bims-proteo Biomed News
on Proteostasis
Issue of 2024‒09‒29
48 papers selected by
Eric Chevet, INSERM
  1. Membrane remodeling via ubiquitin-mediated pathways.
  2. Molecular basis of TMED9 oligomerization and entrapment of misfolded protein cargo in the early secretory pathway.
  3. The Dsc complex and its role in Golgi quality control.
  4. eIF2B localization and its regulation during the integrated stress response is cell-type specific.
  5. Distinct modes of coupling between VCP, an essential unfoldase, and deubiquitinases.
  6. Macrophage NRF1 promotes mitochondrial protein turnover via the ubiquitin proteasome system to limit mitochondrial stress and inflammation.
  7. Chemical tools for probing the Ub/Ubl conjugation cascades.
  8. Efficient PROTAC-ing: combinational use of PROTACs with signaling pathway inhibitors.
  9. Linear poly-ubiquitin remodels the proteome and influences hundreds of regulators in Drosophila.
  10. Remodeling of the secretory pathway is coordinated with de novo membrane formation in budding yeast gametogenesis.
  11. SMURF1 mediates damaged lysosomal homeostasis by ubiquitinating PPP3CB to promote the activation of TFEB.
  12. Designed endocytosis-inducing proteins degrade targets and amplify signals.
  13. PLK2-mediated phosphorylation of SQSTM1 S349 promotes aggregation of polyubiquitinated proteins upon proteasomal dysfunction.
  14. Unveiling the hidden interactome of CRBN molecular glues with chemoproteomics.
  15. DDI2 protease controls embryonic development and inflammation via TCF11/NRF1.
  16. Mass Spectral Feature Analysis of Ubiquitylated Peptides Provides Insights into Probing the Dark Ubiquitylome.
  17. Epigenetic regulation of global proteostasis dynamics by RBBP5 ensures mammalian organismal health.
  18. Kinesin-1 mediates proper ER folding of the CaV1.2 channel and maintains mouse glucose homeostasis.
  19. Large-scale map of RNA-binding protein interactomes across the mRNA life cycle.
  20. Leveraging Covalency to Stabilize Ternary Complex Formation For Cell-Cell "Induced Proximity".
  21. Transferrin receptor targeting chimeras for membrane protein degradation.
  22. ATF6 promotes colorectal cancer growth and stemness by regulating the Wnt pathway.
  23. Biomolecular condensation of ERC1 recruits ATG8 and NBR1 to drive autophagosome formation for plant heat tolerance.
  24. Time-resolved proximity proteomics uncovers a membrane tension-sensitive caveolin-1 interactome at the rear of migrating cells.
  25. Mistranslating tRNA variants have anticodon- and sex-specific impacts on Drosophila melanogaster.
  26. Two distinct regulatory pathways govern Cct2-Atg8 binding in the process of solid aggrephagy.
  27. Specific activation of the integrated stress response uncovers regulation of central carbon metabolism and lipid droplet biogenesis.
  28. Multistate and functional protein design using RoseTTAFold sequence space diffusion.
  29. Integrated multi-omics analysis of zinc-finger proteins uncovers roles in RNA regulation.
  30. Ubiquitylation: Sword and shield in the bacterial arsenal.
  31. Deep neural networks for predicting the affinity landscape of protein-protein interactions.
  32. A rapid in vivo pipeline to identify small molecule inhibitors of amyloid aggregation.
  33. The Role of Protein Disulfide Isomerase Inhibitors in Cancer Therapy.
  34. The genetic architecture of protein stability.
  35. Alternative translation initiation produces synaptic organizer proteoforms with distinct localization and functions.
  36. Arf1-dependent LRBA recruitment to Rab4 endosomes is required for endolysosome homeostasis.
  37. Tau accumulation is cleared by the induced expression of VCP via autophagy.
  38. SURF2 is a MDM2 antagonist in triggering the nucleolar stress response.
  39. New therapies on the horizon: Targeted protein degradation in neuroscience.
  40. Structure of the Nmd4-Upf1 complex supports conservation of the nonsense-mediated mRNA decay pathway between yeast and humans.
  41. Mitochondrial protein heterogeneity stems from the stochastic nature of co-translational protein targeting in cell senescence.
  42. ClickRNA-PROTAC for Tumor-Selective Protein Degradation and Targeted Cancer Therapy.
  43. Identification of proteotoxic and proteoprotective bacteria that non-specifically affect proteins associated with neurodegenerative diseases.
  44. Understanding species-specific and conserved RNA-protein interactions in vivo and in vitro.
  45. Coming in out of the cold: Rho-dependent termination contributes to adaptation to cold shock.
  46. Geranylgeranylated SCFFBXO10 regulates selective outer mitochondrial membrane proteostasis and function.
  47. Translation stalling induced mitochondrial entrapment of ribosomal quality control related proteins offers cancer cell vulnerability.
  48. A novel role for thioredoxin-related transmembrane protein TMX4 in platelet activation and thrombus formation.
  1. Cell Chem Biol. 2024 Sep 19. pii: S2451-9456(24)00356-8. [Epub ahead of print]31(9): 1627-1635
    Membrane remodeling via ubiquitin-mediated pathways.
    Anne-Claire Jacomin, Ivan Dikic.
      The dynamic process of membrane shaping and remodeling plays a vital role in cellular functions, with proteins and cellular membranes interacting intricately to adapt to various cellular needs and environmental cues. Ubiquitination-a posttranslational modification-was shown to be essential in regulating membrane structure and shape. It influences virtually all pathways relying on cellular membranes, such as endocytosis and autophagy by directing protein degradation, sorting, and oligomerization. Ubiquitin is mostly known as a protein modifier; however, it was reported that ubiquitin and ubiquitin-like proteins can associate directly with lipids, affecting membrane curvature and dynamics. In this review, we summarize some of the current knowledge on ubiquitin-mediated membrane remodeling in the context of endocytosis, autophagy, and ER-phagy.
    Keywords:  ER remodeling; ER-phagy; ESCRT; autophagy; membrane remodeling; ubiquitin
    DOI:  https://doi.org/10.1016/j.chembiol.2024.08.007
  2. Sci Adv. 2024 Sep 20. 10(38): eadp2221
    Molecular basis of TMED9 oligomerization and entrapment of misfolded protein cargo in the early secretory pathway.
    Le Xiao, Xiong Pi, Alissa C Goss, Tarick El-Baba, Julian F Ehrmann, Elizabeth Grinkevich, Silvana Bazua-Valenti, Valeria Padovano, Seth L Alper, Dominique Carey, Namrata D Udeshi, Steven A Carr, Juan Lorenzo Pablo, Carol V Robinson, Anna Greka, Hao Wu.
      Intracellular accumulation of misfolded proteins causes serious human proteinopathies. The transmembrane emp24 domain 9 (TMED9) cargo receptor promotes a general mechanism of cytotoxicity by entrapping misfolded protein cargos in the early secretory pathway. However, the molecular basis for this TMED9-mediated cargo retention remains elusive. Here, we report cryo-electron microscopy structures of TMED9, which reveal its unexpected self-oligomerization into octamers, dodecamers, and, by extension, even higher-order oligomers. The TMED9 oligomerization is driven by an intrinsic symmetry mismatch between the trimeric coiled coil domain and the tetrameric transmembrane domain. Using frameshifted Mucin 1 as an example of aggregated disease-related protein cargo, we implicate a mode of direct interaction with the TMED9 luminal Golgi-dynamics domain. The structures suggest and we confirm that TMED9 oligomerization favors the recruitment of coat protein I (COPI), but not COPII coatomers, facilitating retrograde transport and explaining the observed cargo entrapment. Our work thus reveals a molecular basis for TMED9-mediated misfolded protein retention in the early secretory pathway.
    DOI:  https://doi.org/10.1126/sciadv.adp2221
  3. Biochem Soc Trans. 2024 Sep 26. pii: BST20230375. [Epub ahead of print]
    The Dsc complex and its role in Golgi quality control.
    Yannick Weyer, David Teis.
      Membrane proteins play crucial roles in cellular functions. However, processes such as the insertion of membrane proteins into the endoplasmic reticulum (ER), their folding into native structures, the assembly of multi-subunit membrane protein complexes, and their targeting from the ER to specific organelles are prone to errors and have a relatively high failure rate. To prevent the accumulation of defective or orphaned membrane proteins, quality control mechanisms assess folding, quantity, and localization of these proteins. This quality control is vital for preserving organelle integrity and maintaining cellular health. In this mini-review, we will focus on how selective membrane protein quality control at the Golgi apparatus, particularly through the defective for SREBP cleavage (Dsc) ubiquitin ligase complex, detects orphaned proteins and prevents their mis-localization to other organelles.
    Keywords:  Golgi; quality control; ubiquitin
    DOI:  https://doi.org/10.1042/BST20230375
  4. iScience. 2024 Sep 20. 27(9): 110851
    eIF2B localization and its regulation during the integrated stress response is cell-type specific.
    Filipe M Hanson, Madalena I Ribeiro de Oliveira, Alison K Cross, K Elizabeth Allen, Susan G Campbell.
      Eukaryotic initiation factor 2B (eIF2B) controls translation initiation by recycling inactive eIF2-GDP to active eIF2-GTP. Under cellular stress, the integrated stress response (ISR) is activated inhibiting eIF2B activity resulting in the translation attenuation and reprogramming of gene expression to overcome the stress. The ISR can dictate cell fate wherein chronic activation has pathological outcomes. Vanishing white matter disease (VWMD) is a chronic ISR-related disorder with mutations in eIF2B targeting astrocyte and oligodendrocyte cells. Regulation of eIF2B localization (eIF2B bodies) has been implicated in the ISR. We present evidence that neuronal and glial cell types possess distinct patterns of eIF2B bodies which change in a manner correlating to acute and chronic ISR activation. We also demonstrate that while neural and glial cell types respond similarly to the acute induction of the ISR a chronic ISR exerts cell-type specific differences. These findings provide key insights into neural cell responses and adaptation to cellular stress.
    Keywords:  Biological sciences; Cell; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2024.110851
  5. bioRxiv. 2024 Sep 09. pii: 2024.09.08.611915. [Epub ahead of print]
    Distinct modes of coupling between VCP, an essential unfoldase, and deubiquitinases.
    Lauren E Vostal, Noa E Dahan, Wenzhu Zhang, Matthew J Reynolds, Brian T Chait, Tarun M Kapoor.
      Errors in proteostasis, which requires regulated degradation and recycling of diverse proteins, are linked to aging, cancer and neurodegenerative disease (1). In particular, recycling proteins from multiprotein complexes, organelles and membranes is initiated by ubiquitylation, extraction and unfolding by the essential mechanoenzyme VCP (2-4), and ubiquitin removal by deubiquitinases (DUBs), a class of ∼100 ubiquitin-specific proteases in humans (5, 6). As VCP's substrate recognition requires ubiquitylation, the removal of ubiquitins from substrates for recycling must follow extraction and unfolding. How the activities of VCP and different DUBs are coordinated for protein recycling or other fates is unclear. Here, we employ a photochemistry-based approach to profile proteome-wide domain-specific VCP interactions in living cells (7). We identify DUBs that bind near the entry, exit, or both sites of VCP's central pore, the channel for ATP-dependent substrate translocation (8-10). From this set of DUBs, we focus on VCPIP1, required for organelle assembly and DNA repair (11-13), that our chemical proteomics workflow indicates binds the central pore's entry and exit sites. We determine a ∼3Å cryo-EM structure of the VCP-VCPIP1 complex and find up to 3 VCPIP1 protomers interact with the VCP hexamer. VCPIP1's UBX-L domain binds VCP's N-domain in a 'down' conformation, linked to VCP's ADP-bound state (2, 14), and the deubiquitinase domain is positioned at the central pore's exit site, poised to remove ubiquitin following substrate unfolding. We find that VCP stimulates VCPIP1's DUB activity and use mutagenesis and single-molecule mass photometry assays to test the structural model. Together, our data suggest that DUBs bind VCP at distinct sites and reveal how the two enzyme activities can be coordinated to achieve specific downstream outcomes for ubiquitylated proteins.
    DOI:  https://doi.org/10.1101/2024.09.08.611915
  6. Cell Rep. 2024 Sep 25. pii: S2211-1247(24)01131-8. [Epub ahead of print]43(10): 114780
    Macrophage NRF1 promotes mitochondrial protein turnover via the ubiquitin proteasome system to limit mitochondrial stress and inflammation.
    Jiawei Yan, Xin Zhang, Huiying Wang, Xinglong Jia, Ruohong Wang, Shuangyang Wu, Zheng-Jiang Zhu, Minjia Tan, Tiffany Horng.
      Macrophage elaboration of inflammatory responses is dynamically regulated, shifting from acute induction to delayed suppression during the course of infection. Here, we show that such regulation of inflammation is modulated by dynamic shifts in metabolism. In macrophages exposed to the bacterial product lipopolysaccharide (LPS), an initial induction of protein biosynthesis is followed by compensatory induction of the transcription factor nuclear factor erythroid 2-like 1 (NRF1), leading to increased flux through the ubiquitin proteasome system (UPS). A major target of NRF1-mediated UPS flux is the mitochondrial proteome, and in the absence of NRF1, ubiquitinated mitochondrial proteins accumulate to trigger severe mitochondrial stress. Such mitochondrial stress engages the integrated stress response-ATF4 axis, which limits mitochondrial translation to attenuate mitochondrial stress but amplifies inflammatory responses to augment susceptibility to septic shock. Therefore, NRF1 mediates a dynamic regulation of mitochondrial proteostasis in inflammatory macrophages that contributes to curbing inflammatory responses.
    Keywords:  CP: Metabolism; CP: Molecular biology; NRF1; immunometabolism; inflammation; integrated stress response; macrophage; mitochondria; proteostasis
    DOI:  https://doi.org/10.1016/j.celrep.2024.114780
  7. Chembiochem. 2024 Sep 23. e202400659
    Chemical tools for probing the Ub/Ubl conjugation cascades.
    Tomasz Kochanczyk, Michael Fishman, Christopher D Lima.
      Conjugation of ubiquitin (Ub) and structurally related ubiquitin-like proteins (Ubl's), essential for many cellular processes, employs muti-step reactions orchestrated by specific E1, E2 and E3 enzymes. The E1 enzyme activates the Ub/Ubl C-terminus in an ATP-dependent process that results in the formation of a thioester linkage with the E1 active site cysteine. The thioester activated Ub/Ubl is transferred to the active site of an E2 enzyme which then interacts with an E3 enzyme to promote conjugation to the target substrate. The E1-E2-E3 enzymatic cascades utilize labile intermediates, extensive conformational changes, and vast combinatorial diversity of short-lived protein-protein complexes to conjugate Ub/Ubl to various substrates in a regulated manner. In this review, we discuss various chemical tools and methods used to study the consecutive steps of Ub/Ubl activation and conjugation, which are often too elusive for direct studies. We focus on methods developed to probe enzymatic activities and capture and characterize stable mimics of the transient intermediates and transition states thereby providing insights into fundamental mechanisms in the Ub/Ubl conjugation pathways.
    Keywords:  ubiquitin, ubiquitin-like, activity-based probes, ABP, enzymes, inhibitors, analogues, E1 activating enzyme, E2 conjugating enzyme, E3 protein ligase
    DOI:  https://doi.org/10.1002/cbic.202400659
  8. Trends Biochem Sci. 2024 Sep 26. pii: S0968-0004(24)00207-X. [Epub ahead of print]
    Efficient PROTAC-ing: combinational use of PROTACs with signaling pathway inhibitors.
    Yuri Shibata.
      Targeted protein degradation is an innovative therapeutic modality for the degradation of disease-causing proteins. In a recent report combining high-throughput screening of small-molecule compounds and biochemical analyses, Mori et al. identified certain inhibitors of cellular pathways, such as PARylation and proteostatic pathways, which enhance proteolysis-targeting chimera (PROTAC)-induced protein degradation.
    Keywords:  PROTACs; proteasome; targeted protein degradation; ubiquitination
    DOI:  https://doi.org/10.1016/j.tibs.2024.09.002
  9. G3 (Bethesda). 2024 Sep 26. pii: jkae209. [Epub ahead of print]
    Linear poly-ubiquitin remodels the proteome and influences hundreds of regulators in Drosophila.
    Oluwademilade Nuga, Kristin Richardson, Nikhil C Patel, Xusheng Wang, Vishwajeeth Pagala, Anna Stephan, Junmin Peng, Fabio Demontis, Sokol V Todi.
      Ubiquitin controls many cellular processes via its posttranslational conjugation onto substrates. Its use is highly variable due to its ability to form poly-ubiquitin chains with various topologies. Among them, linear chains have emerged as important regulators of immune responses and protein degradation. Previous studies in Drosophila melanogaster found that expression of linear poly-ubiquitin that cannot be dismantled into single moieties leads to their ubiquitination and degradation or, alternatively, to their conjugation onto proteins. However, it remains largely unknown which proteins are sensitive to linear poly-ubiquitin. To address this question, here we expanded the toolkit to modulate linear chains and conducted ultra-deep coverage proteomics from flies that express noncleavable, linear chains comprising 2, 4, or 6 moieties. We found that these chains regulate shared and distinct cellular processes in Drosophila by impacting hundreds of proteins, such as the circadian factor Cryptochrome. Our results provide key insight into the proteome subsets and cellular pathways that are influenced by linear poly-ubiquitin chains with distinct lengths and suggest that the ubiquitin system is exceedingly pliable.
    Keywords:  autophagy; catabolism; genetics; immune system; proteasome; proteomics
    DOI:  https://doi.org/10.1093/g3journal/jkae209
  10. iScience. 2024 Oct 18. 27(10): 110855
    Remodeling of the secretory pathway is coordinated with de novo membrane formation in budding yeast gametogenesis.
    Yasuyuki Suda, Hiroyuki Tachikawa, Tomomi Suda, Kazuo Kurokawa, Akihiko Nakano, Kenji Irie.
      Gametogenesis in budding yeast involves a large-scale rearrangement of membrane traffic to allow the de novo formation of a membrane, called the prospore membrane (PSM). However, the mechanism underlying this event is not fully elucidated. Here, we show that the number of endoplasmic reticulum exit sites (ERES) per cell fluctuates and switches from decreasing to increasing upon the onset of PSM formation. Reduction in ERES number, presumably accompanying a transient stall in membrane traffic, resulting in the loss of preexisting Golgi apparatus from the cell, was followed by local ERES regeneration, leading to Golgi reassembly in nascent spores. We have revealed that protein phosphatase-1 (PP-1) and its development-specific subunit, Gip1, promote ERES regeneration through Sec16 foci formation. Furthermore, sed4Δ, a mutant with impaired ERES formation, showed defects in PSM growth and spore formation. Thus, ERES regeneration in nascent spores facilitates the segregation of membrane traffic organelles, leading to PSM growth.
    Keywords:  Cell biology; Molecular biology
    DOI:  https://doi.org/10.1016/j.isci.2024.110855
  11. Autophagy. 2024 Sep 26.
    SMURF1 mediates damaged lysosomal homeostasis by ubiquitinating PPP3CB to promote the activation of TFEB.
    Qin Xia, Xuan Liu, Lu Zhong, Jun Qu, Lei Dong.
      The calcium-activated phosphatase PPP3/calcineurin dephosphorylates TFEB (transcription factor EB) to trigger its nuclear translocation and the activation of macroautophagic/autophagic targets. However, the detailed molecular mechanism regulating TFEB activation remains poorly understood. Here, we highlighted the importance of SMURF1 (SMAD specific E3 ubiquitin protein ligase 1) in the activation of TFEB for lysosomal homeostasis. SMURF1 deficiency prevents the calcium-triggered ubiquitination of the catalytic subunit of PPP3/calcineurin in a manner consistent with defective autophagic degradation of damaged lysosomes. Mechanically, PPP3CB/CNA2 plays a bridging role in the recruitment of SMURF1 by LGALS3 (galectin 3) upon lysosome damage. Importantly, PPP3CB increases the dissociation of the N-terminal tail (NT) and C-terminal carbohydrate-recognition domain (CRD) of LGALS3, which may promote the formation of open conformers in a PPP3CB dephosphorylation activity-dependent manner. In addition, PPP3CB is ubiquitinated at lysine 146 by the recruited SMURF1 in response to intracellular calcium stimulation. The K63-linked ubiquitination of PPP3CB enhances the recruitment of TFEB. Moreover, TFEB directly interacts with both PPP3CB and the regulatory subunit PPP3R1 which facilitate the conformational correction of TFEB for its activation for the transcription of TFEB-targeted genes. Altogether, our results highlighted a critical mechanism for the regulation of PPP3/calcineurin activity via its ubiquitin ligase SMURF1 in response to lysosomal membrane damage, which may account for a potential target for the treatment of stress-related diseases.
    Keywords:  Autophagy; PPP3/calcineurin; SMURF1; TFEB; lysosomal homeostasis
    DOI:  https://doi.org/10.1080/15548627.2024.2407709
  12. Nature. 2024 Sep 25.
    Designed endocytosis-inducing proteins degrade targets and amplify signals.
    Buwei Huang, Mohamad Abedi, Green Ahn, Brian Coventry, Isaac Sappington, Cong Tang, Rong Wang, Thomas Schlichthaerle, Jason Z Zhang, Yujia Wang, Inna Goreshnik, Ching Wen Chiu, Adam Chazin-Gray, Sidney Chan, Stacey Gerben, Analisa Murray, Shunzhi Wang, Jason O'Neill, Li Yi, Ronald Yeh, Ayesha Misquith, Anitra Wolf, Luke M Tomasovic, Dan I Piraner, Maria J Duran Gonzalez, Nathaniel R Bennett, Preetham Venkatesh, Maggie Ahlrichs, Craig Dobbins, Wei Yang, Xinru Wang, Danny D Sahtoe, Dionne Vafeados, Rubul Mout, Shirin Shivaei, Longxing Cao, Lauren Carter, Lance Stewart, Jamie B Spangler, Kole T Roybal, Per Jr Greisen, Xiaochun Li, Gonçalo J L Bernardes, Carolyn R Bertozzi, David Baker.
      Endocytosis and lysosomal trafficking of cell surface receptors can be triggered by endogenous ligands. Therapeutic approaches such as lysosome-targeting chimaeras1,2 (LYTACs) and cytokine receptor-targeting chimeras3 (KineTACs) have used this to target specific proteins for degradation by fusing modified native ligands to target binding proteins. Although powerful, these approaches can be limited by competition with native ligands and requirements for chemical modification that limit genetic encodability and can complicate manufacturing, and, more generally, there may be no native ligands that stimulate endocytosis through a given receptor. Here we describe computational design approaches for endocytosis-triggering binding proteins (EndoTags) that overcome these challenges. We present EndoTags for insulin-like growth factor 2 receptor (IGF2R) and asialoglycoprotein receptor (ASGPR), sortilin and transferrin receptors, and show that fusing these tags to soluble or transmembrane target protein binders leads to lysosomal trafficking and target degradation. As these receptors have different tissue distributions, the different EndoTags could enable targeting of degradation to different tissues. EndoTag fusion to a PD-L1 antibody considerably increases efficacy in a mouse tumour model compared to antibody alone. The modularity and genetic encodability of EndoTags enables AND gate control for higher-specificity targeted degradation, and the localized secretion of degraders from engineered cells. By promoting endocytosis, EndoTag fusion increases signalling through an engineered ligand-receptor system by nearly 100-fold. EndoTags have considerable therapeutic potential as targeted degradation inducers, signalling activators for endocytosis-dependent pathways, and cellular uptake inducers for targeted antibody-drug and antibody-RNA conjugates.
    DOI:  https://doi.org/10.1038/s41586-024-07948-2
  13. Autophagy. 2024 Oct;20(10): 2221-2237
    PLK2-mediated phosphorylation of SQSTM1 S349 promotes aggregation of polyubiquitinated proteins upon proteasomal dysfunction.
    Yun-Da Chen, Xiu-Ping Lin, Zi-Lun Ruan, Mi Li, Xue-Mei Yi, Xu Zhang, Shu Li, Hong-Bing Shu.
      Dysregulation in protein homeostasis results in accumulation of protein aggregates, which are sequestered into dedicated insoluble compartments so-called inclusion bodies or aggresomes, where they are scavenged through different mechanisms to reduce proteotoxicity. The protein aggregates can be selectively scavenged by macroautophagy/autophagy called aggrephagy, which is mediated by the autophagic receptor SQSTM1. In this study, we have identified PLK2 as an important regulator of SQSTM1-mediated aggregation of polyubiquitinated proteins. PLK2 is upregulated following proteasome inhibition, and then associates with and phosphorylates SQSTM1 at S349. The phosphorylation of SQSTM1 S349 strengthens its binding to KEAP1, which is required for formation of large SQSTM1 aggregates/bodies upon proteasome inhibition. Our findings suggest that PLK2-mediated phosphorylation of SQSTM1 S349 represents a critical regulatory mechanism in SQSTM1-mediated aggregation of polyubiquitinated proteins.
    Keywords:  PLK2; Phosphorylation; SQSTM1/p62; polyubiquitination; proteasome; protein aggregation
    DOI:  https://doi.org/10.1080/15548627.2024.2361574
  14. bioRxiv. 2024 Sep 15. pii: 2024.09.11.612438. [Epub ahead of print]
    Unveiling the hidden interactome of CRBN molecular glues with chemoproteomics.
    Kheewoong Baek, Rebecca J Metivier, Shourya S Roy Burman, Jonathan W Bushman, Ryan J Lumpkin, Dinah M Abeja, Megha Lakshminarayan, Hong Yue, Samuel Ojeda, Alyssa L Verano, Nathanael S Gray, Katherine A Donovan, Eric S Fischer.
      Targeted protein degradation and induced proximity refer to strategies that leverage the recruitment of proteins to facilitate their modification, regulation or degradation. As prospective design of glues remains challenging, unbiased discovery methods are needed to unveil hidden chemical targets. Here we establish a high throughput affinity purification mass spectrometry workflow in cell lysates for the unbiased identification of molecular glue targets. By mapping the targets of 20 CRBN-binding molecular glues, we identify 298 protein targets and demonstrate the utility of enrichment methods for identifying novel targets overlooked using established methods. We use a computational workflow to estimate target confidence and perform a biochemical screen to identify a lead compound for the new non-ZF target PPIL4. Our study provides a comprehensive inventory of targets chemically recruited to CRBN and delivers a robust and scalable workflow for identifying new drug-induced protein interactions in cell lysates.
    DOI:  https://doi.org/10.1101/2024.09.11.612438
  15. iScience. 2024 Oct 18. 27(10): 110893
    DDI2 protease controls embryonic development and inflammation via TCF11/NRF1.
    Monika Nedomova, Stefanie Haberecht-Müller, Sophie Möller, Simone Venz, Michaela Prochazkova, Jan Prochazka, Frantisek Sedlak, Kallayanee Chawengsaksophak, Elke Hammer, Petr Kasparek, Michael Adamek, Radislav Sedlacek, Jan Konvalinka, Elke Krüger, Klara Grantz Saskova.
      DDI2 is an aspartic protease that cleaves polyubiquitinated substrates. Upon proteotoxic stress, DDI2 activates the transcription factor TCF11/NRF1 (NFE2L1), crucial for maintaining proteostasis in mammalian cells, enabling the expression of rescue factors, including proteasome subunits. Here, we describe the consequences of DDI2 ablation in vivo and in cells. DDI2 knock-out (KO) in mice caused embryonic lethality at E12.5 with severe developmental failure. Molecular characterization of embryos showed insufficient proteasome expression with proteotoxic stress, accumulation of high molecular weight ubiquitin conjugates and induction of the unfolded protein response (UPR) and cell death pathways. In DDI2 surrogate KO cells, proteotoxic stress activated the integrated stress response (ISR) and induced a type I interferon (IFN) signature and IFN-induced proliferative signaling, possibly ensuring survival. These results indicate an important role for DDI2 in the cell-tissue proteostasis network and in maintaining a balanced immune response.
    Keywords:  Biological sciences; Developmental biology; Immune respons
    DOI:  https://doi.org/10.1016/j.isci.2024.110893
  16. J Am Soc Mass Spectrom. 2024 Sep 27.
    Mass Spectral Feature Analysis of Ubiquitylated Peptides Provides Insights into Probing the Dark Ubiquitylome.
    Regina M Edgington, Damien B Wilburn.
      Ubiquitylation is a structurally and functionally diverse post-translational modification that involves the covalent attachment of the small protein ubiquitin to other protein substrates. Trypsin-based proteomics is the most common approach for globally identifying ubiquitylation sites. However, we estimate that such methods are unable to detect ∼40% of ubiquitylation sites in the human proteome, i.e., "the dark ubiquitylome", including many important for human health and disease. In this meta-analysis of three large ubiquitylomic data sets, we performed a series of bioinformatic analyses to assess experimental features that could aid in uniquely identifying site-specific ubiquitylation events. Spectral predictions from Prosit were compared to experimental spectra of tryptic ubiquitylated peptides, revealing previously uncharacterized fragmentation of the diGly scar. Analysis of the LysC-derived ubiquitylated peptides reveals systematic, multidimensional peptide fragmentation, including diagnostic b-ions from fragmentation of the LysC ubiquitin scar. Comprehensively, these findings provide diagnostic spectral signatures of modification events that could be applied to new analysis methods for nontryptic ubiquitylomics.
    DOI:  https://doi.org/10.1021/jasms.4c00213
  17. bioRxiv. 2024 Sep 13. pii: 2024.09.13.612812. [Epub ahead of print]
    Epigenetic regulation of global proteostasis dynamics by RBBP5 ensures mammalian organismal health.
    Syeda Kubra, Michelle Sun, William Dion, Ahmet Catak, Hannah Luong, Haokun Wang, Yinghong Pan, Jia-Jun Liu, Aishwarya Ponna, Ian Sipula, Michael J Jurczak, Silvia Liu, Bokai Zhu.
      Proteostasis is vital for cellular health, with disruptions leading to pathologies including aging, neurodegeneration and metabolic disorders. Traditionally, proteotoxic stress responses were studied as acute reactions to various noxious factors; however, recent evidence reveals that many proteostasis stress-response genes exhibit ∼12-hour ultradian rhythms under physiological conditions in mammals. These rhythms, driven by an XBP1s-dependent 12h oscillator, are crucial for managing proteostasis. By exploring the chromatin landscape of the murine 12h hepatic oscillator, we identified RBBP5, a key subunit of the COMPASS complex writing H3K4me3, as an essential epigenetic regulator of proteostasis. RBBP5 is indispensable for regulating both the hepatic 12h oscillator and transcriptional response to acute proteotoxic stress, acting as a co-activator for proteostasis transcription factor XBP1s. RBBP5 ablation leads to increased sensitivity to proteotoxic stress, chronic inflammation, and hepatic steatosis in mice, along with impaired autophagy and reduced cell survival in vitro . In humans, lower RBBP5 expression is associated with reduced adaptive stress-response gene expression and hepatic steatosis. Our findings establish RBBP5 as a central regulator of proteostasis, essential for maintaining mammalian organismal health.
    DOI:  https://doi.org/10.1101/2024.09.13.612812
  18. EMBO Rep. 2024 Sep 25.
    Kinesin-1 mediates proper ER folding of the CaV1.2 channel and maintains mouse glucose homeostasis.
    Yosuke Tanaka, Atena Farkhondeh, Wenxing Yang, Hitoshi Ueno, Mitsuhiko Noda, Nobutaka Hirokawa.
      Glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells is a principal mechanism for systemic glucose homeostasis, of which regulatory mechanisms are still unclear. Here we show that kinesin molecular motor KIF5B is essential for GSIS through maintaining the voltage-gated calcium channel CaV1.2 levels, by facilitating an Hsp70-to-Hsp90 chaperone exchange to pass through the quality control in the endoplasmic reticulum (ER). Phenotypic analyses of KIF5B conditional knockout (cKO) mouse beta cells revealed significant abolishment of glucose-stimulated calcium transients, which altered the behaviors of insulin granules via abnormally stabilized cortical F-actin. KIF5B and Hsp90 colocalize to microdroplets on ER sheets, where CaV1.2 but not Kir6.2 is accumulated. In the absence of KIF5B, CaV1.2 fails to be transferred from Hsp70 to Hsp90 via STIP1, and is likely degraded via the proteasomal pathway. KIF5B and Hsc70 overexpression increased CaV1.2 expression via enhancing its chaperone binding. Thus, ER sheets may serve as the place of KIF5B- and Hsp90-dependent chaperone exchange, which predominantly facilitates CaV1.2 production in beta cells and properly enterprises GSIS against diabetes.
    Keywords:  Calcium Channel; ER Sheets; Hsp90; Insulin Secretion; Kinesin
    DOI:  https://doi.org/10.1038/s44319-024-00246-y
  19. Mol Cell. 2024 Sep 17. pii: S1097-2765(24)00726-3. [Epub ahead of print]
    Large-scale map of RNA-binding protein interactomes across the mRNA life cycle.
    Lena A Street, Katherine L Rothamel, Kristopher W Brannan, Wenhao Jin, Benjamin J Bokor, Kevin Dong, Kevin Rhine, Assael Madrigal, Norah Al-Azzam, Jenny Kim Kim, Yanzhe Ma, Darvesh Gorhe, Ahmed Abdou, Erica Wolin, Orel Mizrahi, Joshua Ahdout, Mayuresh Mujumdar, Ella Doron-Mandel, Marko Jovanovic, Gene W Yeo.
      mRNAs interact with RNA-binding proteins (RBPs) throughout their processing and maturation. While efforts have assigned RBPs to RNA substrates, less exploration has leveraged protein-protein interactions (PPIs) to study proteins in mRNA life-cycle stages. We generated an RNA-aware, RBP-centric PPI map across the mRNA life cycle in human cells by immunopurification-mass spectrometry (IP-MS) of ∼100 endogenous RBPs with and without RNase, augmented by size exclusion chromatography-mass spectrometry (SEC-MS). We identify 8,742 known and 20,802 unreported interactions between 1,125 proteins and determine that 73% of the IP-MS-identified interactions are RNA regulated. Our interactome links many proteins, some with unknown functions, to specific mRNA life-cycle stages, with nearly half associated with multiple stages. We demonstrate the value of this resource by characterizing the splicing and export functions of enhancer of rudimentary homolog (ERH), and by showing that small nuclear ribonucleoprotein U5 subunit 200 (SNRNP200) interacts with stress granule proteins and binds cytoplasmic RNA differently during stress.
    Keywords:  ERH; IP-MS; RBP; RNA-binding proteins; RNA-dependent protein interactions; SEC-MS; SNRNP200; interactome; mRNA life-cycle; protein-protein interactions
    DOI:  https://doi.org/10.1016/j.molcel.2024.08.030
  20. ACS Chem Biol. 2024 Sep 26.
    Leveraging Covalency to Stabilize Ternary Complex Formation For Cell-Cell "Induced Proximity".
    Karolina Krygier, Anjalee N Wijetunge, Arthur Srayeddin, Harrison Mccann, Anthony F Rullo.
      Recent advances in the field of translational chemical biology use diverse "proximity-inducing" synthetic modalities to elicit new modes of "event driven" pharmacology. These include mechanisms of targeted protein degradation and immune clearance of pathogenic cells. Heterobifunctional "chimeric" compounds like Proteolysis TArgeting Chimeras (PROTACs) and Antibody Recruiting Molecules (ARMs) leverage these mechanisms, respectively. Both systems function through the formation of reversible "ternary" or higher-order biomolecular complexes. Critical to function are key parameters, such as bifunctional molecule affinity for endogenous proteins, target residence time, and turnover. To probe the mechanism and enhance function, covalent chemical approaches have been developed to kinetically stabilize ternary complexes. These include electrophilic PROTACs and Covalent Immune Recruiters (CIRs), the latter designed to uniquely enforce cell-cell induced proximity. Inducing cell-cell proximity is associated with key challenges arising from a combination of steric and/or mechanical based destabilizing forces on the ternary complex. These factors can attenuate the formation of ternary complexes driven by high affinity bifunctional/proximity inducing molecules. This Account describes initial efforts in our lab to address these challenges using the CIR strategy in antibody recruitment or receptor engineered T cell model systems of cell-cell induced proximity. ARMs form ternary complexes with serum antibodies and surface protein antigens on tumor cells that subsequently engage immune cells via Fc receptors. Binding and clustering of Fc receptors trigger immune cell killing of the tumor cell. We applied the CIR strategy to convert ARMs to covalent chimeras, which "irreversibly" recruit serum antibodies to tumor cells. These covalent chimeras leverage electrophile preorganization and kinetic effective molarity to achieve fast and selective covalent engagement of the target ternary complex protein, e.g., serum antibody. Importantly, covalent engagement can proceed via diverse binding site amino acids beyond cysteine. Covalent chimeras demonstrated striking functional enhancements compared to noncovalent ARM analogs in functional immune assays. We revealed this enhancement was in fact due to the increased kinetic stability and not concentration, of ternary complexes. This finding was recapitulated using analogous CIR modalities that integrate peptidic or carbohydrate binding ligands with Sulfur(VI) Fluoride Exchange (SuFEx) electrophiles to induce cell-cell proximity. Mechanistic studies in a distinct model system that uses T cells engineered with receptors that recognize covalent chimeras or ARMs, revealed covalent receptor engagement uniquely enforces downstream activation signaling. Finally, this Account discusses potential challenges and future directions for adapting and optimizing covalent chimeric/bifunctional molecules for diverse applications in cell-cell induced proximity.
    Keywords:  ARMs; SuFEx; affinity electrophiles; affinity labeling; bifunctional molecules; covalent chimera; immunotherapy; induced proximity; preorganization; ternary complex
    DOI:  https://doi.org/10.1021/acschembio.4c00286
  21. Nature. 2024 Sep 25.
    Transferrin receptor targeting chimeras for membrane protein degradation.
    Dingpeng Zhang, Jhoely Duque-Jimenez, Francesco Facchinetti, Garyk Brixi, Kaitlin Rhee, William W Feng, Pasi A Jänne, Xin Zhou.
      Cancer cells require high levels of iron for rapid proliferation, leading to significant upregulation of cell-surface transferrin receptor 1 (TfR1), which mediates iron uptake by binding to the iron-carrying protein transferrin1-3. Leveraging this phenomenon and the fast endocytosis rate of TfR1 (refs. 4,5), we developed transferrin receptor targeting chimeras (TransTACs), a heterobispecific antibody modality for membrane protein degradation. TransTACs are engineered to drive rapid co-internalization of a target protein of interest and TfR1 from the cell surface, and to enable target protein entry into the lysosomal degradation pathway. We show that TransTACs can efficiently degrade a diverse range of single-pass, multi-pass, native or synthetic membrane proteins, including epidermal growth factor receptor, programmed cell death 1 ligand 1, cluster of differentiation 20 and chimeric antigen receptor. In example applications, TransTACs enabled the reversible control of human primary chimeric antigen receptor T cells and the targeting of drug-resistant epidermal growth factor receptor-driven lung cancer with the exon 19 deletion/T790M/C797S mutations in a mouse xenograft model. TransTACs represent a promising new family of bifunctional antibodies for precise manipulation of membrane proteins and targeted cancer therapy.
    DOI:  https://doi.org/10.1038/s41586-024-07947-3
  22. Cancer Res Commun. 2025 Sep 26.
    ATF6 promotes colorectal cancer growth and stemness by regulating the Wnt pathway.
    Jeffrey J Rodvold, Matthew Grimmer, Karen Ruiz, Scot A Marsters, Ioanna Oikonomidi, Eileen Tan-Aristy, Victoria C Pham, Tamal Sarkar, Jonathan M Harnoss, Whitney Shatz-Binder, Zora D Modrusan, Thomas D Wu, Jennie R Lill, Elisia Villemure, Joachim Rudolph, Felipe de Sousa E Melo, Avi Ashkenazi.
      The unfolded protein response maintains endoplasmic reticulum (ER) homeostasis by sensing protein-folding stress and orchestrating cellular adaptation via the ER-transmembrane proteins IRE1, PERK and ATF6. Malignant cells can co-opt IRE1 and PERK to sustain growth; however, the importance of ATF6 in cancer remains poorly deciphered. We observed elevated ATF6 transcriptional activity in several cancers including colorectal carcinoma (CRC). Genetic silencing or small molecule inhibition of ATF6 blocked cell cycle progression and reduced viability of several human CRC cell lines in vitro and disrupted tumor progression in vivo. Unexpectedly, ATF6 interference disabled Wnt and Myc signaling and reduced stemness. ATF6 inhibition attenuated growth of organoids derived from malignant but not normal human intestinal tissue, decreasing Wnt-pathway activity and driving cellular differentiation. Wnt-surrogate agonism in a Wnt ligand-dependent CRC organoid restored pathway activity and rescued growth under ATF6 blockade. Our findings identify ATF6 as an unexpected facilitator of oncogenic Wnt signaling in CRC.
    DOI:  https://doi.org/10.1158/2767-9764.CRC-24-0268
  23. bioRxiv. 2024 Sep 10. pii: 2024.09.09.611939. [Epub ahead of print]
    Biomolecular condensation of ERC1 recruits ATG8 and NBR1 to drive autophagosome formation for plant heat tolerance.
    Ka Kit Chung, Ziwei Zhao, Kai Ching Law, Juncai Ma, Cheuk Him Chiang, Kwan Ho Leung, Ruben Shrestha, Yixin Wu, Chaorui Li, Ka Ming Lee, Lei Feng, Xibao Li, Kam Bo Wong, Shou-Ling Xu, Caiji Gao, Xiaohong Zhuang.
      Macroautophagy (hereafter autophagy) is essential for cells to respond to nutrient stress by delivering cytosolic contents to vacuoles for degradation via the formation of a multi-layer vesicle named autophagosome. A set of autophagy-related (ATG) regulators are recruited to the phagophore assembly site for the initiation of phagophore, as well as its expansion and closure and subsequent delivery into the vacuole. However, it remains elusive that how the phagophore assembly is regulated under different stress conditions. Here, we described an unknown Arabidopsis (Arabidopsis thaliana) cytosolic ATG8-interaction protein family (ERC1/2), that binds ATG8 and NBR1 to promote autophagy. ERC1 proteins translocate to the phagophore membrane and develop into classical ring-like autophagosomes upon autophagic induction. However, ERC1 proteins form large droplets together with ATG8e proteins when in the absence of ATG8 lipidation activity. We described the property of these structures as phase-separated membraneless condensates by solving the in vivo organization with spatial and temporal resolution. Moreover, ERC1 condensates elicits a strong recruitment of the autophagic receptor NBR1. Loss of ERC1 suppressed NBR1 turnover and attenuated plant tolerance to heat stress condition. This work provides novel insights into the mechanical principle of phagophore initiation via an unreported ERC1-mediated biomolecular condensation for heat tolerance in Arabidopsis .
    DOI:  https://doi.org/10.1101/2024.09.09.611939
  24. Elife. 2024 Sep 24. pii: e85601. [Epub ahead of print]13
    Time-resolved proximity proteomics uncovers a membrane tension-sensitive caveolin-1 interactome at the rear of migrating cells.
    Eleanor Martin, Rossana Girardello, Gunnar Dittmar, Alexander Ludwig.
      Caveolae are small membrane pits with fundamental roles in mechanotransduction. Several studies have shown that caveolae flatten out in response to an increase in membrane tension, thereby acting as a mechanosensitive membrane reservoir that buffers acute mechanical stress. The dynamic assembly and disassembly of caveolae has also been implicated in the control of RhoA/ROCK-mediated actomyosin contractility at the rear of migrating cells. However, how membrane tension controls the organisation of caveolae and caveolae-mediated mechanotransduction is poorly understood. To address this, we systematically quantified protein-protein interactions of caveolin-1 in migrating RPE1 cells at steady state and in response to an acute increase in membrane tension using biotin-based proximity labelling and quantitative mass spectrometry. Our data show that caveolae are highly enriched at the rear of migrating RPE1 cells and that membrane tension rapidly and reversibly disassembles the caveolar protein coat. Membrane tension also dislodges caveolin-1 from focal adhesion proteins and several mechanosensitive cortical actin regulators including filamins and cortactin. In addition, we present evidence that ROCK and the RhoGAP ARHGAP29 are associated with caveolin-1 in a membrane tension-dependent manner, and that ARHGAP29 regulates caveolin-1 Y14 phosphorylation, caveolae rear localisation, and RPE1 cell migration. Taken together, our work uncovers a membrane tension-sensitive coupling between caveolae and the rear-localised F-actin cytoskeleton. This provides a framework for dissecting the molecular mechanisms underlying caveolae-regulated mechanotransduction pathways.
    Keywords:  cell biology; human
    DOI:  https://doi.org/10.7554/eLife.85601
  25. G3 (Bethesda). 2024 Sep 23. pii: jkae230. [Epub ahead of print]
    Mistranslating tRNA variants have anticodon- and sex-specific impacts on Drosophila melanogaster.
    Joshua R Isaacson, Matthew D Berg, Jessica Jagiello, William Yeung, Brendan Charles, Judit Villén, Christopher J Brandl, Amanda J Moehring.
      Transfer RNAs (tRNAs) are vital in determining the specificity of translation. Mutations in tRNA genes can result in the misincorporation of amino acids into nascent polypeptides in a process known as mistranslation. Since mistranslation has different impacts, depending on the type of amino acid substitution, our goal here was to compare the impact of different mistranslating tRNASer variants on fly development, lifespan, and behaviour. We established two mistranslating fly lines, one with a tRNASer variant that misincorporates serine at valine codons (V→S) and the other that misincorporates serine at threonine codons (T→S). While both mistranslating tRNAs increased development time and developmental lethality, the severity of the impacts differed depending on amino acid substitution and sex. The V→S variant extended embryonic, larval, and pupal development whereas the T→S only extended larval and pupal development. Females, but not males, containing either mistranslating tRNA presented with significantly more anatomical deformities than controls. Since mistranslation disrupts cellular translation and proteostasis, we also tested the hypothesis that tRNA variants impact fly lifespan. Interestingly, mistranslating females experienced extended lifespan whereas mistranslating male lifespan was unaffected. Consistent with delayed neurodegeneration and beneficial effects of mistranslation, mistranslating flies from both sexes showed improved locomotion as they aged. The ability of mistranslating tRNA variants to have both positive and negative effects on fly physiology and behaviour has important implications for human health given the prevalence of tRNA variants in humans.
    Keywords:   Drosophila melanogaster ; aging; development; longevity; mistranslation; neurodegeneration; proteostasis; tRNA
    DOI:  https://doi.org/10.1093/g3journal/jkae230
  26. EMBO Rep. 2024 Sep 25.
    Two distinct regulatory pathways govern Cct2-Atg8 binding in the process of solid aggrephagy.
    Yuting Chen, Zhaojie Liu, Yi Zhang, Miao Ye, Yingcong Chen, Jianhua Gao, Juan Song, Huan Yang, Choufei Wu, Weijing Yao, Xue Bai, Mingzhu Fan, Shan Feng, Yigang Wang, Liqin Zhang, Liang Ge, Du Feng, Cong Yi.
      CCT2 serves as an aggrephagy receptor that plays a crucial role in the clearance of solid aggregates, yet the underlying molecular mechanisms by which CCT2 regulates solid aggrephagy are not fully understood. Here we report that the binding of Cct2 to Atg8 is governed by two distinct regulatory mechanisms: Atg1-mediated Cct2 phosphorylation and the interaction between Cct2 and Atg11. Atg1 phosphorylates Cct2 at Ser412 and Ser470, and disruption of these phosphorylation sites impairs solid aggrephagy by hindering Cct2-Atg8 binding. Additionally, we observe that Atg11, an adaptor protein involved in selective autophagy, directly associates with Cct2 through its CC4 domain. Deficiency in this interaction significantly weakens the association of Cct2 with Atg8. The requirement of Atg1-mediated Cct2 phosphorylation and of Atg11 for CCT2-LC3C binding and subsequent aggrephagy is conserved in mammalian cells. These findings provide insights into the crucial roles of Atg1-mediated Cct2 phosphorylation and Atg11-Cct2 binding as key mediators governing the interaction between Cct2 and Atg8 during the process of solid aggrephagy.
    Keywords:  Atg1; Atg11; Cct2-Atg8 Binding; Phosphorylation; Solid Aggrephagy
    DOI:  https://doi.org/10.1038/s44319-024-00275-7
  27. Nat Commun. 2024 Sep 27. 15(1): 8301
    Specific activation of the integrated stress response uncovers regulation of central carbon metabolism and lipid droplet biogenesis.
    Katherine Labbé, Lauren LeBon, Bryan King, Ngoc Vu, Emily H Stoops, Nina Ly, Austin E Y T Lefebvre, Phillip Seitzer, Swathi Krishnan, Jin-Mi Heo, Bryson Bennett, Carmela Sidrauski.
      The integrated stress response (ISR) enables cells to cope with a variety of insults, but its specific contribution to downstream cellular outputs remains unclear. Using a synthetic tool, we selectively activate the ISR without co-activation of parallel pathways and define the resulting cellular state with multi-omics profiling. We identify time- and dose-dependent gene expression modules, with ATF4 driving only a small but sensitive subgroup that includes amino acid metabolic enzymes. This ATF4 response affects cellular bioenergetics, rerouting carbon utilization towards amino acid production and away from the tricarboxylic acid cycle and fatty acid synthesis. We also find an ATF4-independent reorganization of the lipidome that promotes DGAT-dependent triglyceride synthesis and accumulation of lipid droplets. While DGAT1 is the main driver of lipid droplet biogenesis, DGAT2 plays an essential role in buffering stress and maintaining cell survival. Together, we demonstrate the sufficiency of the ISR in promoting a previously unappreciated metabolic state.
    DOI:  https://doi.org/10.1038/s41467-024-52538-5
  28. Nat Biotechnol. 2024 Sep 25.
    Multistate and functional protein design using RoseTTAFold sequence space diffusion.
    Sidney Lyayuga Lisanza, Jacob Merle Gershon, Samuel W K Tipps, Jeremiah Nelson Sims, Lucas Arnoldt, Samuel J Hendel, Miriam K Simma, Ge Liu, Muna Yase, Hongwei Wu, Claire D Tharp, Xinting Li, Alex Kang, Evans Brackenbrough, Asim K Bera, Stacey Gerben, Bruce J Wittmann, Andrew C McShan, David Baker.
      Protein denoising diffusion probabilistic models are used for the de novo generation of protein backbones but are limited in their ability to guide generation of proteins with sequence-specific attributes and functional properties. To overcome this limitation, we developed ProteinGenerator (PG), a sequence space diffusion model based on RoseTTAFold that simultaneously generates protein sequences and structures. Beginning from a noised sequence representation, PG generates sequence and structure pairs by iterative denoising, guided by desired sequence and structural protein attributes. We designed thermostable proteins with varying amino acid compositions and internal sequence repeats and cage bioactive peptides, such as melittin. By averaging sequence logits between diffusion trajectories with distinct structural constraints, we designed multistate parent-child protein triples in which the same sequence folds to different supersecondary structures when intact in the parent versus split into two child domains. PG design trajectories can be guided by experimental sequence-activity data, providing a general approach for integrated computational and experimental optimization of protein function.
    DOI:  https://doi.org/10.1038/s41587-024-02395-w
  29. Mol Cell. 2024 Sep 16. pii: S1097-2765(24)00664-6. [Epub ahead of print]
    Integrated multi-omics analysis of zinc-finger proteins uncovers roles in RNA regulation.
    Maya L Gosztyla, Lijun Zhan, Sara Olson, Xintao Wei, Jack Naritomi, Grady Nguyen, Lena Street, Grant A Goda, Francisco F Cavazos, Jonathan C Schmok, Manya Jain, Easin Uddin Syed, Eunjeong Kwon, Wenhao Jin, Eric Kofman, Alexandra T Tankka, Allison Li, Valerie Gonzalez, Eric Lécuyer, Daniel Dominguez, Marko Jovanovic, Brenton R Graveley, Gene W Yeo.
      RNA interactome studies have revealed that hundreds of zinc-finger proteins (ZFPs) are candidate RNA-binding proteins (RBPs), yet their RNA substrates and functional significance remain largely uncharacterized. Here, we present a systematic multi-omics analysis of the DNA- and RNA-binding targets and regulatory roles of more than 100 ZFPs representing 37 zinc-finger families. We show that multiple ZFPs are previously unknown regulators of RNA splicing, alternative polyadenylation, stability, or translation. The examined ZFPs show widespread sequence-specific RNA binding and preferentially bind proximal to transcription start sites. Additionally, several ZFPs associate with their targets at both the DNA and RNA levels. We highlight ZNF277, a C2H2 ZFP that binds thousands of RNA targets and acts as a multi-functional RBP. We also show that ZNF473 is a DNA/RNA-associated protein that regulates the expression and splicing of cell cycle genes. Our results reveal diverse roles for ZFPs in transcriptional and post-transcriptional gene regulation.
    Keywords:  Cut&Run; RNA binding protein; RNA-seq; Ribo-STAMP; SLAM-seq; ZNF277; ZNF473; eCLIP; multi-omics; zinc finger
    DOI:  https://doi.org/10.1016/j.molcel.2024.08.010
  30. Mol Cell. 2024 Sep 19. pii: S1097-2765(24)00730-5. [Epub ahead of print]84(18): 3378-3380
    Ubiquitylation: Sword and shield in the bacterial arsenal.
    Jonathan N Pruneda, Felix Randow.
      In two recent studies in Nature, Hör et al.1 and Chambers et al.2 report that ubiquitin-like conjugation in bacteria antagonizes phage replication.
    DOI:  https://doi.org/10.1016/j.molcel.2024.08.034
  31. iScience. 2024 Sep 20. 27(9): 110772
    Deep neural networks for predicting the affinity landscape of protein-protein interactions.
    Reut Meiri, Shay-Lee Aharoni Lotati, Yaron Orenstein, Niv Papo.
      Studies determining protein-protein interactions (PPIs) by deep mutational scanning have focused mainly on a narrow range of affinities within complexes and thus include only partial coverage of the mutation space of given proteins. By inserting an affinity-reducing N-terminal alanine in the N-terminal domain of the tissue inhibitor of metalloproteinases-2 (N-TIMP2), we overcame the limitation of its narrow affinity range for matrix metalloproteinase 9 (MMP9CAT). We trained deep neural networks (DNNs) to quantitatively predict the binding affinity of unobserved wild-type variants and variants carrying an N-terminal alanine. Good correlation was obtained between predicted and observed log2 enrichment ratio (ER) values, which also correlated with the affinity of N-TIMP2 variants to MMP9CAT. Our ability to predict affinities of unobserved N-TIMP2 variants was confirmed on an independent dataset of experimentally validated N-TIMP2 proteins. This ability is of significant importance in the field of PPI prediction and for developing therapies targeting these interactions.
    Keywords:  Biochemical engineering; Machine learning; Protein
    DOI:  https://doi.org/10.1016/j.isci.2024.110772
  32. Nat Commun. 2024 Sep 27. 15(1): 8311
    A rapid in vivo pipeline to identify small molecule inhibitors of amyloid aggregation.
    Muntasir Kamal, Jessica Knox, Robert I Horne, Om Shanker Tiwari, Andrew R Burns, Duhyun Han, Davide Levy, Dana Laor Bar-Yosef, Ehud Gazit, Michele Vendruscolo, Peter J Roy.
      Amyloids are associated with over 50 human diseases and have inspired significant effort to identify small molecule remedies. Here, we present an in vivo platform that efficiently yields small molecule inhibitors of amyloid formation. We previously identified small molecules that kill the nematode C. elegans by forming membrane-piercing crystals in the pharynx cuticle, which is rich in amyloid-like material. We show here that many of these molecules are known amyloid-binders whose crystal-formation in the pharynx can be blocked by amyloid-binding dyes. We asked whether this phenomenon could be exploited to identify molecules that interfere with the ability of amyloids to seed higher-order structures. We therefore screened 2560 compounds and found 85 crystal suppressors, 47% of which inhibit amyloid formation. This hit rate far exceeds other screening methodologies. Hence, in vivo screens for suppressors of crystal formation in C. elegans can efficiently reveal small molecules with amyloid-inhibiting potential.
    DOI:  https://doi.org/10.1038/s41467-024-52480-6
  33. ChemMedChem. 2024 Sep 25. e202400590
    The Role of Protein Disulfide Isomerase Inhibitors in Cancer Therapy.
    Qiuying Nie, Junwei Yang, Xiedong Zhou, Na Li, Junmin Zhang.
      Protein disulfide isomerase (PDI) is a member of the mercaptan isomerase family, primarily located in the endoplasmic reticulum (ER). At least 21 PDI family members have been identified. PDI plays a key role in protein folding, correcting misfolded proteins, and catalyzing disulfide bond formation, rearrangement, and breaking. It also acts as a molecular chaperone. Dysregulation of PDI activity is thus linked to diseases such as cancer, infections, immune disorders, thrombosis, neurodegenerative diseases, and metabolic disorders. In particular, elevated intracellular PDI levels can enhance cancer cell proliferation, metastasis, and invasion, making it a potential cancer marker. Cancer cells require extensive protein synthesis, with disulfide bond formation by PDI being a critical producer. Thus, cancer cells have higher PDI levels than normal cells. Targeting PDI can induce ER stress and activate the Unfolded Protein Response (UPR) pathway, leading to cancer cell apoptosis. This review discusses the structure and function of PDI, PDI inhibitors in cancer therapy, and the limitations of current inhibitors, proposing especially future directions for developing new PDI inhibitors.
    Keywords:  Protein disulfide isomerase (PDI), PDI structure, PDI function, PDI inhibitor, Cancer therapy
    DOI:  https://doi.org/10.1002/cmdc.202400590
  34. Nature. 2024 Sep 25.
    The genetic architecture of protein stability.
    Andre J Faure, Aina Martí-Aranda, Cristina Hidalgo-Carcedo, Antoni Beltran, Jörn M Schmiedel, Ben Lehner.
      There are more ways to synthesize a 100-amino acid (aa) protein (20100) than there are atoms in the universe. Only a very small fraction of such a vast sequence space can ever be experimentally or computationally surveyed. Deep neural networks are increasingly being used to navigate high-dimensional sequence spaces1. However, these models are extremely complicated. Here, by experimentally sampling from sequence spaces larger than 1010, we show that the genetic architecture of at least some proteins is remarkably simple, allowing accurate genetic prediction in high-dimensional sequence spaces with fully interpretable energy models. These models capture the nonlinear relationships between free energies and phenotypes but otherwise consist of additive free energy changes with a small contribution from pairwise energetic couplings. These energetic couplings are sparse and associated with structural contacts and backbone proximity. Our results indicate that protein genetics is actually both rather simple and intelligible.
    DOI:  https://doi.org/10.1038/s41586-024-07966-0
  35. Mol Cell. 2024 Sep 17. pii: S1097-2765(24)00728-7. [Epub ahead of print]
    Alternative translation initiation produces synaptic organizer proteoforms with distinct localization and functions.
    Paul Jongseo Lee, Yu Sun, Alexa R Soares, Caroline Fai, Marina R Picciotto, Junjie U Guo.
      While many mRNAs contain more than one translation initiation site (TIS), the functions of most alternative TISs and their corresponding protein isoforms (proteoforms) remain undetermined. Here, we showed that alternative usage of CUG and AUG TISs in neuronal pentraxin receptor (NPR) mRNA produced two proteoforms, of which the ratio was regulated by RNA secondary structure and neuronal activity. Downstream AUG initiation truncated the N-terminal transmembrane domain and produced a secreted NPR proteoform sufficient in promoting synaptic clustering of AMPA-type glutamate receptors. Mutations that altered the ratio of NPR proteoforms reduced AMPA receptors in parvalbumin-positive interneurons and affected learning behaviors in mice. In addition to NPR, upstream AUU-initiated N-terminal extension of C1q-like synaptic organizers anchored these otherwise secreted factors to the membrane. Together, these results uncovered the plasticity of N-terminal signal sequences regulated by alternative TIS usage as a potentially widespread mechanism in diversifying protein localization and functions.
    Keywords:  AMPA receptor; RNA structure; neuronal pentraxin; proteoform; ribosome; secretory protein; signal sequence; synaptic organizer; translation initiation; transmembrane domain
    DOI:  https://doi.org/10.1016/j.molcel.2024.08.032
  36. J Cell Biol. 2024 Nov 04. pii: e202401167. [Epub ahead of print]223(11):
    Arf1-dependent LRBA recruitment to Rab4 endosomes is required for endolysosome homeostasis.
    Viktória Szentgyörgyi, Leon Maximilian Lueck, Daan Overwijn, Danilo Ritz, Nadja Zoeller, Alexander Schmidt, Maria Hondele, Anne Spang, Shahrzad Bakhtiar.
      Deleterious mutations in the lipopolysaccharide responsive beige-like anchor protein (LRBA) gene cause severe childhood immune dysregulation. The complexity of the symptoms involving multiple organs and the broad range of unpredictable clinical manifestations of LRBA deficiency complicate the choice of therapeutic interventions. Although LRBA has been linked to Rab11-dependent trafficking of the immune checkpoint protein CTLA-4, its precise cellular role remains elusive. We show that LRBA, however, only slightly colocalizes with Rab11. Instead, LRBA is recruited by members of the small GTPase Arf protein family to the TGN and to Rab4+ endosomes, where it controls intracellular traffic. In patient-derived fibroblasts, loss of LRBA led to defects in the endosomal pathway promoting the accumulation of enlarged endolysosomes and lysosome secretion. Thus, LRBA appears to regulate flow through the endosomal system on Rab4+ endosomes. Our data strongly suggest functions of LRBA beyond CTLA-4 trafficking and provide a conceptual framework to develop new therapies for LRBA deficiency.
    DOI:  https://doi.org/10.1083/jcb.202401167
  37. Acta Neuropathol. 2024 Sep 24. 148(1): 46
    Tau accumulation is cleared by the induced expression of VCP via autophagy.
    Hoi-Khoanh Giong, Seung Jae Hyeon, Jae-Geun Lee, Hyun-Ju Cho, Uiyeol Park, Thor D Stein, Junghee Lee, Kweon Yu, Hoon Ryu, Jeong-Soo Lee.
      Tauopathy, including frontotemporal lobar dementia and Alzheimer's disease, describes a class of neurodegenerative diseases characterized by the aberrant accumulation of Tau protein due to defects in proteostasis. Upon generating and characterizing a stable transgenic zebrafish that expresses the human TAUP301L mutant in a neuron-specific manner, we found that accumulating Tau protein was efficiently cleared via an enhanced autophagy activity despite constant Tau mRNA expression; apparent tauopathy-like phenotypes were revealed only when the autophagy was genetically or chemically inhibited. We performed RNA-seq analysis, genetic knockdown, and rescue experiments with clinically relevant point mutations of valosin-containing protein (VCP), and showed that induced expression of VCP, an essential cytosolic chaperone for the protein quality system, was a key factor for Tau degradation via its facilitation of the autophagy flux. This novel function of VCP in Tau clearance was further confirmed in a tauopathy mouse model where VCP overexpression significantly decreased the level of phosphorylated and oligomeric/aggregate Tau and rescued Tau-induced cognitive behavioral phenotypes, which were reversed when the autophagy was blocked. Importantly, VCP expression in the brains of human Alzheimer's disease patients was severely downregulated, consistent with its proposed role in Tau clearance. Taken together, these results suggest that enhancing the expression and activity of VCP in a spatiotemporal manner to facilitate the autophagy pathway is a potential therapeutic approach for treating tauopathy.
    Keywords:  Autophagy; Tau clearance; Tau-overexpressing animal models; VCP/p97
    DOI:  https://doi.org/10.1007/s00401-024-02804-z
  38. Nat Commun. 2024 Sep 27. 15(1): 8404
    SURF2 is a MDM2 antagonist in triggering the nucleolar stress response.
    Sophie Tagnères, Paulo Espirito Santo, Julie Radermecker, Dana Rinaldi, Carine Froment, Quentin Provost, Manon Bongers, Solemne Capeille, Nick Watkins, Julien Marcoux, Pierre-Emmanuel Gleizes, Virginie Marcel, Célia Plisson-Chastang, Simon Lebaron.
      Cancer cells rely on high ribosome production to sustain their proliferation rate. Many chemotherapies impede ribosome production which is perceived by cells as "nucleolar stress" (NS), triggering p53-dependent and independent pathways leading to cell cycle arrest and/or apoptosis. The 5S ribonucleoprotein (RNP) particle, a sub-ribosomal particle, is instrumental to NS response. Upon ribosome assembly defects, the 5S RNP accumulates as free form. This free form is able to sequester and inhibit MDM2, thus promoting p53 stabilization. To investigate how cancer cells can resist to NS, here we purify free 5S RNP and uncover an interaction partner, SURF2. Functional characterization of SURF2 shows that its depletion increases cellular sensitivity to NS, while its overexpression promotes their resistance to it. Consistently, SURF2 is overexpressed in many cancers and its expression level is an independent marker of prognosis for adrenocortical cancer. Our data demonstrate that SURF2 buffers free 5S RNP particles, and can modulate their activity, paving the way for the research of new molecules that can finely tune the response to nucleolar stress in the framework of cancer therapies.
    DOI:  https://doi.org/10.1038/s41467-024-52659-x
  39. Cell Chem Biol. 2024 Sep 19. pii: S2451-9456(24)00359-3. [Epub ahead of print]31(9): 1688-1698
    New therapies on the horizon: Targeted protein degradation in neuroscience.
    James A Gregory, Christopher M Hickey, Juan Chavez, Angela M Cacace.
      This minireview explores the burgeoning field of targeted protein degradation (TPD) and its promising applications in neuroscience and clinical development. TPD offers innovative strategies for modulating protein levels, presenting a paradigm shift in small-molecule drug discovery and therapeutic interventions. Importantly, small-molecule protein degraders specifically target and remove pathogenic proteins from central nervous system cells without the drug delivery challenges of genomic and antibody-based modalities. Here, we review recent advancements in TPD technologies, highlight proteolysis targeting chimera (PROTAC) protein degrader molecules with proximity-induced degradation event-driven and iterative pharmacology, provide applications in neuroscience research, and discuss the high potential for translation of TPD into clinical settings.
    DOI:  https://doi.org/10.1016/j.chembiol.2024.08.010
  40. PLoS Biol. 2024 Sep 27. 22(9): e3002821
    Structure of the Nmd4-Upf1 complex supports conservation of the nonsense-mediated mRNA decay pathway between yeast and humans.
    Irène Barbarin-Bocahu, Nathalie Ulryck, Amandine Rigobert, Nadia Ruiz Gutierrez, Laurence Decourty, Mouna Raji, Bhumika Garkhal, Hervé Le Hir, Cosmin Saveanu, Marc Graille.
      The nonsense-mediated mRNA decay (NMD) pathway clears eukaryotic cells of mRNAs containing premature termination codons (PTCs) or normal stop codons located in specific contexts. It therefore plays an important role in gene expression regulation. The precise molecular mechanism of the NMD pathway has long been considered to differ substantially from yeast to metazoa, despite the involvement of universally conserved factors such as the central ATP-dependent RNA-helicase Upf1. Here, we describe the crystal structure of the yeast Upf1 bound to its recently identified but yet uncharacterized partner Nmd4, show that Nmd4 stimulates Upf1 ATPase activity and that this interaction contributes to the elimination of NMD substrates. We also demonstrate that a region of Nmd4 critical for the interaction with Upf1 in yeast is conserved in the metazoan SMG6 protein, another major NMD factor. We show that this conserved region is involved in the interaction of SMG6 with UPF1 and that mutations in this region affect the levels of endogenous human NMD substrates. Our results support the universal conservation of the NMD mechanism in eukaryotes.
    DOI:  https://doi.org/10.1371/journal.pbio.3002821
  41. Nat Commun. 2024 Sep 27. 15(1): 8274
    Mitochondrial protein heterogeneity stems from the stochastic nature of co-translational protein targeting in cell senescence.
    Abdul Haseeb Khan, Xuefang Gu, Rutvik J Patel, Prabha Chuphal, Matheus P Viana, Aidan I Brown, Brian M Zid, Tatsuhisa Tsuboi.
      A decline in mitochondrial function is a hallmark of aging and neurodegenerative diseases. It has been proposed that changes in mitochondrial morphology, including fragmentation of the tubular mitochondrial network, can lead to mitochondrial dysfunction, yet the mechanism of this loss of function is unclear. Most proteins contained within mitochondria are nuclear-encoded and must be properly targeted to the mitochondria. Here, we report that sustained mRNA localization and co-translational protein delivery leads to a heterogeneous protein distribution across fragmented mitochondria. We find that age-induced mitochondrial fragmentation drives a substantial increase in protein expression noise across fragments. Using a translational kinetic and molecular diffusion model, we find that protein expression noise is explained by the nature of stochastic compartmentalization and that co-translational protein delivery is the main contributor to increased heterogeneity. We observed that cells primarily reduce the variability in protein distribution by utilizing mitochondrial fission-fusion processes rather than relying on the mitophagy pathway. Furthermore, we are able to reduce the heterogeneity of the protein distribution by inhibiting co-translational protein targeting. This research lays the framework for a better understanding of the detrimental impact of mitochondrial fragmentation on the physiology of cells in aging and disease.
    DOI:  https://doi.org/10.1038/s41467-024-52183-y
  42. J Am Chem Soc. 2024 Sep 25.
    ClickRNA-PROTAC for Tumor-Selective Protein Degradation and Targeted Cancer Therapy.
    Xucong Teng, Xuan Zhao, Yicong Dai, Xiangdong Zhang, Qiushuang Zhang, Yuncong Wu, Difei Hu, Jinghong Li.
      Proteolysis-targeting chimeras (PROTACs) show promise in tumor treatment. However, the E3 ligases VHL and CRBN, commonly used in PROTAC, are highly expressed in only a few tumors, thus limiting the application scope and efficacy of PROTAC drugs. Furthermore, the lack of tumor specificity in PROTAC drugs can result in toxic side effects. Therefore, there is an urgent need to develop tumor-selective PROTAC drugs that do not rely on endogenous E3 ligases. In this study, we introduce the ClickRNA-PROTAC system, which involves the expression of a fusion protein of the E3 ubiquitin ligase SIAH1 and SNAPTag through mRNA transfection and recruits the protein of interest (POI) using bio-orthogonal click chemistry. ClickRNA-PROTAC can effectively degrade various proteins such as BRD4, KRAS, and NFκB simply by replacing the warhead molecules. By employing a tumor-specific mRNA-responsive translation strategy, ClickRNA-PROTAC can selectively degrade POIs in tumor cells. Furthermore, ClickRNA-PROTAC demonstrated strong efficacy in targeted cancer therapy in a xenograft mouse model of adrenocortical carcinoma. In conclusion, this approach offers several advantages, including independence from endogenous E3 ubiquitin ligases, tumor specificity, and programmability, thereby paving the way for the development of PROTAC drugs.
    DOI:  https://doi.org/10.1021/jacs.4c06402
  43. iScience. 2024 Sep 20. 27(9): 110828
    Identification of proteotoxic and proteoprotective bacteria that non-specifically affect proteins associated with neurodegenerative diseases.
    Alyssa C Walker, Rohan Bhargava, Michael J Bucher, Yoan M Argote, Amanda S Brust, Daniel M Czyż.
      There are no cures for neurodegenerative protein conformational diseases (PCDs), such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Emerging evidence suggests the gut microbiota plays a role in their pathogenesis, though the influences of specific bacteria on disease-associated proteins remain elusive. Here, we reveal the effects of 229 human bacterial isolates on the aggregation and toxicity of Aβ1-42, α-synuclein, and polyglutamine tracts in Caenorhabditis elegans expressing these culprit proteins. Our findings demonstrate that bacterial effects on host protein aggregation are consistent across different culprit proteins, suggesting that microbes affect protein stability by modulating host proteostasis rather than selectively targeting disease-associated proteins. Furthermore, we found that feeding C. elegans proteoprotective Prevotella corporis activates the heat shock response, revealing an unexpected discovery of a microbial influence on host proteostasis. Insight into how individual bacteria affect PCD proteins could open new strategies for prevention and treatment by altering the abundance of microbes.
    Keywords:  Bacteriology; Biological sciences; Molecular neuroscience; Neuroscience
    DOI:  https://doi.org/10.1016/j.isci.2024.110828
  44. Nat Commun. 2024 Sep 27. 15(1): 8400
    Understanding species-specific and conserved RNA-protein interactions in vivo and in vitro.
    Sarah E Harris, Maria S Alexis, Gilbert Giri, Francisco F Cavazos, Yue Hu, Jernej Murn, Maria M Aleman, Christopher B Burge, Daniel Dominguez.
      While evolution is often considered from a DNA- and protein-centric view, RNA-based regulation can also impact gene expression and protein sequences. Here we examine interspecies differences in RNA-protein interactions using the conserved neuronal RNA-binding protein, Unkempt (UNK) as model. We find that roughly half of mRNAs bound in human are also bound in mouse. Unexpectedly, even when transcript-level binding was conserved across species differential motif usage was prevalent. To understand the biochemical basis of UNK-RNA interactions, we reconstitute the human and mouse UNK-RNA interactomes using a high-throughput biochemical assay. We uncover detailed features driving binding, show that in vivo patterns are captured in vitro, find that highly conserved sites are the strongest bound, and associate binding strength with downstream regulation. Furthermore, subtle sequence differences surrounding motifs are key determinants of species-specific binding. We highlight the complex features driving protein-RNA interactions and how these evolve to confer species-specific regulation.
    DOI:  https://doi.org/10.1038/s41467-024-52231-7
  45. Mol Cell. 2024 Sep 19. pii: S1097-2765(24)00729-9. [Epub ahead of print]84(18): 3373-3374
    Coming in out of the cold: Rho-dependent termination contributes to adaptation to cold shock.
    Teppei Morita, Susan Gottesman.
      During cold shock, bacteria shut down translation of all but a set of cold-shock proteins critical for recovery; in this issue of Molecular Cell, Delaleau et al.1 show that Rho-dependent transcription termination plays an important role in cold adaptation, via temperature-regulated termination of the cold-shock protein mRNAs.
    DOI:  https://doi.org/10.1016/j.molcel.2024.08.033
  46. Cell Rep. 2024 Sep 21. pii: S2211-1247(24)01134-3. [Epub ahead of print]43(10): 114783
    Geranylgeranylated SCFFBXO10 regulates selective outer mitochondrial membrane proteostasis and function.
    Sameer Ahmed Bhat, Zahra Vasi, Liping Jiang, Shruthi Selvaraj, Rachel Ferguson, Sanaz Salarvand, Anish Gudur, Ritika Adhikari, Veronica Castillo, Hagar Ismail, Avantika Dhabaria, Beatrix Ueberheide, Shafi Kuchay.
      Compartment-specific cellular membrane protein turnover is not well understood. We show that FBXO10, the interchangeable component of the cullin-RING-ligase 1 complex, undergoes lipid modification with geranylgeranyl isoprenoid at cysteine953, facilitating its dynamic trafficking to the outer mitochondrial membrane (OMM). FBXO10 polypeptide lacks a canonical mitochondrial targeting sequence (MTS); instead, its geranylgeranylation at C953 and interaction with two cytosolic factors, cytosolic factor-like δ subunit of type 6 phosphodiesterase (PDE6δ; a prenyl-group-binding protein) and heat shock protein 90 (HSP90; a chaperone), orchestrate specific OMM targeting of prenyl-FBXO10. The FBXO10(C953S) mutant redistributes away from the OMM, impairs mitochondrial ATP production and membrane potential, and increases fragmentation. Phosphoglycerate mutase-5 (PGAM5) was identified as a potential substrate of FBXO10 at the OMM using comparative quantitative proteomics of enriched mitochondria. FBXO10 loss or expression of prenylation-deficient FBXO10(C953S) inhibited PGAM5 degradation, disrupted mitochondrial homeostasis, and impaired myogenic differentiation of human induced pluripotent stem cells (iPSCs) and murine myoblasts. Our studies identify a mechanism for FBXO10-mediated regulation of selective mitochondrial proteostasis potentially amenable to therapeutic intervention.
    Keywords:  CP: Metabolism; CP: Molecular biology; E3-ligase; F-box protein; FBXO10; HSP90; PDE6δ; mitochondria; prenylation; trafficking; ubiquitination
    DOI:  https://doi.org/10.1016/j.celrep.2024.114783
  47. Res Sq. 2024 Sep 13. pii: rs.3.rs-4899860. [Epub ahead of print]
    Translation stalling induced mitochondrial entrapment of ribosomal quality control related proteins offers cancer cell vulnerability.
    Rani Ojha, Ishaq Tantray, Shouryarudra Banerjee, Suman Rimal, Sandiya Thirunavukkarasu, Saripella Srikris, Wah Chiu, Uttam Mete, Aditya Sharma, Nandita Kakkar, Bingwei Lu.
      Ribosome-associated quality control (RQC) monitors ribosomes for aberrant translation. While the role of RQC in neurodegenerative disease is beginning to be appreciated, its involvement in cancer is understudied. Here, we show a positive correlation between RQC proteins ABCE1 and ZNF598 and high-grade muscle-invasive bladder cancer. Translational stalling by the inhibitor emetine (EME) leads to increased mitochondrial localization of RQC factors including ABCE1, ZNF598, and NEMF, which are continuously imported into mitochondria facilitated by increased mitochondrial membrane potential caused by EME. This reduces the availability of these factors in the cytosol, compromising the effectiveness of RQC in handling stalled ribosomes in the cytosol and those associated with the mitochondrial outer membrane (MOM). Imported RQC factors form aggregates inside the mitochondria in a process we term stalling-induced mitochondrial stress (SIMS). ABCE1 plays a crucial role in maintaining mitochondrial health during SIMS. Notably, cancer stem cells (CSCs) exhibit increased expression of ABCE1 and consequently are more resistant to EME-induced mitochondrial dysfunction. This points to a potential mechanism of drug resistance by CSCs. Our study highlights the significance of mitochondrial entrapment of RQC factors such as ABCE1 in determining the fate of cancer cells versus CSCs. Targeting ABCE1 or other RQC factors in translational inhibition cancer therapy may help overcome drug resistance.
    DOI:  https://doi.org/10.21203/rs.3.rs-4899860/v1
  48. J Thromb Haemost. 2024 Sep 20. pii: S1538-7836(24)00549-X. [Epub ahead of print]
    A novel role for thioredoxin-related transmembrane protein TMX4 in platelet activation and thrombus formation.
    Zhenzhen Zhao, Yucan Wang, Aizhen Yang, Yi Lu, Xiaofeng Yan, Meinan Peng, Yue Han, Chao Fang, Depei Wu, Yi Wu.
      BACKGROUND: The functions of critical platelet proteins are controlled by thiol-disulfide exchanges, which are mediated by the protein disulfide isomerase (PDI) family. It has been shown that some PDI family members are important in platelet activation and thrombosis with distinct functions. TMX4, a membrane-type PDI family member, is expressed in platelets, whether it has a role in platelet activation remains unknown.OBJECTIVES: To determine the role of TMX4 in platelet activation and thrombosis.
    METHODS: The phenotypes of TMX4-deficient mice were evaluated in tail bleeding time assay and laser-induced and FeCl3-induced arterial injury models. The functions of TMX4 in platelets were assessed in vitro using TMX4-null platelets, recombinant TMX4 protein and anti-TMX4 antibody.
    RESULTS: Compared with the control mice, Tie2-Cre/TMX4fl/fl mice deficient of hematopoietic and endothelial TMX4 exhibited prolonged tail bleeding times and reduced platelet thrombus formation. Pf4-Cre/TMX4fl/fl mice deficient of platelet TMX4 also had prolonged tail bleeding times and decreased thrombus formation, which was rescued by injection of recombinant TMX4 protein. Consistently, TMX4 deficiency inhibited platelet aggregation, integrin αIIbβ3 activation, P-selectin expression, phosphatidylserine exposure and thrombin generation, without affecting tyrosine phosphorylation of intracellular signaling molecules Syk, LAT and PLCγ2 and calcium mobilization. Recombinant TMX4 protein enhanced platelet aggregation and reduced integrin αIIbβ3 disulfide bond, and TMX4 deficiency decreased free thiols of integrin αIIbβ3, consistent with a potent reductase activity of TMX4. In contrast, an inactive TMX4 protein and a specific anti-TMX4 antibody inhibited platelet aggregation.
    CONCLUSIONS: TMX4 is a novel PDI family member that enhances platelet activation and thrombosis.
    Keywords:  TMX4; integrin; platelet; protein disulfide isomerase; thiol-disulfide exchange; thrombosis
    DOI:  https://doi.org/10.1016/j.jtha.2024.09.007
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