesis. While inhibitors of TOPORS are unavailable, we show that inhibition of protein SUMOylation with TAK-981 partially phenocopies HMA-sensitivity and DDR impairment. Overall, our data suggest that the combination of HMAs with inhibition of SUMOylation or TOPORS is a rational treatment option for High-Risk MDS (HR-MDS) or AML.
    DOI:  https://doi.org/10.1038/s41467-024-51646-6
  29. Cell Chem Biol. 2024 Aug 20. pii: S2451-9456(24)00320-9. [Epub ahead of print]
    Chemoproteomics reveals immunogenic and tumor-associated cell surface substrates of ectokinase CK2α.
    Corleone S Delaveris, Sophie Kong, Jeff Glasgow, Rita P Loudermilk, Lisa L Kirkemo, Fangzhu Zhao, Fernando Salangsang, Paul Phojanakong, Juan Antonio Camara Serrano, Veronica Steri, James A Wells.
      Foreign epitopes for immune recognition provide the basis of anticancer immunity. Due to the high concentration of extracellular adenosine triphosphate in the tumor microenvironment, we hypothesized that extracellular kinases (ectokinases) could have dysregulated activity and introduce aberrant phosphorylation sites on cell surface proteins. We engineered a cell-tethered version of the extracellular kinase CK2α, demonstrated it was active on cells under tumor-relevant conditions, and profiled its substrate scope using a chemoproteomic workflow. We then demonstrated that mice developed polyreactive antisera in response to syngeneic tumor cells that had been subjected to surface hyperphosphorylation with CK2α. Interestingly, these mice developed B cell and CD4+ T cell responses in response to these antigens but failed to develop a CD8+ T cell response. This work provides a workflow for probing the extracellular phosphoproteome and demonstrates that extracellular phosphoproteins are immunogenic even in a syngeneic system.
    DOI:  https://doi.org/10.1016/j.chembiol.2024.07.018
  30. Cell Rep. 2024 Aug 23. pii: S2211-1247(24)01017-9. [Epub ahead of print]43(9): 114666
    The RNA-binding protein EIF4A3 promotes axon development by direct control of the cytoskeleton.
    Fernando C Alsina, Bianca M Lupan, Lydia J Lin, Camila M Musso, Federica Mosti, Carly R Newman, Lisa M Wood, Aussie Suzuki, Mark Agostino, Jeffrey K Moore, Debra L Silver.
      The exon junction complex (EJC), nucleated by EIF4A3, is indispensable for mRNA fate and function throughout eukaryotes. We discover that EIF4A3 directly controls microtubules, independent of RNA, which is critical for neural wiring. While neuronal survival in the developing mouse cerebral cortex depends upon an intact EJC, axonal tract development requires only Eif4a3. Using human cortical organoids, we show that EIF4A3 disease mutations also impair neuronal growth, highlighting conserved functions relevant for neurodevelopmental pathology. Live imaging of growing neurons shows that EIF4A3 is essential for microtubule dynamics. Employing biochemistry and competition experiments, we demonstrate that EIF4A3 directly binds to microtubules, mutually exclusive of the EJC. Finally, in vitro reconstitution assays and rescue experiments demonstrate that EIF4A3 is sufficient to promote microtubule polymerization and that EIF4A3-microtubule association is a major contributor to axon growth. This reveals a fundamental mechanism by which neurons re-utilize core gene expression machinery to directly control the cytoskeleton.
    Keywords:  CP: Cell biology; CP: Neuroscience; EIF4A3; Eif4a3; RNA; axon growth; axonal tracts; cortical development; cortical organoids; exon junction complex; microtubules; neural development; neuronal maturation
    DOI:  https://doi.org/10.1016/j.celrep.2024.114666
  31. Nat Commun. 2024 Aug 27. 15(1): 7378
    Release of mitochondrial dsRNA into the cytosol is a key driver of the inflammatory phenotype of senescent cells.
    Vanessa López-Polo, Mate Maus, Emmanouil Zacharioudakis, Miguel Lafarga, Camille Stephan-Otto Attolini, Francisco D M Marques, Marta Kovatcheva, Evripidis Gavathiotis, Manuel Serrano.
      The escape of mitochondrial double-stranded dsRNA (mt-dsRNA) into the cytosol has been recently linked to a number of inflammatory diseases. Here, we report that the release of mt-dsRNA into the cytosol is a general feature of senescent cells and a critical driver of their inflammatory secretome, known as senescence-associated secretory phenotype (SASP). Inhibition of the mitochondrial RNA polymerase, the dsRNA sensors RIGI and MDA5, or the master inflammatory signaling protein MAVS, all result in reduced expression of the SASP, while broadly preserving other hallmarks of senescence. Moreover, senescent cells are hypersensitized to mt-dsRNA-driven inflammation due to their reduced levels of PNPT1 and ADAR1, two proteins critical for mitigating the accumulation of mt-dsRNA and the inflammatory potency of dsRNA, respectively. We find that mitofusin MFN1, but not MFN2, is important for the activation of the mt-dsRNA/MAVS/SASP axis and, accordingly, genetic or pharmacologic MFN1 inhibition attenuates the SASP. Finally, we report that senescent cells within fibrotic and aged tissues present dsRNA foci, and inhibition of mitochondrial RNA polymerase reduces systemic inflammation associated to senescence. In conclusion, we uncover the mt-dsRNA/MAVS/MFN1 axis as a key driver of the SASP and we identify novel therapeutic strategies for senescence-associated diseases.
    DOI:  https://doi.org/10.1038/s41467-024-51363-0
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