bims-proteo Biomed News
on Proteostasis
Issue of 2024‒01‒21
forty papers selected by
Eric Chevet, INSERM
  1. The integrated stress response effector GADD34 is repurposed by neurons to promote stimulus-induced translation.
  2. Decoding the function of Atg13 phosphorylation reveals a role of Atg11 in bulk autophagy initiation.
  3. Simultaneous substrate and ubiquitin modification recognition by bispecific antibodies enables detection of ubiquitinated RIP1 and RIP2.
  4. Interaction of PINK1 with nucleotides and kinetin.
  5. The phosphatase DUSP22 inhibits UBR2-mediated K63-ubiquitination and activation of Lck downstream of TCR signalling.
  6. Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion.
  7. Sculpting nuclear envelope identity from the endoplasmic reticulum during the cell cycle.
  8. The V-ATPase-ATG16L1 axis recruits LRRK2 to facilitate the lysosomal stress response.
  9. TRIB3-TRIM8 complex drives NAFLD progression by regulating HNF4α stability.
  10. Transcriptional profile of ribosome-associated quality control components and their associated phenotypes in mammalian cells.
  11. Hypomorphic human SEL1L and HRD1 variants uncouple multilayered ER-associated degradation machinery.
  12. Mechanisms of RNF168 nucleosome recognition and ubiquitylation.
  13. Molecular glue degraders: exciting opportunities for novel drug discovery.
  14. Glutathione-dependent depalmitoylation of phospholemman by peroxiredoxin 6.
  15. Activation of Ca2+ phosphatase Calcineurin regulates Parkin translocation to mitochondria and mitophagy in flies.
  16. NAC and Zuotin/Hsp70 chaperone systems coexist at the ribosome tunnel exit in vivo.
  17. Induced degradation of lineage-specific oncoproteins drives the therapeutic vulnerability of small cell lung cancer to PARP inhibitors.
  18. miR-217 regulates normal and tumor cell fate following induction of Endoplasmic Reticulum Stress.
  19. Intramembrane protease SPP defines a cholesterol-regulated abundance control of the mevalonate pathway enzyme squalene synthase.
  20. Ribogenesis boosts controlled by HEATR1-MYC interplay promote transition into brain tumour growth.
  21. Selective CK1α degraders exert antiproliferative activity against a broad range of human cancer cell lines.
  22. Riboproteome remodeling during quiescence exit in Saccharomyces cerevisiae.
  23. Covalent inhibition of pro-apoptotic BAX.
  24. Akt enhances the vulnerability of cancer cells to VCP/p97 inhibition-mediated paraptosis.
  25. Cochaperones convey the energy of ATP hydrolysis for directional action of Hsp90.
  26. The dimeric deubiquitinase USP28 integrates 53BP1 and MYC functions to limit DNA damage.
  27. N-Cyanopiperazines as Specific Covalent Inhibitors of the Deubiquitinating Enzyme UCHL1.
  28. STIM1 translocation to the nucleus protects cells from DNA damage.
  29. MITOL deficiency triggers hematopoietic stem cell apoptosis via ER stress response.
  30. MinD-RNase E interplay controls localization of polar mRNAs in E. coli.
  31. From interaction networks to interfaces, scanning intrinsically disordered regions using AlphaFold2.
  32. Protein quality control: from molecular mechanisms to therapeutic intervention-EMBO workshop, May 21-26 2023, Srebreno, Croatia.
  33. Glioblastoma and its treatment are associated with extensive accelerated brain aging.
  34. The beauty and complexity of the small heat shock proteins: a report on the proceedings of the fourth workshop on small heat shock proteins.
  35. Systematic discovery of protein interaction interfaces using AlphaFold and experimental validation.
  36. Structural assembly of the bacterial essential interactome.
  37. Drugging Protein Tyrosine Phosphatases through Targeted Protein Degradation.
  38. DDRGK1-mediated ER-phagy attenuates acute kidney injury through ER-stress and apoptosis.
  39. Recruitment of BAG2 to DNAJ-PKAc scaffolds promotes cell survival and resistance to drug-induced apoptosis in fibrolamellar carcinoma.
  40. Organelle proteomic profiling reveals lysosomal heterogeneity in association with longevity.
  1. Cell Rep. 2024 Jan 13. pii: S2211-1247(23)01681-9. [Epub ahead of print]43(2): 113670
    The integrated stress response effector GADD34 is repurposed by neurons to promote stimulus-induced translation.
    Mauricio M Oliveira, Muhaned Mohamed, Megan K Elder, Keylin Banegas-Morales, Maggie Mamcarz, Emily H Lu, Ela A N Golhan, Nishika Navrange, Snehajyoti Chatterjee, Ted Abel, Eric Klann.
      Neuronal protein synthesis is required for long-lasting plasticity and long-term memory consolidation. Dephosphorylation of eukaryotic initiation factor 2α is one of the key translational control events that is required to increase de novo protein synthesis that underlies long-lasting plasticity and memory consolidation. Here, we interrogate the molecular pathways of translational control that are triggered by neuronal stimulation with brain-derived neurotrophic factor (BDNF), which results in eukaryotic initiation factor 2α (eIF2α) dephosphorylation and increases in de novo protein synthesis. Primary rodent neurons exposed to BDNF display elevated translation of GADD34, which facilitates eIF2α dephosphorylation and subsequent de novo protein synthesis. Furthermore, GADD34 requires G-actin generated by cofilin to dephosphorylate eIF2α and enhance protein synthesis. Finally, GADD34 is required for BDNF-induced translation of synaptic plasticity-related proteins. Overall, we provide evidence that neurons repurpose GADD34, an effector of the integrated stress response, as an orchestrator of rapid increases in eIF2-dependent translation in response to plasticity-inducing stimuli.
    Keywords:  BDNF; CP: Molecular biology; CP: Neuroscience; GADD34; cytoskeleton; eIF2α; local translation; mRNA translation; neuron; neuronal plasticity; protein synthesis; synaptic plasticity
    DOI:  https://doi.org/10.1016/j.celrep.2023.113670
  2. EMBO Rep. 2024 Jan 17.
    Decoding the function of Atg13 phosphorylation reveals a role of Atg11 in bulk autophagy initiation.
    Anuradha Bhattacharya, Raffaela Torggler, Wolfgang Reiter, Natalie Romanov, Mariya Licheva, Akif Ciftci, Muriel Mari, Lena Kolb, Dominik Kaiser, Fulvio Reggiori, Gustav Ammerer, David M Hollenstein, Claudine Kraft.
      Autophagy is initiated by the assembly of multiple autophagy-related proteins that form the phagophore assembly site where autophagosomes are formed. Atg13 is essential early in this process, and a hub of extensive phosphorylation. How these multiple phosphorylations contribute to autophagy initiation, however, is not well understood. Here we comprehensively analyze the role of phosphorylation events on Atg13 during nutrient-rich conditions and nitrogen starvation. We identify and functionally characterize 48 in vivo phosphorylation sites on Atg13. By generating reciprocal mutants, which mimic the dephosphorylated active and phosphorylated inactive state of Atg13, we observe that disrupting the dynamic regulation of Atg13 leads to insufficient or excessive autophagy, which are both detrimental to cell survival. We furthermore demonstrate an involvement of Atg11 in bulk autophagy even during nitrogen starvation, where it contributes together with Atg1 to the multivalency that drives phase separation of the phagophore assembly site. These findings reveal the importance of post-translational regulation on Atg13 early during autophagy initiation, which provides additional layers of regulation to control bulk autophagy activity and integrate cellular signals.
    Keywords:  Atg1 Kinase Complex; Atg11; Atg13; Autophagy; PAS Formation
    DOI:  https://doi.org/10.1038/s44319-023-00055-9
  3. Sci Signal. 2024 Jan 16. 17(819): eabn1101
    Simultaneous substrate and ubiquitin modification recognition by bispecific antibodies enables detection of ubiquitinated RIP1 and RIP2.
    Tatiana Goncharov, László G Kőműves, Matthias Kist, Erick R Castellanos, Axel Witt, Anna V Fedorova, Anita Izrael-Tomasevic, Kebing Yu, Mary Keir, Marissa L Matsumoto, Domagoj Vucic.
      Ubiquitination is a posttranslational modification that is crucial for the dynamic regulation of diverse signaling pathways. To enhance our understanding of ubiquitination-mediated signaling, we generated a new class of bispecific antibodies that combine recognition of ubiquitination substrates and specific polyubiquitin linkages. RIP1-K63 and RIP1-linear (Lin) linkage polyubiquitin bispecific antibodies detected linkage-specific ubiquitination of the proinflammatory kinase RIP1 in cells and in tissues and revealed RIP1 ubiquitination by immunofluorescence. Similarly, ubiquitination of the RIP1-related kinase RIP2 with K63 or linear linkages was specifically detected with the RIP2-K63 and RIP2-Lin bispecific antibodies, respectively. Furthermore, using the RIP2-K63 and RIP2-Lin bispecific antibodies, we found prominent K63-linked and linear RIP2 ubiquitination in samples from patients with ulcerative colitis and Crohn's disease. We also developed a bispecific antibody (K63-Lin) that simultaneously recognizes K63-linked and linear ubiquitination of components of various signaling pathways. Together, these bispecific antibodies represent a new class of reagents with the potential to be developed for the detection of inflammatory biomarkers.
    DOI:  https://doi.org/10.1126/scisignal.abn1101
  4. Sci Adv. 2024 Jan 19. 10(3): eadj7408
    Interaction of PINK1 with nucleotides and kinetin.
    Zhong Yan Gan, Sylvie Callegari, Thanh N Nguyen, Nicholas S Kirk, Andrew Leis, Michael Lazarou, Grant Dewson, David Komander.
      The ubiquitin kinase PINK1 accumulates on damaged mitochondria to trigger mitophagy, and PINK1 loss-of-function mutations cause early onset Parkinson's disease. Nucleotide analogs such as kinetin triphosphate (KTP) were reported to enhance PINK1 activity and may represent a therapeutic strategy for the treatment of Parkinson's disease. Here, we investigate the interaction of PINK1 with nucleotides, including KTP. We establish a cryo-EM platform exploiting the dodecamer assembly of Pediculus humanus corporis (Ph) PINK1 and determine PINK1 structures bound to AMP-PNP and ADP, revealing conformational changes in the kinase N-lobe that help establish PINK1's ubiquitin binding site. Notably, we find that KTP is unable to bind PhPINK1 or human (Hs) PINK1 due to a steric clash with the kinase "gatekeeper" methionine residue, and mutation to Ala or Gly is required for PINK1 to bind and use KTP as a phosphate donor in ubiquitin phosphorylation and mitophagy. HsPINK1 M318G can be used to conditionally uncouple PINK1 stabilization and activity on mitochondria.
    DOI:  https://doi.org/10.1126/sciadv.adj7408
  5. Nat Commun. 2024 Jan 15. 15(1): 532
    The phosphatase DUSP22 inhibits UBR2-mediated K63-ubiquitination and activation of Lck downstream of TCR signalling.
    Ying-Chun Shih, Hsueh-Fen Chen, Chia-Ying Wu, Yi-Ru Ciou, Chia-Wen Wang, Huai-Chia Chuang, Tse-Hua Tan.
      DUSP22 is a dual-specificity phosphatase that inhibits T cell activation by inactivating the kinase Lck. Here we show that the E3 ubiquitin ligase UBR2 is a positive upstream regulator of Lck during T-cell activation. DUSP22 dephosphorylates UBR2 at specific Serine residues, leading to ubiquitin-mediated UBR2 degradation. UBR2 is also modified by the SCF E3 ubiquitin ligase complex via Lys48-linked ubiquitination at multiple Lysine residues. Single-cell RNA sequencing analysis and UBR2 loss of function experiments showed that UBR2 is a positive regulator of proinflammatory cytokine expression. Mechanistically, UBR2 induces Lys63-linked ubiquitination of Lck at Lys99 and Lys276 residues, followed by Lck Tyr394 phosphorylation and activation as part of TCR signalling. Inflammatory phenotypes induced by TCR-triggered Lck activation or knocking out DUSP22, are attenuated by genomic deletion of UBR2. UBR2-Lck interaction and Lck Lys63-linked ubiquitination are induced in the peripheral blood T cells of human SLE patients, which demonstrate the relevance of the UBR2-mediated regulation of inflammation to human pathology. In summary, we show here an important regulatory mechanism of T cell activation, which finetunes the balance between T cell response and aggravated inflammation.
    DOI:  https://doi.org/10.1038/s41467-024-44843-w
  6. bioRxiv. 2023 Dec 29. pii: 2023.12.29.573643. [Epub ahead of print]
    Gene module reconstruction elucidates cellular differentiation processes and the regulatory logic of specialized secretion.
    Yiqun Wang, Jialin Liu, Lucia Y Du, Jannik L Wyss, Jeffrey A Farrell, Alexander F Schier.
      During differentiation, cells become structurally and functionally specialized, but comprehensive views of the underlying remodeling processes are elusive. Here, we leverage scRNA-seq developmental trajectories to reconstruct differentiation using two secretory tissues as a model system: the zebrafish notochord and hatching gland. First, we present an approach to integrate expression and functional similarities for gene module identification, revealing dozens of gene modules representing known and newly associated differentiation processes and their temporal ordering. Second, we focused on the unfolded protein response (UPR) transducer module to study how general versus cell-type specific secretory functions are regulated. By profiling loss- and gain-of-function embryos, we found that the UPR transcription factors creb3l1, creb3l2, and xbp1 are master regulators of a general secretion program. creb3l1/creb3l2 additionally activate an extracellular matrix secretion program, while xbp1 partners with bhlha15 to activate a gland-specific secretion program. Our study offers a multi-source integrated approach for functional gene module identification and illustrates how transcription factors confer general and specialized cellular functions.
    DOI:  https://doi.org/10.1101/2023.12.29.573643
  7. Nucleus. 2024 Dec;15(1): 2299632
    Sculpting nuclear envelope identity from the endoplasmic reticulum during the cell cycle.
    Pallavi Deolal, Julia Scholz, Kaike Ren, Helena Bragulat-Teixidor, Shotaro Otsuka.
      The nuclear envelope (NE) regulates nuclear functions, including transcription, nucleocytoplasmic transport, and protein quality control. While the outer membrane of the NE is directly continuous with the endoplasmic reticulum (ER), the NE has an overall distinct protein composition from the ER, which is crucial for its functions. During open mitosis in higher eukaryotes, the NE disassembles during mitotic entry and then reforms as a functional territory at the end of mitosis to reestablish nucleocytoplasmic compartmentalization. In this review, we examine the known mechanisms by which the functional NE reconstitutes from the mitotic ER in the continuous ER-NE endomembrane system during open mitosis. Furthermore, based on recent findings indicating that the NE possesses unique lipid metabolism and quality control mechanisms distinct from those of the ER, we explore the maintenance of NE identity and homeostasis during interphase. We also highlight the potential significance of membrane junctions between the ER and NE.
    Keywords:  Cell cycle; ER–NE junction; endoplasmic reticulum; mitosis; nuclear assembly; nuclear envelope; nuclear pore complex
    DOI:  https://doi.org/10.1080/19491034.2023.2299632
  8. J Cell Biol. 2024 Mar 04. pii: e202302067. [Epub ahead of print]223(3):
    The V-ATPase-ATG16L1 axis recruits LRRK2 to facilitate the lysosomal stress response.
    Tomoya Eguchi, Maria Sakurai, Yingxue Wang, Chieko Saito, Gen Yoshii, Thomas Wileman, Noboru Mizushima, Tomoki Kuwahara, Takeshi Iwatsubo.
      Leucine-rich repeat kinase 2 (LRRK2), a Rab kinase associated with Parkinson's disease and several inflammatory diseases, has been shown to localize to stressed lysosomes and get activated to regulate lysosomal homeostasis. However, the mechanisms of LRRK2 recruitment and activation have not been well understood. Here, we found that the ATG8 conjugation system regulates the recruitment of LRRK2 as well as LC3 onto single membranes of stressed lysosomes/phagosomes. This recruitment did not require FIP200-containing autophagy initiation complex, nor did it occur on double-membrane autophagosomes, suggesting independence from canonical autophagy. Consistently, LRRK2 recruitment was regulated by the V-ATPase-ATG16L1 axis, which requires the WD40 domain of ATG16L1 and specifically mediates ATG8 lipidation on single membranes. This mechanism was also responsible for the lysosomal stress-induced activation of LRRK2 and the resultant regulation of lysosomal secretion and enlargement. These results indicate that the V-ATPase-ATG16L1 axis serves a novel non-autophagic role in the maintenance of lysosomal homeostasis by recruiting LRRK2.
    DOI:  https://doi.org/10.1083/jcb.202302067
  9. J Hepatol. 2024 Jan 16. pii: S0168-8278(24)00036-9. [Epub ahead of print]
    TRIB3-TRIM8 complex drives NAFLD progression by regulating HNF4α stability.
    Meng-Chao Xiao, Nan Jiang, Li-Lin Chen, Fang Liu, Shu-Qing Liu, Chen-Hong Ding, Si-Han Wu, Ke-Qi Wang, Yuan-Yuan Luo, Yu Peng, Fang-Zhi Yan, Xin Zhang, Hui Qian, Wei-Fen Xie.
      BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress of hepatocytes plays a causative role in non-alcoholic fatty liver disease (NAFLD). Reduced expression of Hepatic nuclear factor 4α (HNF4α) is a critical event in the pathogenesis of NAFLD and other liver diseases. Whether ER stress regulates HNF4α expression remains unknown. The aim of this study was to delineate the machinery of HNF4α protein degradation and explore a therapeutic strategy based on protecting HNF4α stability during NAFLD progression.METHODS: Correlation of HNF4α and tribbles homologue 3 (TRIB3), an ER stress sensor, was evaluated in human and mouse NAFLD tissues. RNA-sequencing, mass spectrometry analysis, co-immunoprecipitation (Co-IP), in vivo and in vitro ubiquitination assays were used to elucidate the mechanisms of TRIB3-mediated HNF4α degradation. Molecular Docking and Co-IP were performed to identify a cell-penetrating peptide (CPP) ablating TRIB3-HNF4α interaction.
    RESULTS: TRIB3 directly interacts with HNF4α and mediates ER stress-induced HNF4α degradation. TRIB3 recruits tripartite motif containing 8 (TRIM8) to form an E3 ligase complex that catalyzes K48-linked polyubiquitination of HNF4α on lysine 470. Abrogating the degradation of HNF4α attenuates the effect of TRIB3 on HFF-induced NAFLD. Moreover, TRIB3 gain-of-function variant p.Q84R is associated with poor NAFLD progression in patients, and induces lower HNF4α levels and more severe hepatic steatosis in mice. Importantly, disrupting TRIB3-HNF4α interaction using a CPP restores HNF4α levels and ameliorates NAFLD progression in mice.
    CONCLUSIONS: Our findings unravel the machinery of HNF4α protein degradation and indicate that targeting TRIB3-TRIM8 E3 complex-mediated HNF4α polyubiquitination may be an ideal strategy for NAFLD therapy.
    IMPACT AND IMPLICATIONS: Reduced expression of Hepatic nuclear factor 4α (HNF4α) is a critical event in the pathogenesis of NAFLD and other liver diseases. However, the mechanism of HNF4α protein degradation remains unknown. Herein, we reveal that TRIB3-TRIM8 E3 ligase complex is responsible for HNF4α degradation during NAFLD. Inhibiting the TRIB3-HNF4α interaction effectively stabilized HNF4α protein levels and transcription factor activity in liver and ameliorated TRIB3-mediated NAFLD progression. Our findings demonstrate that disturbing TRIM8-TRIB3-HNF4α interaction may provide a novel approach to treat NAFLD and even other liver diseases by stabilizing HNF4α protein.
    Keywords:  Hepatic nuclear factor 4α; Non-alcoholic fatty liver disease; Tribbles homologue 3; Tripartite motif containing 8; Ubiquitin
    DOI:  https://doi.org/10.1016/j.jhep.2023.12.029
  10. Sci Rep. 2024 01 16. 14(1): 1439
    Transcriptional profile of ribosome-associated quality control components and their associated phenotypes in mammalian cells.
    Otávio Augusto Leitão Dos Santos, Rodolfo L Carneiro, Rodrigo D Requião, Marcelo Ribeiro-Alves, Tatiana Domitrovic, Fernando L Palhano.
      During protein synthesis, organisms detect translation defects that induce ribosome stalling and result in protein aggregation. The Ribosome-associated Quality Control (RQC) complex, comprising TCF25, LTN1, and NEMF, is responsible for identifying incomplete protein products from unproductive translation events, targeting them for degradation. Although RQC disruption causes adverse effects on vertebrate neurons, data regarding mRNA/protein expression and regulation across tissues are lacking. Employing high-throughput methods, we analyzed public datasets to explore RQC gene expression and phenotypes. Our findings revealed widespread expression of RQC components in human tissues; however, silencing of RQC yielded only mild negative effects on cell growth. Notably, TCF25 exhibited elevated mRNA levels that were not reflected in the protein content. We experimentally demonstrated that this disparity arose from post-translational protein degradation by the proteasome. Additionally, we observed that cellular aging marginally influenced RQC expression, leading to reduced mRNA levels in specific tissues. Our results suggest the necessity of RQC expression in all mammalian tissues. Nevertheless, when RQC falters, alternative mechanisms seem to compensate, ensuring cell survival under nonstress conditions.
    DOI:  https://doi.org/10.1038/s41598-023-50811-z
  11. J Clin Invest. 2024 Jan 16. pii: e175448. [Epub ahead of print]134(2):
    Hypomorphic human SEL1L and HRD1 variants uncouple multilayered ER-associated degradation machinery.
    Katharine Umphred-Wilson, Stanley Adoro.
      The suppressor of lin-12-like-HMG-CoA reductase degradation 1 (SEL1L-HRD1) complex of the endoplasmic reticulum-associated degradation (ERAD) machinery is a key cellular proteostasis pathway. Although previous studies have shown ERAD as promoting the development and maintenance of many cell types in mice, its importance to human physiology remained undetermined. In two articles in this issue of the JCI, Qi and colleagues describe four biallelic hypomorphic SEL1L and HRD1 variants that were associated with neurodevelopment disorders, locomotor dysfunction, impaired immunity, and premature death in patients. These pathogenic SEL1L-HRD1 variants shine a light on the critical importance of ERAD in humans and pave the way for future studies dissecting ERAD mechanisms in specific cell types.
    DOI:  https://doi.org/10.1172/JCI175448
  12. Mol Cell. 2024 Jan 12. pii: S1097-2765(23)01082-1. [Epub ahead of print]
    Mechanisms of RNF168 nucleosome recognition and ubiquitylation.
    Qi Hu, Debiao Zhao, Gaofeng Cui, Janarjan Bhandari, James R Thompson, Maria Victoria Botuyan, Georges Mer.
      RNF168 plays a central role in the DNA damage response (DDR) by ubiquitylating histone H2A at K13 and K15. These modifications direct BRCA1-BARD1 and 53BP1 foci formation in chromatin, essential for cell-cycle-dependent DNA double-strand break (DSB) repair pathway selection. The mechanism by which RNF168 catalyzes the targeted accumulation of H2A ubiquitin conjugates to form repair foci around DSBs remains unclear. Here, using cryoelectron microscopy (cryo-EM), nuclear magnetic resonance (NMR) spectroscopy, and functional assays, we provide a molecular description of the reaction cycle and dynamics of RNF168 as it modifies the nucleosome and recognizes its ubiquitylation products. We demonstrate an interaction of a canonical ubiquitin-binding domain within full-length RNF168, which not only engages ubiquitin but also the nucleosome surface, clarifying how such site-specific ubiquitin recognition propels a signal amplification loop. Beyond offering mechanistic insights into a key DDR protein, our study aids in understanding site specificity in both generating and interpreting chromatin ubiquitylation.
    Keywords:  53BP1; BRCA1-BARD1; DNA damage response; NMR spectroscopy; RNF168; UbcH5c; X-ray crystallography; chromatin; cryo-EM; nucleosome; ubiquitin ligase
    DOI:  https://doi.org/10.1016/j.molcel.2023.12.036
  13. Expert Opin Drug Discov. 2024 Jan 19.
    Molecular glue degraders: exciting opportunities for novel drug discovery.
    Thomas Lemaitre, Marie Cornu, Florian Schwalen, Marc Since, Charline Kieffer, Anne Sophie Voisin-Chiret.
      INTRODUCTION: Molecular Glue Degraders (MGDs) is a concept that refers to a class of compounds that facilitate the interaction between two proteins or molecules within a cell. These compounds act as bridge that enhances specific Protein-Protein Interactions (PPIs). Over the past decade, this technology has gained attention as a potential strategy to target proteins that were traditionally considered undruggable using small molecules.AREAS COVERED: This review presents the concept of cellular homeostasis and the balance between protein synthesis and protein degradation. The concept of protein degradation is concerned with molecular glues, which form part of the broader field of Targeted Protein Degradation (TPD). Next, pharmacochemical strategies for the rational design of MGDs are detailed and illustrated by examples of Ligand-Based (LBDD), Structure-Based (SBDD) and Fragment-Based Drug Design (FBDD).
    EXPERT OPINION: Expanding the scope of what can be effectively targeted in the development of treatments for diseases that are incurable or resistant to conventional therapies offers new therapeutic options. The treatment of microbial infections and neurodegenerative diseases is a major societal challenge, and the discovery of MGDs appears to be a promising avenue. Combining different approaches to discover and exploit a variety of innovative therapeutic agents will create opportunities to treat diseases that are still incurable.
    Keywords:  Chemical biology; E3 ubiquitin ligase; Molecular glue degrader; Targeted protein degradation
    DOI:  https://doi.org/10.1080/17460441.2024.2306845
  14. Cell Rep. 2024 Jan 17. pii: S2211-1247(24)00007-X. [Epub ahead of print]43(2): 113679
    Glutathione-dependent depalmitoylation of phospholemman by peroxiredoxin 6.
    Jacqueline Howie, Lindsay B Tulloch, Elaine Brown, Louise Reilly, Fiona B Ashford, Jennifer Kennedy, Krzysztof J Wypijewski, Karen L Aughton, Jason K C Mak, Michael J Shattock, Niall J Fraser, William Fuller.
      Phospholemman (PLM) regulates the cardiac sodium pump: PLM phosphorylation activates the pump whereas PLM palmitoylation inhibits its activity. Here, we show that the anti-oxidant protein peroxiredoxin 6 (Prdx6) interacts with and depalmitoylates PLM in a glutathione-dependent manner. Glutathione loading cells acutely reduce PLM palmitoylation; glutathione depletion significantly increases PLM palmitoylation. Prdx6 silencing abolishes these effects, suggesting that PLM can be depalmitoylated by reduced Prdx6. In vitro, only recombinant Prdx6, among several peroxiredoxin isoforms tested, removes palmitic acid from recombinant palmitoylated PLM. The broad-spectrum depalmitoylase inhibitor palmostatin B prevents Prdx6-dependent PLM depalmitoylation in cells and in vitro. Our data suggest that Prdx6 is a thioesterase that can depalmitoylate proteins by nucleophilic attack via its reactive thiol, linking PLM palmitoylation and hence sodium pump activity to cellular glutathione status. We show that protein depalmitoylation can occur via a catalytic cysteine in which substrate specificity is determined by a protein-protein interaction.
    Keywords:  CP: Cell biology; FXYD; Na/K ATPase; deacylation; glutathione; peroxiredoxin; phospholemman; sodium pump; thioesterase
    DOI:  https://doi.org/10.1016/j.celrep.2024.113679
  15. Cell Death Differ. 2024 Jan 18.
    Activation of Ca2+ phosphatase Calcineurin regulates Parkin translocation to mitochondria and mitophagy in flies.
    Elena Marchesan, Alice Nardin, Sofia Mauri, Greta Bernardo, Vivek Chander, Simone Di Paola, Monica Chinellato, Sophia von Stockum, Joy Chakraborty, Stephanie Herkenne, Valentina Basso, Emilie Schrepfer, Oriano Marin, Laura Cendron, Diego L Medina, Luca Scorrano, Elena Ziviani.
      Selective removal of dysfunctional mitochondria via autophagy is crucial for the maintenance of cellular homeostasis. This event is initiated by the translocation of the E3 ubiquitin ligase Parkin to damaged mitochondria, and it requires the Serine/Threonine-protein kinase PINK1. In a coordinated set of events, PINK1 operates upstream of Parkin in a linear pathway that leads to the phosphorylation of Parkin, Ubiquitin, and Parkin mitochondrial substrates, to promote ubiquitination of outer mitochondrial membrane proteins. Ubiquitin-decorated mitochondria are selectively recruiting autophagy receptors, which are required to terminate the organelle via autophagy. In this work, we show a previously uncharacterized molecular pathway that correlates the activation of the Ca2+-dependent phosphatase Calcineurin to Parkin translocation and Parkin-dependent mitophagy. Calcineurin downregulation or genetic inhibition prevents Parkin translocation to CCCP-treated mitochondria and impairs stress-induced mitophagy, whereas Calcineurin activation promotes Parkin mitochondrial recruitment and basal mitophagy. Calcineurin interacts with Parkin, and promotes Parkin translocation in the absence of PINK1, but requires PINK1 expression to execute mitophagy in MEF cells. Genetic activation of Calcineurin in vivo boosts basal mitophagy in neurons and corrects locomotor dysfunction and mitochondrial respiratory defects of a Drosophila model of impaired mitochondrial functions. Our study identifies Calcineurin as a novel key player in the regulation of Parkin translocation and mitophagy.
    DOI:  https://doi.org/10.1038/s41418-023-01251-9
  16. Nucleic Acids Res. 2024 Jan 15. pii: gkae005. [Epub ahead of print]
    NAC and Zuotin/Hsp70 chaperone systems coexist at the ribosome tunnel exit in vivo.
    Thomas Ziegelhoffer, Amit K Verma, Wojciech Delewski, Brenda A Schilke, Paige M Hill, Marcin Pitek, Jaroslaw Marszalek, Elizabeth A Craig.
      The area surrounding the tunnel exit of the 60S ribosomal subunit is a hub for proteins involved in maturation and folding of emerging nascent polypeptide chains. How different factors vie for positioning at the tunnel exit in the complex cellular environment is not well understood. We used in vivo site-specific cross-linking to approach this question, focusing on two abundant factors-the nascent chain-associated complex (NAC) and the Hsp70 chaperone system that includes the J-domain protein co-chaperone Zuotin. We found that NAC and Zuotin can cross-link to each other at the ribosome, even when translation initiation is inhibited. Positions yielding NAC-Zuotin cross-links indicate that when both are present the central globular domain of NAC is modestly shifted from the mutually exclusive position observed in cryogenic electron microscopy analysis. Cross-linking results also suggest that, even in NAC's presence, Hsp70 can situate in a manner conducive for productive nascent chain interaction-with the peptide binding site at the tunnel exit and the J-domain of Zuotin appropriately positioned to drive stabilization of nascent chain binding. Overall, our results are consistent with the idea that, in vivo, the NAC and Hsp70 systems can productively position on the ribosome simultaneously.
    DOI:  https://doi.org/10.1093/nar/gkae005
  17. Sci Adv. 2024 Jan 19. 10(3): eadh2579
    Induced degradation of lineage-specific oncoproteins drives the therapeutic vulnerability of small cell lung cancer to PARP inhibitors.
    Chiho Kim, Xu-Dong Wang, Zhengshuai Liu, Jianwei Hao, Shuai Wang, Peng Li, Zhenzhen Zi, Qing Ding, Seoyeon Jang, Jiwoong Kim, Yikai Luo, Kenneth E Huffman, Shreoshi Pal Choudhuri, Sofia Del Rio, Ling Cai, Han Liang, Benjamin J Drapkin, John D Minna, Yonghao Yu.
      Although BRCA1/2 mutations are not commonly found in small cell lung cancer (SCLC), a substantial fraction of SCLC shows clinically relevant response to PARP inhibitors (PARPis). However, the underlying mechanism(s) of PARPi sensitivity in SCLC is poorly understood. We performed quantitative proteomic analyses and identified proteomic changes that signify PARPi responses in SCLC cells. We found that the vulnerability of SCLC to PARPi could be explained by the degradation of lineage-specific oncoproteins (e.g., ASCL1). PARPi-induced activation of the E3 ligase HUWE1 mediated the ubiquitin-proteasome system (UPS)-dependent ASCL1 degradation. Although PARPi induced a general DNA damage response in SCLC cells, this signal generated a cell-specific response in ASCL1 degradation, leading to the identification of HUWE1 expression as a predictive biomarker for PARPi. Combining PARPi with agents targeting these pathways markedly improved therapeutic response in SCLC. The degradation of lineage-specific oncoproteins therefore represents a previously unidentified mechanism for PARPi efficacy in SCLC.
    DOI:  https://doi.org/10.1126/sciadv.adh2579
  18. Mol Cancer Res. 2024 Jan 18.
    miR-217 regulates normal and tumor cell fate following induction of Endoplasmic Reticulum Stress.
    Neekkan Dey, Constantinos Koumenis, Davide Ruggero, Serge Y Fuchs, J Alan Diehl.
      Rapidly proliferating cancer cells must thrive in a microenvironment wherein metabolic nutrients such as glucose, oxygen and growth factors become limiting as tumor volume expands beyond the established vascularity of the tissue. Limits in nutrient availability typically trigger growth arrest and/or apoptosis to prevent cellular expansion. However, tumor cells frequently co-opt cellular survival pathways thereby favoring cell survival under this environmental stress. The Unfolded Protein Response (UPR) pathway is typically engaged by tumor cells to favor adaptation to stress. PERK, an endoplasmic reticulum (ER) protein kinase and UPR effector is activated in tumor cells and contributes tumor cell adaptation by limiting protein translation and balancing redox stress. PERK also induces micro-RNAs that contribute to tumor adaptation. miR-211 and miR-216b were previously identified as a PERK-ATF4 regulated micro-RNAs that regulate cell survival. We have identified another PERK responsive miRNA, miR-217, with increased expression under prolonged ER stress. Key targets of miR-217 are identified as TRPM1, the host gene for miR-211 and EZH2. Evidence is provided that miR-217 expression is essential for the rapid loss of miR-211 in prolonged ER stress and provides a functional link for determining whether cells adapt to stress or commit to apoptosis. Implications: PERK-dependent induction of miR-217 limits accumulation and function of the pro-survival micro-RNA, miR-211, to establish cell fate and promote cell commitment to apoptosis.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-23-0676
  19. J Biol Chem. 2024 Jan 11. pii: S0021-9258(24)00020-6. [Epub ahead of print] 105644
    Intramembrane protease SPP defines a cholesterol-regulated abundance control of the mevalonate pathway enzyme squalene synthase.
    Dönem Avci, Ronny Heidasch, Martina Costa, Christian Lüchtenborg, Dipali Kale, Britta Brügger, Marius K Lemberg.
      Intramembrane proteolysis regulates important processes such as signaling and transcriptional and posttranslational abundance control of proteins with key functions in metabolic pathways. This includes transcriptional control of mevalonate pathway genes, thereby ensuring balanced biosynthesis of cholesterol and other isoprenoids. Our work shows that, at high cholesterol levels, signal peptide peptidase (SPP) cleaves squalene synthase (SQS), an enzyme that defines the branching point for allocation of isoprenoids to the sterol and non-sterol arms of the mevalonate pathway. This intramembrane cleavage releases SQS from the membrane and targets it for proteasomal degradation. Regulation of this mechanism is achieved by the E3 ubiquitin ligase TRC8 that, in addition to ubiquitinating SQS in response to cholesterol levels, acts as an allosteric activator of SPP-catalyzed intramembrane cleavage of SQS. Cellular cholesterol levels increase in the absence of SPP activity. We infer from these results that, SPP-TRC8 mediated abundance control of SQS acts as a regulation step within the mevalonate pathway.
    Keywords:  ER-associated degradation; Intramembrane proteolysis; cholesterol; metabolic regulation; mevalonate pathway
    DOI:  https://doi.org/10.1016/j.jbc.2024.105644
  20. EMBO Rep. 2024 Jan 15.
    Ribogenesis boosts controlled by HEATR1-MYC interplay promote transition into brain tumour growth.
    Laura R Diaz, Jon Gil-Ranedo, Karolina J Jaworek, Nsikan Nsek, Joao Pinheiro Marques, Eleni Costa, David A Hilton, Hubert Bieluczyk, Oliver Warrington, C Oliver Hanemann, Matthias E Futschik, Torsten Bossing, Claudia S Barros.
      Cell commitment to tumourigenesis and the onset of uncontrolled growth are critical determinants in cancer development but the early events directing tumour initiating cell (TIC) fate remain unclear. We reveal a single-cell transcriptome profile of brain TICs transitioning into tumour growth using the brain tumour (brat) neural stem cell-based Drosophila model. Prominent changes in metabolic and proteostasis-associated processes including ribogenesis are identified. Increased ribogenesis is a known cell adaptation in established tumours. Here we propose that brain TICs boost ribogenesis prior to tumour growth. In brat-deficient TICs, we show that this dramatic change is mediated by upregulated HEAT-Repeat Containing 1 (HEATR1) to promote ribosomal RNA generation, TIC enlargement and onset of overgrowth. High HEATR1 expression correlates with poor glioma patient survival and patient-derived glioblastoma stem cells rely on HEATR1 for enhanced ribogenesis and tumourigenic potential. Finally, we show that HEATR1 binds the master growth regulator MYC, promotes its nucleolar localisation and appears required for MYC-driven ribogenesis, suggesting a mechanism co-opted in ribogenesis reprogramming during early brain TIC development.
    Keywords:  Brain Tumourigenesis; HEATR1; MYC; Neural Stem Cells; Ribogenesis
    DOI:  https://doi.org/10.1038/s44319-023-00017-1
  21. Nat Commun. 2024 Jan 16. 15(1): 482
    Selective CK1α degraders exert antiproliferative activity against a broad range of human cancer cell lines.
    Gisele Nishiguchi, Lauren G Mascibroda, Sarah M Young, Elizabeth A Caine, Sherif Abdelhamed, Jeffrey J Kooijman, Darcie J Miller, Sourav Das, Kevin McGowan, Anand Mayasundari, Zhe Shi, Juan M Barajas, Ryan Hiltenbrand, Anup Aggarwal, Yunchao Chang, Vibhor Mishra, Shilpa Narina, Melvin Thomas, Allister J Loughran, Ravi Kalathur, Kaiwen Yu, Suiping Zhou, Xusheng Wang, Anthony A High, Junmin Peng, Shondra M Pruett-Miller, Danette L Daniels, Marjeta Urh, Anang A Shelat, Charles G Mullighan, Kristin M Riching, Guido J R Zaman, Marcus Fischer, Jeffery M Klco, Zoran Rankovic.
      Molecular-glue degraders are small molecules that induce a specific interaction between an E3 ligase and a target protein, resulting in the target proteolysis. The discovery of molecular glue degraders currently relies mostly on screening approaches. Here, we describe screening of a library of cereblon (CRBN) ligands against a panel of patient-derived cancer cell lines, leading to the discovery of SJ7095, a potent degrader of CK1α, IKZF1 and IKZF3 proteins. Through a structure-informed exploration of structure activity relationship (SAR) around this small molecule we develop SJ3149, a selective and potent degrader of CK1α protein in vitro and in vivo. The structure of SJ3149 co-crystalized in complex with CK1α + CRBN + DDB1 provides a rationale for the improved degradation properties of this compound. In a panel of 115 cancer cell lines SJ3149 displays a broad antiproliferative activity profile, which shows statistically significant correlation with MDM2 inhibitor Nutlin-3a. These findings suggest potential utility of selective CK1α degraders for treatment of hematological cancers and solid tumors.
    DOI:  https://doi.org/10.1038/s41467-024-44698-1
  22. iScience. 2024 Jan 19. 27(1): 108727
    Riboproteome remodeling during quiescence exit in Saccharomyces cerevisiae.
    Clara A Solari, María Clara Ortolá Martínez, Juan M Fernandez, Christian Bates, Gerardo Cueto, María Pía Valacco, Fabián Morales-Polanco, Silvia Moreno, Silvia Rossi, Mark P Ashe, Paula Portela.
      The quiescent state is the prevalent mode of cellular life in most cells. Saccharomyces cerevisiae is a useful model for studying the molecular basis of the cell cycle, quiescence, and aging. Previous studies indicate that heterogeneous ribosomes show a specialized translation function to adjust the cellular proteome upon a specific stimulus. Using nano LC-MS/MS, we identified 69 of the 79 ribosomal proteins (RPs) that constitute the eukaryotic 80S ribosome during quiescence. Our study shows that the riboproteome is composed of 444 accessory proteins comprising cellular functions such as translation, protein folding, amino acid and glucose metabolism, cellular responses to oxidative stress, and protein degradation. Furthermore, the stoichiometry of both RPs and accessory proteins on ribosome particles is different depending on growth conditions and among monosome and polysome fractions. Deficiency of different RPs resulted in defects of translational capacity, suggesting that ribosome composition can result in changes in translational activity during quiescence.
    Keywords:  Cell biology
    DOI:  https://doi.org/10.1016/j.isci.2023.108727
  23. Nat Chem Biol. 2024 Jan 17.
    Covalent inhibition of pro-apoptotic BAX.
    Matthew W McHenry, Peiwen Shi, Christina M Camara, Daniel T Cohen, T Justin Rettenmaier, Utsarga Adhikary, Micah A Gygi, Ka Yang, Steven P Gygi, Thomas E Wales, John R Engen, James A Wells, Loren D Walensky.
      BCL-2-associated X protein (BAX) is a promising therapeutic target for activating or restraining apoptosis in diseases of pathologic cell survival or cell death, respectively. In response to cellular stress, BAX transforms from a quiescent cytosolic monomer into a toxic oligomer that permeabilizes the mitochondria, releasing key apoptogenic factors. The mitochondrial lipid trans-2-hexadecenal (t-2-hex) sensitizes BAX activation by covalent derivatization of cysteine 126 (C126). In this study, we performed a disulfide tethering screen to discover C126-reactive molecules that modulate BAX activity. We identified covalent BAX inhibitor 1 (CBI1) as a compound that selectively derivatizes BAX at C126 and inhibits BAX activation by triggering ligands or point mutagenesis. Biochemical and structural analyses revealed that CBI1 can inhibit BAX by a dual mechanism of action: conformational constraint and competitive blockade of lipidation. These data inform a pharmacologic strategy for suppressing apoptosis in diseases of unwanted cell death by covalent targeting of BAX C126.
    DOI:  https://doi.org/10.1038/s41589-023-01537-6
  24. Cell Death Dis. 2024 Jan 13. 15(1): 48
    Akt enhances the vulnerability of cancer cells to VCP/p97 inhibition-mediated paraptosis.
    Dong Min Lee, In Young Kim, Hong Jae Lee, Min Ji Seo, Mi-Young Cho, Hae In Lee, Gyesoon Yoon, Jae-Hoon Ji, Seok Soon Park, Seong-Yun Jeong, Eun Kyung Choi, Yong Hyeon Choi, Chae-Ok Yun, Mirae Yeo, Eunhee Kim, Kyeong Sook Choi.
      Valosin-containing protein (VCP)/p97, an AAA+ ATPase critical for maintaining proteostasis, emerges as a promising target for cancer therapy. This study reveals that targeting VCP selectively eliminates breast cancer cells while sparing non-transformed cells by inducing paraptosis, a non-apoptotic cell death mechanism characterized by endoplasmic reticulum and mitochondria dilation. Intriguingly, oncogenic HRas sensitizes non-transformed cells to VCP inhibition-mediated paraptosis. The susceptibility of cancer cells to VCP inhibition is attributed to the non-attenuation and recovery of protein synthesis under proteotoxic stress. Mechanistically, mTORC2/Akt activation and eIF3d-dependent translation contribute to translational rebound and amplification of proteotoxic stress. Furthermore, the ATF4/DDIT4 axis augments VCP inhibition-mediated paraptosis by activating Akt. Given that hyperactive Akt counteracts chemotherapeutic-induced apoptosis, VCP inhibition presents a promising therapeutic avenue to exploit Akt-associated vulnerabilities in cancer cells by triggering paraptosis while safeguarding normal cells.
    DOI:  https://doi.org/10.1038/s41419-024-06434-x
  25. Nat Commun. 2024 Jan 17. 15(1): 569
    Cochaperones convey the energy of ATP hydrolysis for directional action of Hsp90.
    Leonie Vollmar, Julia Schimpf, Bianca Hermann, Thorsten Hugel.
      The molecular chaperone and heat shock protein Hsp90 is part of many protein complexes in eukaryotic cells. Together with its cochaperones, Hsp90 is responsible for the maturation of hundreds of clients. Although having been investigated for decades, it still is largely unknown which components are necessary for a functional complex and how the energy of ATP hydrolysis is used to enable cyclic operation. Here we use single-molecule FRET to show how cochaperones introduce directionality into Hsp90's conformational changes during its interaction with the client kinase Ste11. Three cochaperones are needed to couple ATP turnover to these conformational changes. All three are therefore essential for a functional cyclic operation, which requires coupling to an energy source. Finally, our findings show how the formation of sub-complexes in equilibrium followed by a directed selection of the functional complex can be the most energy efficient pathway for kinase maturation.
    DOI:  https://doi.org/10.1038/s41467-024-44847-6
  26. Nucleic Acids Res. 2024 Jan 16. pii: gkae004. [Epub ahead of print]
    The dimeric deubiquitinase USP28 integrates 53BP1 and MYC functions to limit DNA damage.
    Chao Jin, Elias Einig, Wenshan Xu, Ravi Babu Kollampally, Andreas Schlosser, Michael Flentje, Nikita Popov.
      DNA replication is a major source of endogenous DNA damage in tumor cells and a key target of cellular response to genotoxic stress. DNA replication can be deregulated by oncoproteins, such as transcription factor MYC, aberrantly activated in many human cancers. MYC is stringently regulated by the ubiquitin system - for example, ubiquitination controls recruitment of the elongation factor PAF1c, instrumental in MYC activity. Curiously, a key MYC-targeting deubiquitinase USP28 also controls cellular response to DNA damage via the mediator protein 53BP1. USP28 forms stable dimers, but the biological role of USP28 dimerization is unknown. We show here that dimerization limits USP28 activity and restricts recruitment of PAF1c by MYC. Expression of monomeric USP28 stabilizes MYC and promotes PAF1c recruitment, leading to ectopic DNA synthesis and replication-associated DNA damage. USP28 dimerization is stimulated by 53BP1, which selectively binds USP28 dimers. Genotoxic stress diminishes 53BP1-USP28 interaction, promotes disassembly of USP28 dimers and stimulates PAF1c recruitment by MYC. This triggers firing of DNA replication origins during early response to genotoxins and exacerbates DNA damage. We propose that dimerization of USP28 prevents ectopic DNA replication at transcriptionally active chromatin to maintain genome stability.
    DOI:  https://doi.org/10.1093/nar/gkae004
  27. Angew Chem Int Ed Engl. 2024 Jan 19. e202318849
    N-Cyanopiperazines as Specific Covalent Inhibitors of the Deubiquitinating Enzyme UCHL1.
    Mirko Schmidt, Christian Grethe, Sarah Recknagel, Gian-Marvin Kipka, Nikolas Klink, Malte Gersch.
      Cyanamides have emerged as privileged scaffolds in covalent inhibitors of deubiquitinating enzymes (DUBs). However, many compounds with a cyanopyrrolidine warhead show cross-reactivity toward small subsets of DUBs or toward the protein deglycase PARK7/DJ-1, hampering their use for the selective perturbation of a single DUB in living cells. Here, we disclose N'-alkyl,N-cyanopiperazines as structures for covalent enzyme inhibition with exceptional specificity for the DUB UCHL1 among 55 human deubiquitinases and with potent target engagement in cells. Notably, transitioning from 5-membered pyrrolidines to 6-membered heterocycles eliminated PARK7 binding and introduced context-dependent reversibility of the isothiourea linkage to the catalytic cysteine of UCHL1. Compound potency and specificity were analysed by a range of biochemical assays and with a crystal structure of a cyanopiperazine in covalent complex with UCHL1. The structure revealed a compound-induced conformational restriction of the cross-over loop, which underlies the observed inhibitory potencies. Through the rationalization of specificities of different cyanamides, we introduce a framework for the investigation of protein reactivity of bioactive nitriles of this compound class. Our results represent an encouraging case study for the refining of electrophilic compounds into chemical probes, emphasizing the potential to engineer specificity through subtle chemical modifications around the warhead.
    Keywords:  Heterocycles; Ubiquitin; covalent inhibitors; proteases; protein structures
    DOI:  https://doi.org/10.1002/anie.202318849
  28. Nucleic Acids Res. 2024 Jan 15. pii: gkae001. [Epub ahead of print]
    STIM1 translocation to the nucleus protects cells from DNA damage.
    Irene Sanchez-Lopez, Yolanda Orantos-Aguilera, Eulalia Pozo-Guisado, Alberto Alvarez-Barientos, Sergio Lilla, Sara Zanivan, Christophe Lachaud, Francisco Javier Martin-Romero.
      DNA damage represents a challenge for cells, as this damage must be eliminated to preserve cell viability and the transmission of genetic information. To reduce or eliminate unscheduled chemical modifications in genomic DNA, an extensive signaling network, known as the DNA damage response (DDR) pathway, ensures this repair. In this work, and by means of a proteomic analysis aimed at studying the STIM1 protein interactome, we have found that STIM1 is closely related to the protection from endogenous DNA damage, replicative stress, as well as to the response to interstrand crosslinks (ICLs). Here we show that STIM1 has a nuclear localization signal that mediates its translocation to the nucleus, and that this translocation and the association of STIM1 to chromatin increases in response to mitomycin-C (MMC), an ICL-inducing agent. Consequently, STIM1-deficient cell lines show higher levels of basal DNA damage, replicative stress, and increased sensitivity to MMC. We show that STIM1 normalizes FANCD2 protein levels in the nucleus, which explains the increased sensitivity of STIM1-KO cells to MMC. This study not only unveils a previously unknown nuclear function for the endoplasmic reticulum protein STIM1 but also expands our understanding of the genes involved in DNA repair.
    DOI:  https://doi.org/10.1093/nar/gkae001
  29. EMBO J. 2024 Jan 18.
    MITOL deficiency triggers hematopoietic stem cell apoptosis via ER stress response.
    Wenjuan Ma, Shah Adil Ishtiyaq Ahmad, Michihiro Hashimoto, Ahad Khalilnezhad, Miho Kataoka, Yuichiro Arima, Yosuke Tanaka, Shigeru Yanagi, Terumasa Umemoto, Toshio Suda.
      Hematopoietic stem cell (HSC) divisional fate and function are determined by cellular metabolism, yet the contribution of specific cellular organelles and metabolic pathways to blood maintenance and stress-induced responses in the bone marrow remains poorly understood. The outer mitochondrial membrane-localized E3 ubiquitin ligase MITOL/MARCHF5 (encoded by the Mitol gene) is known to regulate mitochondrial and endoplasmic reticulum (ER) interaction and to promote cell survival. Here, we investigated the functional involvement of MITOL in HSC maintenance by generating MX1-cre inducible Mitol knockout mice. MITOL deletion in the bone marrow resulted in HSC exhaustion and impairment of bone marrow reconstitution capability in vivo. Interestingly, MITOL loss did not induce major mitochondrial dysfunction in hematopoietic stem and progenitor cells. In contrast, MITOL deletion induced prolonged ER stress in HSCs, which triggered cellular apoptosis regulated by IRE1α. In line, dampening of ER stress signaling by IRE1α inihibitor KIRA6 partially rescued apoptosis of long-term-reconstituting HSC. In summary, our observations indicate that MITOL is a principal regulator of hematopoietic homeostasis and protects blood stem cells from cell death through its function in ER stress signaling.
    Keywords:  Apoptosis; Cell Cycle; ER Stress Response; IRE1; MITOL
    DOI:  https://doi.org/10.1038/s44318-024-00029-0
  30. EMBO J. 2024 Jan 19.
    MinD-RNase E interplay controls localization of polar mRNAs in E. coli.
    Shanmugapriya Kannaiah, Omer Goldberger, Nawsad Alam, Georgina Barnabas, Yair Pozniak, Anat Nussbaum-Shochat, Ora Schueler-Furman, Tamar Geiger, Orna Amster-Choder.
      The E. coli transcriptome at the cell's poles (polar transcriptome) is unique compared to the membrane and cytosol. Several factors have been suggested to mediate mRNA localization to the membrane, but the mechanism underlying polar localization of mRNAs remains unknown. Here, we combined a candidate system approach with proteomics to identify factors that mediate mRNAs localization to the cell poles. We identified the pole-to-pole oscillating protein MinD as an essential factor regulating polar mRNA localization, although it is not able to bind RNA directly. We demonstrate that RNase E, previously shown to interact with MinD, is required for proper localization of polar mRNAs. Using in silico modeling followed by experimental validation, the membrane-binding site in RNase E was found to mediate binding to MinD. Intriguingly, not only does MinD affect RNase E interaction with the membrane, but it also affects its mode of action and dynamics. Polar accumulation of RNase E in ΔminCDE cells resulted in destabilization and depletion of mRNAs from poles. Finally, we show that mislocalization of polar mRNAs may prevent polar localization of their protein products. Taken together, our findings show that the interplay between MinD and RNase E determines the composition of the polar transcriptome, thus assigning previously unknown roles for both proteins.
    Keywords:  Bacterial Cell Organization; Bacterial Cell Poles; MinD; RNA Localization; RNase E
    DOI:  https://doi.org/10.1038/s44318-023-00026-9
  31. Nat Commun. 2024 Jan 18. 15(1): 597
    From interaction networks to interfaces, scanning intrinsically disordered regions using AlphaFold2.
    Hélène Bret, Jinmei Gao, Diego Javier Zea, Jessica Andreani, Raphaël Guerois.
      The revolution brought about by AlphaFold2 opens promising perspectives to unravel the complexity of protein-protein interaction networks. The analysis of interaction networks obtained from proteomics experiments does not systematically provide the delimitations of the interaction regions. This is of particular concern in the case of interactions mediated by intrinsically disordered regions, in which the interaction site is generally small. Using a dataset of protein-peptide complexes involving intrinsically disordered regions that are non-redundant with the structures used in AlphaFold2 training, we show that when using the full sequences of the proteins, AlphaFold2-Multimer only achieves 40% success rate in identifying the correct site and structure of the interface. By delineating the interaction region into fragments of decreasing size and combining different strategies for integrating evolutionary information, we manage to raise this success rate up to 90%. We obtain similar success rates using a much larger dataset of protein complexes taken from the ELM database. Beyond the correct identification of the interaction site, our study also explores specificity issues. We show the advantages and limitations of using the AlphaFold2 confidence score to discriminate between alternative binding partners, a task that can be particularly challenging in the case of small interaction motifs.
    DOI:  https://doi.org/10.1038/s41467-023-44288-7
  32. Cell Stress Chaperones. 2023 Nov;pii: S1355-8145(24)00025-7. [Epub ahead of print]28(6): 631-640
    Protein quality control: from molecular mechanisms to therapeutic intervention-EMBO workshop, May 21-26 2023, Srebreno, Croatia.
    Christian Münch, Janine Kirstein.
      Protein quality control pathways ensure a functional proteome and rely on a complex proteostasis network (PN) that is composed of molecular chaperones and proteases. Failures in the PN can lead to a broad spectrum of diseases, including neurodegenerative disorders like Alzheimer's, Parkinson's, and a range of motor neuron diseases. The EMBO workshop "Protein quality control: from molecular mechanisms to therapeutic intervention" covered all aspects of protein quality control from underlying molecular mechanisms of chaperones and proteases to stress signaling pathways and medical implications. This report summarizes the workshop and highlights selected presentations.
    Keywords:  Condensation; Molecular chaperones; Proteases; Protein aggregation; Proteostasis
    DOI:  https://doi.org/10.1007/s12192-023-01383-4
  33. Aging Cell. 2024 Jan 17. e14066
    Glioblastoma and its treatment are associated with extensive accelerated brain aging.
    Anna P Ainslie, Myrthe Klaver, Daniëlle C Voshart, Emma Gerrits, Wilfred F A den Dunnen, Bart J L Eggen, Steven Bergink, Lara Barazzuol.
      Progressive neurocognitive dysfunction is the leading cause of a reduced quality of life in patients with primary brain tumors. Understanding how the human brain responds to cancer and its treatment is essential to improve the associated cognitive sequelae. In this study, we performed integrated transcriptomic and tissue analysis on postmortem normal-appearing non-tumor brain tissue from glioblastoma (GBM) patients that had received cancer treatments, region-matched brain tissue from unaffected control individuals and Alzheimer's disease (AD) patients. We show that normal-appearing non-tumor brain regions of patients with GBM display hallmarks of accelerated aging, in particular mitochondrial dysfunction, inflammation, and proteostasis deregulation. The extent and spatial pattern of this response decreased with distance from the tumor. Gene set enrichment analyses and a direct comparative analysis with an independent cohort of brain tissue samples from AD patients revealed a significant overlap in differentially expressed genes and a similar biological aging trajectory. Additionally, these responses were validated at the protein level showing the presence of increased lysosomal lipofuscin, phosphorylated microtubule-associated protein Tau, and oxidative DNA damage in normal-appearing brain areas of GBM patients. Overall, our data show that the brain of GBM patients undergoes accelerated aging and shared AD-like features, providing the basis for novel or repurposed therapeutic targets for managing brain tumor-related side effects.
    Keywords:  aging hallmarks; cancer treatment side effects; cognitive decline; glioblastoma; neurodegeneration
    DOI:  https://doi.org/10.1111/acel.14066
  34. Cell Stress Chaperones. 2023 Nov;pii: S1355-8145(24)00003-8. [Epub ahead of print]28(6): 621-629
    The beauty and complexity of the small heat shock proteins: a report on the proceedings of the fourth workshop on small heat shock proteins.
    Heath Ecroyd, Britta Bartelt-Kirbach, Anat Ben-Zvi, Raffaella Bonavita, Yevheniia Bushman, Elena Casarotto, Ciro Cecconi, Wilson Chun Yu Lau, Jonathan D Hibshman, Joep Joosten, Virginia Kimonis, Rachel Klevit, Krzysztof Liberek, Kathryn A McMenimen, Tsukumi Miwa, Axel Mogk, Daniele Montepietra, Carsten Peters, Maria Resa Te Rocchetti, Dominik Saman, Angela Sisto, Valentina Secco, Annika Strauch, Hideki Taguchi, Morgan Tanguay, Barbara Tedesco, Melinda E Toth, Zihao Wang, Justin L P Benesch, Serena Carra.
      The Fourth Cell Stress Society International workshop on small heat shock proteins (sHSPs), a follow-up to successful workshops held in 2014, 2016 and 2018, took place as a virtual meeting on the 17-18 November 2022. The meeting was designed to provide an opportunity for those working on sHSPs to reconnect and discuss their latest work. The diversity of research in the sHSP field is reflected in the breadth of topics covered in the talks presented at this meeting. Here we summarise the presentations at this meeting and provide some perspectives on exciting future topics to be addressed in the field.
    Keywords:  Cellular stress; Molecular chaperones; Protein aggregation; Protein folding; Protein homeostasis; Small heat shock proteins
    DOI:  https://doi.org/10.1007/s12192-023-01360-x
  35. Mol Syst Biol. 2024 Jan 15.
    Systematic discovery of protein interaction interfaces using AlphaFold and experimental validation.
    Chop Yan Lee, Dalmira Hubrich, Julia K Varga, Christian Schäfer, Mareen Welzel, Eric Schumbera, Milena Djokic, Joelle M Strom, Jonas Schönfeld, Johanna L Geist, Feyza Polat, Toby J Gibson, Claudia Isabelle Keller Valsecchi, Manjeet Kumar, Ora Schueler-Furman, Katja Luck.
      Structural resolution of protein interactions enables mechanistic and functional studies as well as interpretation of disease variants. However, structural data is still missing for most protein interactions because we lack computational and experimental tools at scale. This is particularly true for interactions mediated by short linear motifs occurring in disordered regions of proteins. We find that AlphaFold-Multimer predicts with high sensitivity but limited specificity structures of domain-motif interactions when using small protein fragments as input. Sensitivity decreased substantially when using long protein fragments or full length proteins. We delineated a protein fragmentation strategy particularly suited for the prediction of domain-motif interfaces and applied it to interactions between human proteins associated with neurodevelopmental disorders. This enabled the prediction of highly confident and likely disease-related novel interfaces, which we further experimentally corroborated for FBXO23-STX1B, STX1B-VAMP2, ESRRG-PSMC5, PEX3-PEX19, PEX3-PEX16, and SNRPB-GIGYF1 providing novel molecular insights for diverse biological processes. Our work highlights exciting perspectives, but also reveals clear limitations and the need for future developments to maximize the power of Alphafold-Multimer for interface predictions.
    Keywords:  AlphaFold; Benchmarking; Experimental Validation; Linear Motifs; Protein Interaction Interface Prediction
    DOI:  https://doi.org/10.1038/s44320-023-00005-6
  36. Elife. 2024 Jan 16. pii: e94919. [Epub ahead of print]13
    Structural assembly of the bacterial essential interactome.
    Jordi Gomez Borrego, Marc Torrent.
      The study of protein interactions in living organisms is fundamental for understanding biological processes and central metabolic pathways. Yet, our knowledge of the bacterial interactome remains limited. Here, we combined gene deletion mutant analysis with deep learning protein folding using Alphafold2 to predict the core bacterial essential interactome. We predicted and modeled 1402 interactions between essential proteins in bacteria and generated 146 high-accuracy models. Our analysis reveals previously unknown details about the assembly mechanisms of these complexes, highlighting the importance of specific structural features in their stability and function. Our work provides a framework for predicting the essential interactomes of bacteria and highlight the potential of deep learning algorithms in advancing our understanding of the complex biology of living organisms. Also, the results presented here offer a promising approach to identify novel antibiotic targets.
    Keywords:  B. subtilis; E. coli; computational biology; systems biology
    DOI:  https://doi.org/10.7554/eLife.94919
  37. ChemMedChem. 2024 Jan 17. e202300669
    Drugging Protein Tyrosine Phosphatases through Targeted Protein Degradation.
    Jinmin Miao, Zhong-Yin Zhang.
      Protein tyrosine phosphatases (PTPs) are an important class of enzymes that regulate protein tyrosine phosphorylation levels of a large variety of proteins in cells. Anomalies in protein tyrosine phosphorylation have been associated with the development of numerous human diseases, leading to a heightened interest in PTPs as promising targets for drug development. However, therapeutic targeting of PTPs has faced skepticism about their druggability. Besides the conventional small molecule inhibitors, proteolysis-targeting chimera (PROTAC) technology offers an alternative approach to target PTPs. PROTAC molecules utilize the ubiquitin-proteasome system to degrade specific proteins and have unique advantages compared with inhibitors: 1) PROTACs are highly efficient and can work at much lower concentrations than that expected based on their biophysical binding affinity; 2) PROTACs may achieve higher selectivity for the targeted protein than that dictated by their binding affinity alone; and 3) PROTACs may engage any region of the target protein in addition to the functional site. This review focuses on the latest advancement in the development of targeted PTP degraders and deliberates on the obstacles and prospective paths of harnessing this technology for therapeutic targeting of the PTPs.
    Keywords:  Cancer; diabetes; obesity; protein tyrosine phosphatase; targeted protein degradation
    DOI:  https://doi.org/10.1002/cmdc.202300669
  38. Cell Death Dis. 2024 Jan 17. 15(1): 63
    DDRGK1-mediated ER-phagy attenuates acute kidney injury through ER-stress and apoptosis.
    Haijiao Jin, Yuanting Yang, Xuying Zhu, Yin Zhou, Yao Xu, Jialin Li, Chaojun Qi, Xinghua Shao, Jingkui Wu, Shan Wu, Hong Cai, Leyi Gu, Shan Mou, Zhaohui Ni, Shu Li, Qisheng Lin.
      Acute kidney injury (AKI) constitutes a prevalent clinical syndrome characterized by elevated morbidity and mortality rates, emerging as a significant public health issue. This study investigates the interplay between endoplasmic reticulum (ER) stress, unfolded protein response (UPR), and ER-associated degradation (ER-phagy) in the pathogenesis of AKI. We employed four distinct murine models of AKI-induced by contrast media, ischemia-reperfusion injury, cisplatin, and folic acid-to elucidate the relationship between ER-phagy, ER stress, and apoptosis. Our findings reveal a marked decrease in ER-phagy coinciding with an accumulation of damaged ER, elevated ER stress, and increased apoptosis across all AKI models. Importantly, overexpression of DDRGK1 in HK-2 cells enhanced ER-phagy levels, ameliorating contrast-induced ER stress and apoptosis. These findings unveil a novel protective mechanism in AKI, wherein DDRGK1-UFL1-mediated ER-phagy mitigates ER stress and apoptosis in renal tubular epithelial cells. Our results thereby contribute to understanding the molecular underpinnings of AKI and offer potential therapeutic targets for its treatment.
    DOI:  https://doi.org/10.1038/s41419-024-06449-4
  39. Cell Rep. 2024 Jan 17. pii: S2211-1247(24)00006-8. [Epub ahead of print]43(2): 113678
    Recruitment of BAG2 to DNAJ-PKAc scaffolds promotes cell survival and resistance to drug-induced apoptosis in fibrolamellar carcinoma.
    Sophia M Lauer, Mitchell H Omar, Martin G Golkowski, Heidi L Kenerson, Kyung-Soon Lee, Bryan C Pascual, Huat C Lim, Katherine Forbush, F Donelson Smith, John D Gordan, Shao-En Ong, Raymond S Yeung, John D Scott.
      The DNAJ-PKAc fusion kinase is a defining feature of fibrolamellar carcinoma (FLC). FLC tumors are notoriously resistant to standard chemotherapies, with aberrant kinase activity assumed to be a contributing factor. By combining proximity proteomics, biochemical analyses, and live-cell photoactivation microscopy, we demonstrate that DNAJ-PKAc is not constrained by A-kinase anchoring proteins. Consequently, the fusion kinase phosphorylates a unique array of substrates, including proteins involved in translation and the anti-apoptotic factor Bcl-2-associated athanogene 2 (BAG2), a co-chaperone recruited to the fusion kinase through association with Hsp70. Tissue samples from patients with FLC exhibit increased levels of BAG2 in primary and metastatic tumors. Furthermore, drug studies implicate the DNAJ-PKAc/Hsp70/BAG2 axis in potentiating chemotherapeutic resistance. We find that the Bcl-2 inhibitor navitoclax enhances sensitivity to etoposide-induced apoptosis in cells expressing DNAJ-PKAc. Thus, our work indicates BAG2 as a marker for advanced FLC and a chemotherapeutic resistance factor in DNAJ-PKAc signaling scaffolds.
    Keywords:  CP: Cancer; CP: Molecular biology
    DOI:  https://doi.org/10.1016/j.celrep.2024.113678
  40. Elife. 2024 Jan 19. pii: e85214. [Epub ahead of print]13
    Organelle proteomic profiling reveals lysosomal heterogeneity in association with longevity.
    Yong Yu, Shihong M Gao, Youchen Guan, Pei-Wen Hu, Qinghao Zhang, Jiaming Liu, Bentian Jing, Qian Zhao, David M Sabatini, Monther Abu-Remaileh, Sung Yun Jung, Meng C Wang.
      Lysosomes are active sites to integrate cellular metabolism and signal transduction. A collection of proteins associated with the lysosome mediate these metabolic and signaling functions. Both lysosomal metabolism and lysosomal signaling have been linked to longevity regulation; however, how lysosomes adjust their protein composition to accommodate this regulation remains unclear. Using deep proteomic profiling, we systemically profiled lysosome-associated proteins linked with four different longevity mechanisms. We discovered the lysosomal recruitment of AMPK and nucleoporin proteins and their requirements for longevity in response to increased lysosomal lipolysis. Through comparative proteomic analyses of lysosomes from different tissues and labeled with different markers, we further elucidated lysosomal heterogeneity across tissues as well as the increased enrichment of the Ragulator complex on Cystinosin positive lysosomes. Together, this work uncovers lysosomal proteome heterogeneity across multiple scales and provides resources for understanding the contribution of lysosomal protein dynamics to signal transduction, organelle crosstalk and organism longevity.
    Keywords:  C. elegans; cell biology
    DOI:  https://doi.org/10.7554/eLife.85214
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