bims-proteo Biomed News
on Proteostasis
Issue of 2024–12–01
48 papers selected by
Eric Chevet, INSERM
  1. ER quality control through reticulophagy and protein secretion.
  2. E3 ubiquitin ligase RNF10 promotes dissociation of stalled ribosomes and responds to ribosomal subunit imbalance.
  3. Unveiling the physiological impact of ESCRT-dependent autophagosome closure by targeting the VPS37A ubiquitin E2 variant-like domain.
  4. Identification of a factor that accelerates substrate release from the signal recognition particle.
  5. Leveraging Dual-Ligase Recruitment to Enhance Protein Degradation via a Heterotrivalent Proteolysis Targeting Chimera.
  6. UBAC2 serves as a reticulophagy receptor to suppress inflammatory responses.
  7. Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53.
  8. Pin1 promotes human CaV2.1 channel polyubiquitination by RNF138: pathophysiological implication for episodic ataxia type 2.
  9. Intersectin1 promotes clathrin-mediated endocytosis by organizing and stabilizing endocytic protein interaction networks.
  10. UPR deficiency in budding yeast reveals a trade-off between ER folding capacity and maintenance of euploidy.
  11. Ubiquitin-Independent Degradation: An Emerging PROTAC Approach?
  12. Crystal structures of DCAF1-PROTAC-WDR5 ternary complexes provide insight into DCAF1 substrate specificity.
  13. Investigating Protein Degradability through Site-Specific Ubiquitin Ligase Recruitment.
  14. Master regulators governing protein abundance across ten human cancer types.
  15. An engineered cereblon optimized for high throughput screening and molecular glue discovery.
  16. Dynamic remodelling of the endoplasmic reticulum for mitosis.
  17. Gene module reconstruction identifies cellular differentiation processes and the regulatory logic of specialized secretion in zebrafish.
  18. Selective autophagy impedes KSHV entry after recruiting the membrane damage sensor galectin-8 to virus-containing endosomes.
  19. Features of membrane protein sequence direct post-translational insertion.
  20. Coordination between the eIF2 kinase GCN2 and p53 signaling supports purine metabolism and the progression of prostate cancer.
  21. Cryo-EM Structure of AAA + ATPase Thorase Reveals Novel Helical Filament Formation.
  22. Dynamic fibrillar assembly of αB-crystallin induced by perturbation of the conserved NT-IXI motif resolved by cryo-EM.
  23. HSP90 buffers deleterious genetic variations in BRCA1.
  24. Programmable protein degraders enable selective knockdown of pathogenic β-catenin subpopulations in vitro and in vivo.
  25. ER-tethered stress sensor CREBH regulates mitochondrial unfolded protein response to maintain energy homeostasis.
  26. diaPASEF-Powered Chemoproteomics Enables Deep Kinome Interaction Profiling.
  27. UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation.
  28. A Kinetic Scout Approach Accelerates Targeted Protein Degrader Development.
  29. GOLPH3 and GOLPH3L maintain Golgi localization of LYSET and a functional mannose 6-phosphate transport pathway.
  30. Cancers adapt to their mutational load by buffering protein misfolding stress.
  31. snoRNA-facilitated protein secretion revealed by transcriptome-wide snoRNA target identification.
  32. The contribution of mitochondria-associated ER membranes to cholesterol homeostasis.
  33. Development of Oral, Potent, and Selective CK1α Degraders for AML Therapy.
  34. Global analysis of endogenous protein disorder in cells.
  35. Proteolethargy is a pathogenic mechanism in chronic disease.
  36. Covalent targeting of splicing in T cells.
  37. Accurate de novo design of high-affinity protein binding macrocycles using deep learning.
  38. Molecular basis of mRNA delivery to the bacterial ribosome.
  39. Stress Changes the Bacterial Biomolecular Condensate Material State and Shifts Function from mRNA Decay to Storage.
  40. Thioredoxin 1 moonlights as a chaperone for an interbacterial ADP-ribosyltransferase toxin.
  41. Structural basis for Retriever-SNX17 assembly and endosomal sorting.
  42. Interformer: an interaction-aware model for protein-ligand docking and affinity prediction.
  43. SurfDock is a surface-informed diffusion generative model for reliable and accurate protein-ligand complex prediction.
  44. Reveal the autophagic degradation of glutaredoxin Grx4 in Schizosaccharomyces pombe.
  45. Molecular insights into the interaction between a disordered protein and a folded RNA.
  46. IFN-γ-dependent regulation of intestinal epithelial homeostasis by NKT cells.
  47. Structural diversity and oligomerization of bacterial ubiquitin-like proteins.
  48. Stereochemistry in the disorder-order continuum of protein interactions.
  1. Autophagy. 2024 Nov 27. 1-2
    ER quality control through reticulophagy and protein secretion.
    Cathena Meiling Li, Yong-Keun Jung.
      The endoplasmic reticulum (ER) is the site of multiple cellular events and maintaining its quality control is thus crucial for cell homeostasis. Through a morphology-based gain-of-function screen, we identified the cytosolic protein FKBPL as a regulator of reticulophagy. With multiple protein-binding domains, FKBPL binds to the ER-resident CKAP4, acting as a bridge that connects the ER to the phagophore and facilitating the delivery of ER contents for lysosomal degradation. The FKBPL-CKAP4 axis is essential for both basal and stress-induced reticulophagy. Loss of the FKBPL-CKAP4 interaction attenuates reticulophagy and enhances protein secretion via microvesicle shedding. Here, we propose a dual role for the FKBPL-CKAP4 axis in regulating reticulophagy and protein secretion.
    Keywords:  ER quality control; ER stress; microvesicle shedding; protein secretion; reticulophagy
    DOI:  https://doi.org/10.1080/15548627.2024.2431340
  2. Nat Commun. 2024 Nov 28. 15(1): 10350
    E3 ubiquitin ligase RNF10 promotes dissociation of stalled ribosomes and responds to ribosomal subunit imbalance.
    Janina A Lehmann, Doris Lindner, Hsu-Min Sung, Georg Stoecklin.
      Aberrant translation causes ribosome stalling, which leads to the ubiquitination of ribosomal proteins and induces ribosome-associated quality control. As part of this quality control process, the E3 ubiquitin ligase RNF10 monoubiquitinates ribosomal protein RPS3. Here, we demonstrate that RNF10-mediated RPS3 monoubiquitination antagonizes ribosomal half-mer formation by promoting dissociation of 40S subunits from ribosomes stalled during translation elongation. Interestingly, RNF10 also promotes dissociation of 40S subunits stalled during aberrant translation initiation. Moreover, RNF10 levels are tightly coupled to the amount of 40S subunits. Knockdown of RPS proteins, which abrogates 40S ribosome biogenesis, results in proteasomal degradation of RNF10. Vice versa, knockdown of RPL proteins, which abrogates 60S biogenesis, leads to the accumulation of stalled initiating 40S subunits, increased RNF10 levels, and RPS3 monoubiquitination. As a factor required for the resolution of stalled translation events, RNF10 is part of a fundamental mechanism by which cells respond to imbalances in ribosomal subunit stoichiometry.
    DOI:  https://doi.org/10.1038/s41467-024-54411-x
  3. Cell Rep. 2024 Nov 27. pii: S2211-1247(24)01367-6. [Epub ahead of print]43(12): 115016
    Unveiling the physiological impact of ESCRT-dependent autophagosome closure by targeting the VPS37A ubiquitin E2 variant-like domain.
    Kouta Hamamoto, Xinwen Liang, Ayako Ito, Matthew Lanza, Van Bui, Jiawen Zhang, David M Opozda, Tatsuya Hattori, Longgui Chen, David Haddock, Fumiaki Imamura, Hong-Gang Wang, Yoshinori Takahashi.
      Macroautophagy (autophagy) involves the formation of phagophores that mature into autophagosomes. The impact of inhibiting autophagosome closure remains unclear. Here, we report the generation and analysis of mice with impaired autophagosome closure by targeting the ubiquitin E2 variant-like (UEVL) β strands of the endosomal sorting complex required for transport (ESCRT) I subunit VPS37A. The VPS37A UEVL mutation (Δ43-139) impairs bulk autophagic flux without disrupting ESCRT-I complex assembly and endosomal function. Homozygous mutant mice exhibit signs of autophagy impairment, including p62/SQSTM1 and ubiquitinated protein accumulation, neuronal dysfunction, growth retardation, antioxidant gene upregulation, and tissue abnormalities. However, about half of the mutant neonates survive to adulthood without severe liver injury. LC3 proximity proteomics reveals that the VPS37A UEVL mutation leads to active TANK-binding kinase 1 (TBK1) accumulation on phagophores, resulting in increased p62 phosphorylation and inclusion formation. These findings reveal a previously unappreciated role of LC3-conjugated phagophores in facilitating protein aggregation and sequestration, potentially alleviating proteotoxicity.
    Keywords:  CP: Cell biology; LC3-conjugated phagophore; TBK1; UEVL; VPS37A; autophagosome closure; mouse; neonatal survival; p62 phosphorylation; protein aggregation; tissue abnormalities; ubiquitin E2 variant-like domain
    DOI:  https://doi.org/10.1016/j.celrep.2024.115016
  4. Science. 2024 Nov 29. 386(6725): 996-1003
    Identification of a factor that accelerates substrate release from the signal recognition particle.
    Huping Wang, Ramanujan S Hegde.
      The eukaryotic signal recognition particle (SRP) cotranslationally recognizes the first hydrophobic segment of nascent secretory and membrane proteins and delivers them to a receptor at the endoplasmic reticulum (ER). How substrates are released from SRP at the ER to subsequently access translocation factors is not well understood. We found that TMEM208 can engage the substrate binding domain of SRP to accelerate release of its bound cargo. Without TMEM208, slow cargo release resulted in excessive synthesis of downstream polypeptide before engaging translocation factors. Delayed access to translocation machinery caused progressive loss of insertion competence, particularly for multipass membrane proteins, resulting in their impaired biogenesis. Thus, TMEM208 facilitates prompt cargo handover from the targeting to translocation machinery to minimize biogenesis errors and maintain protein homeostasis.
    DOI:  https://doi.org/10.1126/science.adp0787
  5. J Am Chem Soc. 2024 Nov 28.
    Leveraging Dual-Ligase Recruitment to Enhance Protein Degradation via a Heterotrivalent Proteolysis Targeting Chimera.
    Adam G Bond, Miquel Muñoz I Ordoño, Celia M Bisbach, Conner Craigon, Nikolai Makukhin, Elizabeth A Caine, Manjula Nagala, Marjeta Urh, Georg E Winter, Kristin M Riching, Alessio Ciulli.
      Proteolysis targeting chimera (PROTAC) degraders are typically bifunctional with one E3 ligase ligand connected to one target protein ligand via a linker. While augmented valency has been shown with trivalent PROTACs targeting two binding sites within a given target protein, or used to recruit two different targets, the possibility of recruiting two different E3 ligases within the same compound has not been demonstrated. Here we present dual-ligase recruitment as a strategy to enhance targeted protein degradation. We designed heterotrivalent PROTACs composed of CRBN, VHL and BET targeting ligands, separately tethered via a branched trifunctional linker. Structure-activity relationships of 12 analogues qualifies AB3067 as the most potent and fastest degrader of BET proteins, with minimal E3 ligase cross-degradation. Comparative kinetic analyses in wild-type and ligase single and double knockout cell lines revealed that protein ubiquitination and degradation induced by AB3067 was contributed to by both CRBN and VHL in an additive fashion. We further expand the scope of the dual-ligase approach by developing a heterotrivalent CRBN/VHL-based BromoTag degrader and a tetravalent PROTAC comprising of two BET ligand moieties. In summary, we provide proof-of-concept for dual-E3 ligase recruitment as a strategy to boost degradation fitness by recruiting two E3 ligases with a single degrader molecule. This approach could potentially delay the outset of resistance mechanisms involving loss of E3 ligase functionality.
    DOI:  https://doi.org/10.1021/jacs.4c11556
  6. Autophagy. 2024 Nov 27. 1-2
    UBAC2 serves as a reticulophagy receptor to suppress inflammatory responses.
    Xing He, Shouheng Jin.
      Reticulophagy selectively degrades fragments of the endoplasmic reticulum (ER) through macroautophagy/autophagy to maintain ER homeostasis. The deficiency of reticulophagy results in the unfolded protein response (UPR), which is a crucial clue to the pathogenesis of inflammatory diseases. However, the detailed mechanism underlying the cross-regulation between reticulophagy and inflammatory diseases remains largely unclear. Recently, we have revealed that UBAC2 (UBA domain containing 2) is essential for controlling ER homeostasis as a novel reticulophagy receptor. MARK2 catalyzes the phosphorylation of UBAC2 at serine (S) 223, hence facilitating the progression of reticulophagy and inhibiting ER stress-induced inflammatory responses.
    Keywords:  Inflammatory responses; MARK2; UBAC2; UPR; reticulophagy
    DOI:  https://doi.org/10.1080/15548627.2024.2431341
  7. Nat Chem Biol. 2024 Nov 25.
    Discovery and mechanism of K63-linkage-directed deubiquitinase activity in USP53.
    Kim Wendrich, Kai Gallant, Sarah Recknagel, Stavroula Petroulia, Nafizul Haque Kazi, Jan André Hane, Siska Führer, Karel Bezstarosti, Rachel O'Dea, Jeroen Demmers, Malte Gersch.
      Ubiquitin-specific proteases (USPs) represent the largest class of human deubiquitinases (DUBs) and comprise its phylogenetically most distant members USP53 and USP54, which are annotated as catalytically inactive pseudoenzymes. Conspicuously, mutations within the USP domain of USP53 cause progressive familial intrahepatic cholestasis. Here, we report the discovery that USP53 and USP54 are active DUBs with high specificity for K63-linked polyubiquitin. We demonstrate how USP53 mutations abrogate catalytic activity, implicating loss of DUB activity in USP53-mediated pathology. Depletion of USP53 increases K63-linked ubiquitination of tricellular junction components. Assays with substrate-bound polyubiquitin reveal that USP54 cleaves within K63-linked chains, whereas USP53 can en bloc deubiquitinate substrate proteins in a K63-linkage-dependent manner. Biochemical and structural analyses uncover underlying K63-specific S2 ubiquitin-binding sites within their catalytic domains. Collectively, our work revises the annotation of USP53 and USP54, provides reagents and a mechanistic framework to investigate K63-linked polyubiquitin decoding and establishes K63-linkage-directed deubiquitination as a new DUB activity.
    DOI:  https://doi.org/10.1038/s41589-024-01777-0
  8. Cell Commun Signal. 2024 Nov 28. 22(1): 571
    Pin1 promotes human CaV2.1 channel polyubiquitination by RNF138: pathophysiological implication for episodic ataxia type 2.
    Ssu-Ju Fu, Kai-Min Cheng, Cheng-Tsung Hsiao, Ya-Ching Fang, Chung-Jiuan Jeng, Chih-Yung Tang.
      Loss-of-function mutations in the human gene encoding the neuron-specific Ca2+ channel CaV2.1 are linked to the neurological disease episodic ataxia type 2 (EA2), as well as neurodevelopmental disorders such as developmental delay and developmental epileptic encephalopathy. Disease-associated CaV2.1 mutants may exhibit defective proteostasis and promote endoplasmic reticulum (ER)-associated degradation of their wild-type (WT) counterpart in a dominant-negative manner. The E3 ubiquitin ligase RNF138 was previously shown to mediate EA2-related aberrant degradation of CaV2.1 at the ER. Herein we aimed to elucidate the ER proteostasis mechanism of CaV2.1. The peptidyl-prolyl cis/trans isomerase, NIMA-interacting 1 (Pin1) was identified as a novel neuronal CaV2.1 binding partner that promoted polyubiquitination and proteasomal degradation of CaV2.1. Suppression of endogenous Pin1 level with either shRNA knockdown or the Pin1 inhibitor all-trans retinoic acid (ATRA) enhanced endogenous CaV2.1 protein level in neurons, and attenuated ER-associated degradation of CaV2.1 WT and EA2-causing mutants. Detailed mutation analyses suggested that Pin1 interacted with specific phosphorylated serine/threonine-proline motifs in the intracellular II-III loop and the distal carboxy-terminal region of human CaV2.1. We further generated Pin1-insensitive CaV2.1 constructs and demonstrated that, during ER quality control, Pin1 served as an upstream regulator of CaV2.1 polyubiquitination and degradation by RNF138. Pin1 regulation was required for the dominant-negative effect of EA2 missense mutants, but not nonsense mutants, on CaV2.1 WT protein expression. Our data are consistent with the idea that CaV2.1 proteostasis at the ER, as well as dominant-negative suppression of disease-causing loss-of-function mutants on CaV2.1 WT, entail both Pin1/RNF138-dependent and -independent mechanisms.
    Keywords:  Channelopathy; E3 ubiquitin ligase; ER quality control; Peptidyl-prolyl cis/trans isomerase; Proteasomal degradation; Proteostasis
    DOI:  https://doi.org/10.1186/s12964-024-01960-9
  9. Cell Rep. 2024 Nov 22. pii: S2211-1247(24)01340-8. [Epub ahead of print]43(12): 114989
    Intersectin1 promotes clathrin-mediated endocytosis by organizing and stabilizing endocytic protein interaction networks.
    Meiyan Jin, Yuichiro Iwamoto, Cyna Shirazinejad, David G Drubin.
      During clathrin-mediated endocytosis (CME), dozens of proteins are recruited to nascent CME sites on the plasma membrane, and their spatial and temporal coordination is crucial for efficient CME. Here, we show that the scaffold protein intersectin1 (ITSN1) promotes CME by organizing and stabilizing endocytic protein interaction networks. Live-cell imaging of genome-edited cells revealed that endogenously labeled ITSN1 is recruited during CME site stabilization and growth and that ITSN1 knockdown impairs endocytic protein recruitment during this stage. Targeting ITSN1 to the mitochondrial surface was sufficient to assemble puncta consisting of the EPS15 and FCHO2 initiation proteins, the AP2 and epsin1 (EPN1) adaptor proteins, and the dynamin2 (DNM2) vesicle scission GTPase. ITSN1 can form puncta and recruit DNM2 independent of EPS15/FCHO2 or EPN1. Our findings redefine ITSN1's primary endocytic role as organizing and stabilizing CME protein interaction networks rather than initiation, providing deeper insights into the multi-step and multi-zone organization of CME site assembly.
    Keywords:  CP: Cell biology; SH3; clathrin-coated pit; clathrin-mediated endocytosis; dynamin; intersectin
    DOI:  https://doi.org/10.1016/j.celrep.2024.114989
  10. bioRxiv. 2024 Nov 24. pii: 2024.11.22.624941. [Epub ahead of print]
    UPR deficiency in budding yeast reveals a trade-off between ER folding capacity and maintenance of euploidy.
    Constantine Bartolutti, Allison J Kim, Gloria A Brar.
      The Unfolded Protein Response (UPR) was discovered in budding yeast as a mechanism that allows cells to adapt to ER stress. While the Ire1 branch of this pathway is highly conserved, it is not thought to be important for cellular homeostasis in the absence of stress. Surprisingly, we found that removal of UPR activity led to pervasive aneuploidy in budding yeast cells, suggesting selective pressure resulting from UPR-deficiency. Aneuploid UPR-deficient cells grew better than euploid cells, but exhibited heightened general proteostatic stress, a hallmark of aneuploidy in wild-type cells. Modulation of key genes involved in ER proteostasis that were encoded on aneuploid chromosomes, could phenocopy the effects of aneuploidy, indicating that the reason cells require UPR activity to maintain euploidy is to counteract protein folding stress in the ER. In support of this model, aneuploidy in UPR-deficient cells can be prevented by expression of a UPR-independent general ER chaperone. Overall, our results indicate an unexpected role for the UPR in basal cell growth that is sufficiently important for cells to accept the costly trade-off of aneuploidy in the absence of UPR activity.
    DOI:  https://doi.org/10.1101/2024.11.22.624941
  11. Bioessays. 2024 Nov 26. e202400161
    Ubiquitin-Independent Degradation: An Emerging PROTAC Approach?
    Tiantian Li, Saskia A Hogenhout, Weijie Huang.
      Targeted protein degradation (TPD) has emerged as a highly promising approach for eliminating disease-associated proteins in the field of drug discovery. Among the most advanced TPD technologies, PROteolysis TArgeting Chimera (PROTAC), functions by bringing a protein of interest (POI) into proximity with an E3 ubiquitin ligase, leading to ubiquitin (Ub)-dependent proteasomal degradation. However, the designs of most PROTACs are based on the utilization of a limited number of available E3 ligases, which significantly restricts their potential. Recent studies have shown that phytoplasmas, a group of bacterial plant pathogens, have developed several E3- and ubiquitin-independent proteasomal degradation (UbInPD) mechanisms for breaking down host targets. This suggests an alternative approach for substrate recruitment and TPD. Here, we present existing evidence that supports the feasibility of UbInPD in eukaryotic cells and propose candidate proteins that can serve as docking sites for the development of E3-independent PROTACs.
    DOI:  https://doi.org/10.1002/bies.202400161
  12. Nat Commun. 2024 Nov 23. 15(1): 10165
    Crystal structures of DCAF1-PROTAC-WDR5 ternary complexes provide insight into DCAF1 substrate specificity.
    Mark F Mabanglo, Brian Wilson, Mahmoud Noureldin, Serah W Kimani, Ahmed Mamai, Chiara Krausser, Héctor González-Álvarez, Smriti Srivastava, Mohammed Mohammed, Laurent Hoffer, Manuel Chan, Jamie Avrumutsoae, Alice Shi Ming Li, Taraneh Hajian, Sarah Tucker, Stuart Green, Magdalena Szewczyk, Dalia Barsyte-Lovejoy, Vijayaratnam Santhakumar, Suzanne Ackloo, Peter Loppnau, Yanjun Li, Almagul Seitova, Taira Kiyota, Jue George Wang, Gilbert G Privé, Douglas A Kuntz, Bhashant Patel, Vaibhavi Rathod, Anand Vala, Bhimsen Rout, Ahmed Aman, Gennady Poda, David Uehling, Jailall Ramnauth, Levon Halabelian, Richard Marcellus, Rima Al-Awar, Masoud Vedadi.
      Proteolysis-targeting chimeras (PROTACs) have been explored for the degradation of drug targets for more than two decades. However, only a handful of E3 ligase substrate receptors have been efficiently used. Downregulation and mutation of these receptors would reduce the effectiveness of such PROTACs. We recently developed potent ligands for DCAF1, a substrate receptor of EDVP and CUL4 E3 ligases. Here, we focus on DCAF1 toward the development of PROTACs for WDR5, a drug target in various cancers. We report four DCAF1-based PROTACs with endogenous and exogenous WDR5 degradation effects and high-resolution crystal structures of the ternary complexes of DCAF1-PROTAC-WDR5. The structures reveal detailed insights into the interaction of DCAF1 with various WDR5-PROTACs, indicating a significant role of DCAF1 loops in providing needed surface plasticity, and reflecting the mechanism by which DCAF1 functions as a substrate receptor for E3 ligases with diverse sets of substrates.
    DOI:  https://doi.org/10.1038/s41467-024-54500-x
  13. bioRxiv. 2024 Nov 12. pii: 2024.11.11.623099. [Epub ahead of print]
    Investigating Protein Degradability through Site-Specific Ubiquitin Ligase Recruitment.
    Olivia Shade, Amy Ryan, Gabriella Belsito, Alexander Deiters.
      We report targeted protein degradation through the site-specific recruitment of native ubiquitin ligases to a protein of interest via conjugation of E3 ligase ligands. Direct comparison of degradation ability of proteins displaying the corresponding bioconjugation handle at different regions of protein surfaces was explored. We demonstrate the benefit of proximal lysine residues and investigate flexibility in linker length for the design of optimal degraders. Two proteins without known small molecule ligands, EGFP and DUSP6, were differentially degraded when modified at different locations on their protein surfaces. Further, the cereblon-mediated degradation of the known PROTAC target ERRα was improved through the recruitment of the E3 ligase to regions different from the known ligand binding site. This new methodology will provide insight into overall protein degradability, even in the absence of a known small molecule ligand and inform the process of new ligand and PROTAC development to achieve optimal protein degradation. Furthermore, this approach represents a new, small molecule-based conditional OFF switch of protein function with complete genetic specificity. Importantly, the protein of interest is only modified with a minimal surface modification (< 200 Da) and does not require any protein domain fusions.
    DOI:  https://doi.org/10.1101/2024.11.11.623099
  14. bioRxiv. 2024 Nov 12. pii: 2024.11.11.619147. [Epub ahead of print]
    Master regulators governing protein abundance across ten human cancer types.
    Zishan Wang, Megan Wojciechowicz, Jordan Rosen, Abdulkadir Elmas, Won-Min Song, Yansheng Liu, Kuan-Lin Huang.
      Protein abundance correlates only moderately with mRNA levels, and are modulated post-transcriptionally by a network of regulators including ribosomes, RNA-binding proteins (RBPs), and the proteasome. Here, we identified Ma ster P rotein abundance R egulators (MaPRs) across ten cancer types by devising a new computational pipeline that jointly analyzed transcriptomes and proteomes from 1,305 tumor samples. We identified 232 to 1,394 MaPRs per cancer type, mediating up to 79% of post- transcriptional regulatory networks. MaPRs exhibit high network connectivity, strong genetic dependency in cancer cells, and significant enrichment for RBPs. Combining tumor up-regulation, druggability, and target network analyses identified cancer-specific vulnerabilities. MaPRs predict tumor proteomic subtypes more accurately than other proteins. Finally, significant portions of RBP MaPR-target relationships were validated by experimental evidence from eCLIP binding and knockdown assays. Our findings uncover central MaPRs that govern post-transcriptional networks, highlighting diverse processes underlying human proteome regulation and identifying key regulators in cancer biology.
    DOI:  https://doi.org/10.1101/2024.11.11.619147
  15. Cell Chem Biol. 2024 Nov 20. pii: S2451-9456(24)00460-4. [Epub ahead of print]
    An engineered cereblon optimized for high throughput screening and molecular glue discovery.
    Henry J Bailey, Jonathan Eisert, Rubina Kazi, Jan Gerhartz, Dominika Ewa Pieńkowska, Ina Dressel, Joshua Vollrath, Ivan Kondratov, Tetiana Matviyuk, Nataliya Tolmachova, Varun Jayeshkumar Shah, Giulio Giuliani, Thorsten Mosler, Thomas M Geiger, Ana M Esteves, Sandra P Santos, Raquel L Sousa, Tiago M Bandeiras, Eva-Maria Leibrock, Ulrike Bauer, Birgitta Leuthner, Julian D Langer, Ansgar A Wegener, Radosław P Nowak, Fiona J Sorrell, Ivan Dikic.
      The majority of clinical degraders utilize an immunomodulatory imide drug (IMiD)-based derivative that directs their target to the E3 ligase receptor cereblon (CRBN); however, identification of IMiD molecular glue substrates has remained underexplored. To tackle this, we design human CRBN constructs, which retain all features for ternary complex formation, while allowing generation of homogenous and cost-efficient expression in E. coli. Extensive profiling of the construct shows it to be the "best of both worlds" in terms of binding activity and ease of production. We next designed the "Enamine focused IMiD library" and demonstrated applicability of the construct to high-throughput screening, identifying binders with high potency, ligand efficiency, and specificity. Finally, we adapt our construct for proof of principle glue screening approaches enabling IMiD cellular interactome determination. Coupled with our IMiD binding landscape the methods described here should serve as valuable tools to assist discovery of next generation CRBN glues.
    Keywords:  IMiDs; PROTACs; cereblon; degraders; molecular glues; targeted protein degradation; ubiquitin
    DOI:  https://doi.org/10.1016/j.chembiol.2024.11.002
  16. J Cell Sci. 2024 Nov 15. pii: jcs261444. [Epub ahead of print]137(22):
    Dynamic remodelling of the endoplasmic reticulum for mitosis.
    Suzan Kors, Anne-Lore Schlaitz.
      The endoplasmic reticulum (ER) is a dynamic and continuous membrane network with roles in many cellular processes. The importance and maintenance of ER structure and function have been extensively studied in interphase cells, yet recent findings also indicate crucial roles of the ER in mitosis. During mitosis, the ER is remodelled significantly with respect to composition and morphology but persists as a continuous network. The ER interacts with microtubules, actin and intermediate filaments, and concomitant with the mitotic restructuring of all cytoskeletal systems, ER dynamics and distribution change. The ER is a metabolic hub and several examples of altered ER functions during mitosis have been described. However, we lack an overall understanding of the ER metabolic pathways and functions that are active during mitosis. In this Review, we will discuss mitotic changes to the ER at different organizational levels to explore how the mitotic ER, with its distinct properties, might support cell division.
    Keywords:  Cell division; ER; ER dynamics and morphology; ER–cytoskeleton contacts; Endoplasmic reticulum; Membrane contact sites; Mitosis
    DOI:  https://doi.org/10.1242/jcs.261444
  17. Dev Cell. 2024 Nov 22. pii: S1534-5807(24)00634-8. [Epub ahead of print]
    Gene module reconstruction identifies cellular differentiation processes and the regulatory logic of specialized secretion in zebrafish.
    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 single-cell RNA sequencing (scRNA-seq) developmental trajectories to reconstruct differentiation using two secretory tissues as models-the zebrafish notochord and hatching gland. First, we integrated expression and functional similarities to identify gene modules, revealing dozens of modules representing known and newly associated differentiation processes and their dynamics. Second, we focused on the unfolded protein response (UPR) transducer module to study how general versus cell-type-specific secretory functions are regulated. Profiling loss- and gain-of-function embryos identified 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-like secretion program. Our study presents module identification via multi-source integration for reconstructing differentiation (MIMIR) and illustrates how transcription factors confer general and specialized cellular functions.
    Keywords:  UPR; bhlha15; creb3l; differentiation processes; functional gene module; hatching gland; notochord; regulatory logic; secretion; xbp1
    DOI:  https://doi.org/10.1016/j.devcel.2024.10.015
  18. Cell Rep. 2024 Nov 26. pii: S2211-1247(24)01370-6. [Epub ahead of print]43(12): 115019
    Selective autophagy impedes KSHV entry after recruiting the membrane damage sensor galectin-8 to virus-containing endosomes.
    Katarina Wendy Schmidt, Charlotte Montespan, Danielle Thompson, Miriam S Lucas, Laure-Anne Ligeon, Harald Wodrich, Alexander S Hahn, Urs F Greber, Christian Münz.
      Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic γ-herpesvirus. Autophagy during KSHV entry has remained unexplored. We show that LC3 lipidation as a hallmark of autophagy is induced shortly after KSHV entry. LC3 co-localizes with KSHV in amphisomes during entry and loss of LC3 lipidation increases infection. Accordingly, NDP52, a receptor of selective autophagy, was recruited to endocytosed viral particles, and its reduction increased KSHV infection. Additionally, virus particles co-localized with the endolysosome damage sensor galectin-8 upon KSHV entry and depletion of galectin-8 promoted KSHV infection. Compared with herpes simplex virus, listeriolysin, adenovirus, and influenza virus, and in contrast to what was previously thought about enveloped viruses, KSHV binding to EphA2 by its envelope protein gH causes endolysosomal membrane damage, akin to non-enveloped viruses and bacteria. Taken together, our study identifies an important anti-viral role for galectin-8, NDP52, and the autophagy machinery at virus-damaged endosomes, restricting KSHV entry by selective autophagy.
    Keywords:  CP: Immunology; CP: Microbiology; KSHV; Kaposi sarcoma-associated herpesvirus; NDP52; endosomal damage; galectin-8; macroautophagy
    DOI:  https://doi.org/10.1016/j.celrep.2024.115019
  19. Nat Commun. 2024 Nov 25. 15(1): 10198
    Features of membrane protein sequence direct post-translational insertion.
    Ilya A Kalinin, Hadas Peled-Zehavi, Alon B D Barshap, Shai A Tamari, Yarden Weiss, Reinat Nevo, Nir Fluman.
      The proper folding of multispanning membrane proteins (MPs) hinges on the accurate insertion of their transmembrane helices (TMs) into the membrane. Predominantly, TMs are inserted during protein translation, via a conserved mechanism centered around the Sec translocon. Our study reveals that the C-terminal TMs (cTMs) of numerous MPs across various organisms bypass this cotranslational route, necessitating an alternative posttranslational insertion strategy. We demonstrate that evolution has refined the hydrophilicity and length of the C-terminal tails of these proteins to optimize cTM insertion. Alterations in the C-tail sequence disrupt cTM insertion in both E. coli and human, leading to protein defects, loss of function, and genetic diseases. In E. coli, we identify YidC, a member of the widespread Oxa1 family, as the insertase facilitating cTMs insertion, with C-tail mutations disrupting the productive interaction of cTMs with YidC. Thus, MP sequences are fine-tuned for effective collaboration with the cellular biogenesis machinery, ensuring proper membrane protein folding.
    DOI:  https://doi.org/10.1038/s41467-024-54575-6
  20. Sci Signal. 2024 Nov 26. 17(864): eadp1375
    Coordination between the eIF2 kinase GCN2 and p53 signaling supports purine metabolism and the progression of prostate cancer.
    Ricardo A Cordova, Noah R Sommers, Andrew S Law, Angela J Klunk, Katherine E Brady, David W Goodrich, Tracy G Anthony, Jeffrey J Brault, Roberto Pili, Ronald C Wek, Kirk A Staschke.
      Cancers invoke various pathways to mitigate external and internal stresses to continue their growth and progression. We previously reported that the eIF2 kinase GCN2 and the integrated stress response are constitutively active in prostate cancer (PCa) and are required to maintain amino acid homeostasis needed to fuel tumor growth. However, although loss of GCN2 function reduces intracellular amino acid availability and PCa growth, there is no appreciable cell death. Here, we discovered that the loss of GCN2 in PCa induces prosenescent p53 signaling. This p53 activation occurred through GCN2 inhibition-dependent reductions in purine nucleotides that impaired ribosome biogenesis and, consequently, induced the impaired ribosome biogenesis checkpoint. p53 signaling induced cell cycle arrest and senescence that promoted the survival of GCN2-deficient PCa cells. Depletion of GCN2 combined with loss of p53 or pharmacological inhibition of de novo purine biosynthesis reduced proliferation and enhanced cell death in PCa cell lines, organoids, and xenograft models. Our findings highlight the coordinated interplay between GCN2 and p53 regulation during nutrient stress and provide insight into how they could be targeted in developing new therapeutic strategies for PCa.
    DOI:  https://doi.org/10.1126/scisignal.adp1375
  21. bioRxiv. 2024 Nov 22. pii: 2024.11.22.624887. [Epub ahead of print]
    Cryo-EM Structure of AAA + ATPase Thorase Reveals Novel Helical Filament Formation.
    Mohamad Aasif Dar, Robert Louder, Marisol Cortes, Rong Chen, Qianqian Ma, Mayukh Chakrabarti, George K E Umanah, Ted M Dawson, Valina L Dawson.
      The AAA+ ( A TPases a ssociated with a variety of cellular a ctivities) ATPase, Thorase, also known as ATAD1, plays multiple roles in synaptic plasticity, mitochondrial quality control and mTOR signaling through disassembling protein complexes like AMPAR and mTORC1 in an ATP-dependent manner. The Oligomerization of Thorase is crucial for its disassembly and remodeling functions. We show that wild-type Thorase forms long helical filaments in vitro , dependent on ATP binding but not hydrolysis. We report the Cryogenic Electron Microscopy (cryo-EM) structure of the Thorase filament at a resolution of 4 Å, revealing the dimeric arrangement of the basic repeating unit that is formed through a distinct interface compared to the hexameric MSP1/ATAD1E193Q assembly. Structure-guided mutagenesis confirms the role of critical amino acid residues required for filament formation, oligomerization and disassembly of mTORC1 protein complex. Together, our data reveals a novel filament structure of Thorase and provides critical information that elucidates the mechanism underlying Thorase filament formation and Thorase-mediated disassembly of the mTORC1 complex.
    DOI:  https://doi.org/10.1101/2024.11.22.624887
  22. Nat Commun. 2024 Nov 28. 15(1): 10336
    Dynamic fibrillar assembly of αB-crystallin induced by perturbation of the conserved NT-IXI motif resolved by cryo-EM.
    Russell McFarland, Rozhan Noroozi, Adam P Miller, Steve L Reichow.
      αB-crystallin is an archetypical member of the small heat shock proteins (sHSPs) vital for cellular proteostasis and mitigating protein misfolding diseases. Gaining insights into the principles defining their molecular organization and chaperone function have been hindered by intrinsic dynamic properties and limited high-resolution structural analysis. To disentangle the mechanistic underpinnings of these dynamical properties, we ablate a conserved IXI-motif located within the N-terminal (NT) domain of human αB-crystallin implicated in subunit exchange dynamics and client sequestration. This results in a profound structural transformation, from highly polydispersed caged-like native assemblies into an elongated fibril state amenable to high-resolution cryo-EM analysis. The reversible nature of this variant facilitates interrogation of functional effects due to perturbation of the NT-IXI motif in both the native-like oligomer and fibril states. Together, our investigations unveil several features thought to be key mechanistic attributes to sHSPs and point to a critical significance of the NT-IXI motif in αB-crystallin assembly, polydispersity, and chaperone activity.
    DOI:  https://doi.org/10.1038/s41467-024-54647-7
  23. bioRxiv. 2024 Nov 17. pii: 2024.11.15.623783. [Epub ahead of print]
    HSP90 buffers deleterious genetic variations in BRCA1.
    Brant Gracia, Patricia Montes, Min Huang, Junjie Chen, Georgios Ioannis Karras.
      Protein-folding chaperone HSP90 buffers genetic variation in diverse organisms, but the clinical significance of HSP90 buffering in disease remains unclear. Here, we show that HSP90 buffers mutations in the BRCT domain of BRCA1. HSP90-buffered BRCA1 mutations encode protein variants that retain interactions with partner proteins and rely on HSP90 for protein stability and function in cell survival. Moreover, HSP90-buffered BRCA1 variants confer PARP inhibitor resistance in cancer cell lines. Low-level HSP90 inhibition alleviates this resistance, revealing a cryptic and mutant-specific HSP90-contingent synthetic lethality. Hence, by stabilizing metastable variants across the entirety of the BRCT domain, HSP90 reduces the clinical severity of BRCA1 mutations allowing them to accumulate in populations. We estimate that HSP90 buffers 11% to 28% of known human BRCA1- BRCT missense mutations. Our work extends the clinical significance of HSP90 buffering to a prevalent class of variations in BRCA1 , pioneering its importance in cancer predisposition and therapy resistance.
    DOI:  https://doi.org/10.1101/2024.11.15.623783
  24. bioRxiv. 2024 Nov 11. pii: 2024.11.10.622803. [Epub ahead of print]
    Programmable protein degraders enable selective knockdown of pathogenic β-catenin subpopulations in vitro and in vivo.
    Tianzheng Ye, Azmain Alamgir, Cara M Robertus, Darianna Colina, Connor Monticello, Thomas Connor Donahue, Lauren Hong, Sophia Vincoff, Shrey Goel, Peter Fekkes, Luis Miguel Camargo, Kieu Lam, James Heyes, David Putnam, Christopher A Alabi, Pranam Chatterjee, Matthew P DeLisa.
      Aberrant activation of Wnt signaling results in unregulated accumulation of cytosolic β-catenin, which subsequently enters the nucleus and promotes transcription of genes that contribute to cellular proliferation and malignancy. Here, we sought to eliminate pathogenic β-catenin from the cytosol using designer ubiquibodies (uAbs), chimeric proteins composed of an E3 ubiquitin ligase and a target-binding domain that redirect intracellular proteins to the proteasome for degradation. To accelerate uAb development, we leveraged a protein language model (pLM)-driven algorithm called SaLT&PepPr to computationally design "guide" peptides with affinity for β-catenin, which were subsequently fused to the catalytic domain of a human E3 called C-terminus of Hsp70-interacting protein (CHIP). Expression of the resulting peptide-guided uAbs in colorectal cancer cells led to the identification of several designs that significantly reduced the abnormally stable pool of free β-catenin in the cytosol and nucleus while preserving the normal membrane-associated subpopulation. This selective knockdown of pathogenic β-catenin suppressed Wnt/β-catenin signaling and impaired tumor cell survival and proliferation. Furthermore, one of the best degraders selectively decreased cytosolic but not membrane-associated β-catenin levels in livers of BALB/c mice following delivery as a lipid nanoparticle (LNP)-encapsulated mRNA. Collectively, these findings reveal the unique ability of uAbs to selectively eradicate abnormal proteins in vitro and in vivo and open the door to peptide-programmable biologic modulators of other disease-causing proteins.
    DOI:  https://doi.org/10.1101/2024.11.10.622803
  25. Proc Natl Acad Sci U S A. 2024 Dec 03. 121(49): e2410486121
    ER-tethered stress sensor CREBH regulates mitochondrial unfolded protein response to maintain energy homeostasis.
    Hyunbae Kim, Qi Chen, Donghong Ju, Neeraja Purandare, Xuequn Chen, Lobelia Samavati, Li Li, Ren Zhang, Lawrence I Grossman, Kezhong Zhang.
      The Mitochondrial Unfolded Protein Response (UPRmt), a mitochondria-originated stress response to altered mitochondrial proteostasis, plays important roles in various pathophysiological processes. In this study, we revealed that the endoplasmic reticulum (ER)-tethered stress sensor CREBH regulates UPRmt to maintain mitochondrial homeostasis and function in the liver. CREBH is enriched in and required for hepatic Mitochondria-Associated Membrane (MAM) expansion induced by energy demands. Under a fasting challenge or during the circadian cycle, CREBH is activated to promote expression of the genes encoding the key enzymes, chaperones, and regulators of UPRmt in the liver. Activated CREBH, cooperating with peroxisome proliferator-activated receptor α (PPARα), activates expression of Activating Transcription Factor (ATF) 5 and ATF4, two major UPRmt transcriptional regulators, independent of the ER-originated UPR (UPRER) pathways. Hepatic CREBH deficiency leads to accumulation of mitochondrial unfolded proteins, decreased mitochondrial membrane potential, and elevated cellular redox state. Dysregulation of mitochondrial function caused by CREBH deficiency coincides with increased hepatic mitochondrial oxidative phosphorylation (OXPHOS) but decreased glycolysis. CREBH knockout mice display defects in fatty acid oxidation and increased reliance on carbohydrate oxidation for energy production. In summary, our studies uncover that hepatic UPRmt is activated through CREBH under physiological challenges, highlighting a molecular link between ER and mitochondria in maintaining mitochondrial proteostasis and energy homeostasis under stress conditions.
    Keywords:  ER-mitochondria contact; cell metabolism; michondrial UPR; transcriptional regulation; unfolded protein response
    DOI:  https://doi.org/10.1073/pnas.2410486121
  26. bioRxiv. 2024 Nov 22. pii: 2024.11.22.624841. [Epub ahead of print]
    diaPASEF-Powered Chemoproteomics Enables Deep Kinome Interaction Profiling.
    Kathryn Woods, Thankhoe A Rants'o, Alexandria M Chan, Tanmay Sapre, Grace E Mastin, Kathleen M Maguire, Shao-En Ong, Martin Golkowski.
      Protein-protein interactions (PPIs) underlie most biological functions. Devastating human conditions like cancers, neurological disorders, and infections, hijack PPI networks to initiate disease, and to drive disease progression. Understanding precisely how diseases remodel PPI networks can, therefore, help clarify disease mechanisms and identify therapeutic targets. Protein kinases control most cellular processes through protein phosphorylation. The 518 human kinases, known as the kinome, are frequently dysregulated in disease and highly druggable with ATP-competitive inhibitors. Kinase activity, localization, and substrate recognition are regulated by dynamic PPI networks composed of scaffolding and adapter proteins, other signaling enzymes like small GTPases and E3 ligases, and phospho-substrates. Accordingly, mapping kinase PPI networks can help determine kinome activation states, and, in turn, cellular activation states; this information can be used for studying kinase-mediated cell signaling, and for prioritizing kinases for drug discovery. Previously, we have developed a high-throughput method for kinome PPI mapping based on mass spectrometry (MS)-based chemoproteomics that we named kinobead competition and correlation analysis (kiCCA). Here, we introduce 2 nd generation (gen) kiCCA which utilizes data-independent acquisition (dia) with parallel accumulation serial fragmentation (PASEF) MS and a re-designed CCA algorithm with improved selection criteria and the ability to predict multiple kinase interaction partners of the same proteins. Using neuroblastoma cell line models of the noradrenergic-mesenchymal transition (NMT), we demonstrate that 2 nd gen kiCCA (1) identified 6.1-times more kinase PPIs in native cell extracts compared to our 1 st gen approach, (2) determined kinase-mediated signaling pathways that underly the neuroblastoma NMT, and (3) accurately predicted pharmacological targets for manipulating NMT states. Our 2 nd gen kiCCA method is broadly useful for cell signaling research and kinase drug discovery.
    DOI:  https://doi.org/10.1101/2024.11.22.624841
  27. Nucleic Acids Res. 2024 Nov 28. pii: gkae1105. [Epub ahead of print]
    UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation.
    Rochelle L Tiedemann, Joel Hrit, Qian Du, Ashley K Wiseman, Hope E Eden, Bradley M Dickson, Xiangqian Kong, Alison A Chomiak, Robert M Vaughan, Bailey M Tibben, Jakob M Hebert, Yael David, Wanding Zhou, Stephen B Baylin, Peter A Jones, Susan J Clark, Scott B Rothbart.
      The RING E3 ubiquitin ligase UHRF1 is an established cofactor for DNA methylation inheritance. The model posits that nucleosomal engagement through histone and DNA interactions directs UHRF1 ubiquitin ligase activity toward lysines on histone H3 tails, creating binding sites for DNMT1 through ubiquitin interacting motifs (UIM1 and UIM2). However, the extent to which DNMT1 relies on ubiquitin signaling through UHRF1 in support of DNA methylation maintenance remains unclear. Here, with integrative epigenomic and biochemical analyses, we reveal that DNA methylation maintenance at low-density cytosine-guanine dinucleotides (CpGs) is particularly vulnerable to disruption of UHRF1 ubiquitin ligase activity and DNMT1 ubiquitin reading activity through UIM1. Hypomethylation of low-density CpGs in this manner induces formation of partially methylated domains (PMDs), a methylation signature observed across human cancers. In contrast, UIM2 disruption completely abolishes the DNA methylation maintenance function of DNMT1 in a CpG density-independent manner. In the context of DNA methylation recovery following acute DNMT1 depletion, we further reveal a 'bookmarking' function for UHRF1 ubiquitin ligase activity in support of DNA re-methylation. Collectively, these studies show that DNMT1-dependent DNA methylation inheritance is a ubiquitin-regulated process that is partially reliant on UHRF1 and suggest a disrupted UHRF1-DNMT1 ubiquitin signaling axis contributes to PMD formation in cancers.
    DOI:  https://doi.org/10.1093/nar/gkae1105
  28. Angew Chem Int Ed Engl. 2024 Nov 27. e202417272
    A Kinetic Scout Approach Accelerates Targeted Protein Degrader Development.
    Angela T Fan, Gillian E Gadbois, Hai-Tsang Huang, Charu Chaudhry, Jiewei Jiang, Logan H Sigua, Emily R Smith, Sitong Wu, Grace J Poirier, Kara Dunne-Dombrink, Pavitra Goyal, Andrew J Tao, William R Sellers, Eric S Fischer, Katherine A Donovan, Fleur M Ferguson.
      Bifunctional molecules such as targeted protein degraders induce proximity to promote gain-of-function pharmacology. These powerful approaches have gained broad traction across academia and the pharmaceutical industry, leading to an intensive focus on strategies that can accelerate their identification and optimization. We and others have previously used chemical proteomics to map degradable target space, and these datasets have been used to develop and train multiparameter models to extend degradability predictions across the proteome. In this study, we now turn our attention to develop generalizable chemistry strategies to accelerate the development of new bifunctional degraders. We implement lysine-targeted reversible-covalent chemistry to rationally tune the binding kinetics at the protein-of-interest across a set of 25 targets. We define an unbiased workflow consisting of global proteomics analysis, IP/MS of ternary complexes and the E-STUB assay, to mechanistically characterize the effects of ligand residence time on targeted protein degradation and formulate hypotheses about the rate-limiting step of degradation for each target. Our key finding is that target residence time is a major determinant of degrader activity, and this can be rapidly and rationally tuned through the synthesis of a minimal number of analogues to accelerate early degrader discovery and optimization.
    Keywords:  Targeted protein degradation * PROTACs * compound residence time * proteomics * proteasomal degradation
    DOI:  https://doi.org/10.1002/anie.202417272
  29. EMBO J. 2024 Nov 25.
    GOLPH3 and GOLPH3L maintain Golgi localization of LYSET and a functional mannose 6-phosphate transport pathway.
    Berit K Brauer, Zilei Chen, Felix Beirow, Jiaran Li, Daniel Meisinger, Emanuela Capriotti, Michaela Schweizer, Lea Wagner, Jascha Wienberg, Laura Hobohm, Lukas Blume, Wenjie Qiao, Yoshiki Narimatsu, Jan E Carette, Henrik Clausen, Dominic Winter, Thomas Braulke, Sabrina Jabs, Matthias Voss.
      Glycosylation, which plays an important role in modifying lipids and sorting of proteins, is regulated by asymmetric intra-Golgi distribution and SPPL3-mediated cleavage of Golgi enzymes. We found that cells lacking LYSET/TMEM251, a retention factor for Golgi N-acetylglucosamine-1-phosphotransferase (GNPT), display SPPL3-dependent hypersecretion of the Golgi membrane protein B4GALT5. We demonstrate that in wild-type cells B4GALT5 is tagged with mannose 6-phosphate (M6P), a sorting tag typical of soluble lysosomal hydrolases. Hence, M6P-tagging of B4GALT5 may represent a novel degradative lysosomal pathway. We also observed B4GALT5 hypersecretion and prominent destabilization of LYSET-GNPT complexes, impaired M6P-tagging, and disturbed maturation and trafficking of lysosomal enzymes in multiple human cell lines lacking the COPI adaptors GOLPH3 and GOLPH3L. Mechanistically, we identified LYSET as a novel, atypical client of GOLPH3/GOLPH3L. Thus, by ensuring the cis-Golgi localization of the LYSET-GNPT complex and maintaining its Golgi polarity, GOLPH3/GOLPH3L is essential for the integrity of the M6P-tagging machinery and homeostasis of lysosomes.
    Keywords:  Glycosyltransferase Secretion; Golgi Apparatus; Intramembrane Proteolysis; Lysosomes; Mannose 6-Phophate Tagging
    DOI:  https://doi.org/10.1038/s44318-024-00305-z
  30. Elife. 2024 Nov 25. pii: RP87301. [Epub ahead of print]12
    Cancers adapt to their mutational load by buffering protein misfolding stress.
    Susanne Tilk, Judith Frydman, Christina Curtis, Dmitri A Petrov.
      In asexual populations that don't undergo recombination, such as cancer, deleterious mutations are expected to accrue readily due to genome-wide linkage between mutations. Despite this mutational load of often thousands of deleterious mutations, many tumors thrive. How tumors survive the damaging consequences of this mutational load is not well understood. Here, we investigate the functional consequences of mutational load in 10,295 human tumors by quantifying their phenotypic response through changes in gene expression. Using a generalized linear mixed model (GLMM), we find that high mutational load tumors up-regulate proteostasis machinery related to the mitigation and prevention of protein misfolding. We replicate these expression responses in cancer cell lines and show that the viability in high mutational load cancer cells is strongly dependent on complexes that degrade and refold proteins. This indicates that the upregulation of proteostasis machinery is causally important for high mutational burden tumors and uncovers new therapeutic vulnerabilities.
    Keywords:  cancer; cancer biology; evolutionary biology; human; mutational load; proteostasis; somatic evolution
    DOI:  https://doi.org/10.7554/eLife.87301
  31. Cell. 2024 Nov 21. pii: S0092-8674(24)01269-8. [Epub ahead of print]
    snoRNA-facilitated protein secretion revealed by transcriptome-wide snoRNA target identification.
    Bei Liu, Tong Wu, Bernadette A Miao, Fei Ji, Shun Liu, Pingluan Wang, Yutao Zhao, Yuhao Zhong, Arunkumar Sundaram, Tie-Bo Zeng, Marta Majcherska-Agrawal, Robert J Keenan, Tao Pan, Chuan He.
      Small nucleolar RNAs (snoRNAs) are non-coding RNAs known for guiding RNA modifications, including 2'-O-methylation (Nm) and pseudouridine (Ψ). While snoRNAs may also interact with other RNAs, such as mRNA, the full repertoire of RNAs targeted by snoRNA remains elusive due to the lack of effective technologies that identify snoRNA targets transcriptome wide. Here, we develop a chemical crosslinking-based approach that comprehensively detects cellular RNA targets of snoRNAs, yielding thousands of previously unrecognized snoRNA-mRNA interactions in human cells and mouse brain tissues. Many interactions occur outside of snoRNA-guided RNA modification sites, hinting at non-canonical functions beyond RNA modification. We find that one of these snoRNAs, SNORA73, targets mRNAs that encode secretory proteins and membrane proteins. SNORA73 also interacts with 7SL RNA, part of the signal recognition particle (SRP) required for protein secretion. The mRNA-SNORA73-7SL RNA interactions enhance the association of the SNORA73-target mRNAs with SRP, thereby facilitating the secretion of encoded proteins.
    Keywords:  RNA modifications Ψ and N(m); protein secretion; protein translocation; snoRNA molecular glue; snoRNA-7SL RNA interaction; snoRNA-mRNA interaction
    DOI:  https://doi.org/10.1016/j.cell.2024.10.046
  32. bioRxiv. 2024 Nov 11. pii: 2024.11.11.622945. [Epub ahead of print]
    The contribution of mitochondria-associated ER membranes to cholesterol homeostasis.
    J Montesinos, K Kabra, M Uceda, D Larrea, R R Agrawal, K A Tamucci, M Pera, A C Ferre, N Gomez-Lopez, T D Yun, K R Velasco, E A Schon, E Area-Gomez.
      Cellular demands for cholesterol are met by a balance between its biosynthesis in the endoplasmic reticulum (ER) and its uptake from lipoproteins. Cholesterol levels in intracellular membranes form a gradient maintained by a complex network of mechanisms including the control of the expression, compartmentalization and allosteric modulation of the enzymes that balance endogenous and exogenous sources of cholesterol. Low-density lipoproteins (LDLs) are internalized and delivered to lysosomal compartments to release their cholesterol content, which is then distributed within cellular membranes. High-density lipoproteins (HDLs), on the other hand, can transfer their cholesterol content directly into cellular membranes through the action of receptors such as the scavenger receptor B type 1 (SR-B1; gene SCARB1 ). We show here that SR-B1-mediated exogenous cholesterol internalization from HDL stimulates the formation of lipid-raft subdomains in the ER known as mitochondria-associated ER membranes (MAM), that, in turn, suppress de novo cholesterol biosynthesis machinery. We propose that MAM is a regulatory hub for cholesterol homeostasis that offers a novel dimension for understanding the intracellular regulation of this important lipid.
    DOI:  https://doi.org/10.1101/2024.11.11.622945
  33. JACS Au. 2024 Nov 25. 4(11): 4423-4434
    Development of Oral, Potent, and Selective CK1α Degraders for AML Therapy.
    Lu Huang, Lu Chen, Lu Chen, Bo Peng, Lixin Zhou, Yanli Sun, Taiting Shi, Jiayi Lu, Weiye Lin, Yuhang Liu, Linhui Cao, Lanlan Li, Qiangqiang Han, Xi Chen, Ping Yang, Shuo Zhang, Zhe Wang, Jing Yang, Zhixiang Guo, Baishan Jiang, Wenchao Lu.
      Molecular glue degraders (MGDs) are proximity-inducing agents that mediate the cooperative interaction between a target protein and an E3 ligase, introducing an additional layer of specificity beyond that afforded by traditional small molecules. Historically, molecular glues that stabilize protein-protein interactions were often discovered serendipitously. In this study, we leveraged the reprogramming potential of cereblon (CRBN)-based ligands and conducted a CRBN-dependent proliferation screen to identify CRBN-based MGDs capable of inducing the degradation of proteins essential for cell viability. Through our screening and subsequent medicinal chemistry optimization, we identified dCK1α-1 as a potent and selective CK1α-targeting molecular glue degrader. Furthermore, we synthesized an orally active derivative, dCK1α-2, with enhanced pharmacokinetic properties, which exhibited pronounced degradation activity and demonstrated efficacy in mouse models following oral gavage. These findings indicate that phenotypic drug discovery campaigns, in combination with chemically distinct CRBN ligand libraries, can accelerate the development of therapeutically relevant MGDs. Furthermore, the development of dCK1α-1 and dCK1α-2 provides new therapeutic options for cancers with functional p53 signaling and offers valuable chemical tools for future investigations into the role of CK1α.
    DOI:  https://doi.org/10.1021/jacsau.4c00762
  34. Nat Methods. 2024 Nov 25.
    Global analysis of endogenous protein disorder in cells.
    Shouxiang Zhang, Tze Cin Owyong, Oana Sanislav, Lukas Englmaier, Xiaojing Sui, Geqing Wang, David W Greening, Nicholas A Williamson, Andreas Villunger, Jonathan M White, Begoña Heras, Wallace W H Wong, Paul R Fisher, Yuning Hong.
      Disorder and flexibility in protein structures are essential for biological function but can also contribute to diseases, such as neurodegenerative disorders. However, characterizing protein folding on a proteome-wide scale within biological matrices remains challenging. Here we present a method using a bifunctional chemical probe, named TME, to capture in situ, enrich and quantify endogenous protein disorder in cells. TME exhibits a fluorescence turn-on effect upon selective conjugation with proteins with free cysteines in surface-exposed and flexible environments-a distinctive signature of protein disorder. Using an affinity-based proteomic approach, we identify both basal disordered proteins and those whose folding status changes under stress, with coverage to proteins even of low abundance. In lymphoblastoid cells from individuals with Parkinson's disease and healthy controls, our TME-based strategy distinguishes the two groups more effectively than lysate profiling methods. High-throughput TME fluorescence and proteomics further reveal a universal cellular quality-control mechanism in which cells adapt to proteostatic stress by adopting aggregation-prone distributions and sequestering disordered proteins, as illustrated in Huntington's disease cell models.
    DOI:  https://doi.org/10.1038/s41592-024-02507-z
  35. Cell. 2024 Nov 25. pii: S0092-8674(24)01274-1. [Epub ahead of print]
    Proteolethargy is a pathogenic mechanism in chronic disease.
    Alessandra Dall'Agnese, Ming M Zheng, Shannon Moreno, Jesse M Platt, An T Hoang, Deepti Kannan, Giuseppe Dall'Agnese, Kalon J Overholt, Ido Sagi, Nancy M Hannett, Hailey Erb, Olivia Corradin, Arup K Chakraborty, Tong Ihn Lee, Richard A Young.
      The pathogenic mechanisms of many diseases are well understood at the molecular level, but there are prevalent syndromes associated with pathogenic signaling, such as diabetes and chronic inflammation, where our understanding is more limited. Here, we report that pathogenic signaling suppresses the mobility of a spectrum of proteins that play essential roles in cellular functions known to be dysregulated in these chronic diseases. The reduced protein mobility, which we call proteolethargy, was linked to cysteine residues in the affected proteins and signaling-related increases in excess reactive oxygen species. Diverse pathogenic stimuli, including hyperglycemia, dyslipidemia, and inflammation, produce similar reduced protein mobility phenotypes. We propose that proteolethargy is an overlooked cellular mechanism that may account for various pathogenic features of diverse chronic diseases.
    Keywords:  chronic disease; cysteine; protein mobility; proteolethargy; reactive oxygen species; signaling
    DOI:  https://doi.org/10.1016/j.cell.2024.10.051
  36. Cell Chem Biol. 2024 Nov 22. pii: S2451-9456(24)00445-8. [Epub ahead of print]
    Covalent targeting of splicing in T cells.
    Kevin A Scott, Hiroyuki Kojima, Nathalie Ropek, Charles D Warren, Tiffany L Zhang, Simon J Hogg, Henry Sanford, Caroline Webster, Xiaoyu Zhang, Jahan Rahman, Bruno Melillo, Benjamin F Cravatt, Jiankun Lyu, Omar Abdel-Wahab, Ekaterina V Vinogradova.
      Despite significant interest in therapeutic targeting of splicing, few chemical probes are available for the proteins involved in splicing. Here, we show that elaborated stereoisomeric acrylamide EV96 and its analogues lead to a selective T cell state-dependent loss of interleukin 2-inducible T cell kinase (ITK) by targeting one of the core splicing factors SF3B1. Mechanistic investigations suggest that the state-dependency stems from a combination of differential protein turnover rates and extensive ITK mRNA alternative splicing. We further introduce the most comprehensive list to date of proteins involved in splicing and leverage cysteine- and protein-directed activity-based protein profiling with electrophilic scout fragments to demonstrate covalent ligandability for many classes of splicing factors and splicing regulators in T cells. Taken together, our findings show how chemical perturbation of splicing can lead to immune state-dependent changes in protein expression and provide evidence for the broad potential to target splicing factors with covalent chemistry.
    Keywords:  activity-based protein profiling; alternative splicing; covalent immunomodulators; interleukin-2-inducible T-cell kinase; splicing factor 3b subunit 1
    DOI:  https://doi.org/10.1016/j.chembiol.2024.10.010
  37. bioRxiv. 2024 Nov 18. pii: 2024.11.18.622547. [Epub ahead of print]
    Accurate de novo design of high-affinity protein binding macrocycles using deep learning.
    Stephen A Rettie, David Juergens, Victor Adebomi, Yensi Flores Bueso, Qinqin Zhao, Alexandria N Leveille, Andi Liu, Asim K Bera, Joana A Wilms, Alina Üffing, Alex Kang, Evans Brackenbrough, Mila Lamb, Stacey R Gerben, Analisa Murray, Paul M Levine, Maika Schneider, Vibha Vasireddy, Sergey Ovchinnikov, Oliver H Weiergräber, Dieter Willbold, Joshua A Kritzer, Joseph D Mougous, David Baker, Frank DiMaio, Gaurav Bhardwaj.
      The development of macrocyclic binders to therapeutic proteins typically relies on large-scale screening methods that are resource-intensive and provide little control over binding mode. Despite considerable progress in physics-based methods for peptide design and deep-learning methods for protein design, there are currently no robust approaches for de novo design of protein-binding macrocycles. Here, we introduce RFpeptides, a denoising diffusion-based pipeline for designing macrocyclic peptide binders against protein targets of interest. We test 20 or fewer designed macrocycles against each of four diverse proteins and obtain medium to high-affinity binders against all selected targets. Designs against MCL1 and MDM2 demonstrate K D between 1-10 μM, and the best anti-GABARAP macrocycle binds with a K D of 6 nM and a sub-nanomolar IC 50 in vitro . For one of the targets, RbtA, we obtain a high-affinity binder with K D < 10 nM despite starting from the target sequence alone due to the lack of an experimentally determined target structure. X-ray structures determined for macrocycle-bound MCL1, GABARAP, and RbtA complexes match very closely with the computational design models, with three out of the four structures demonstrating Ca RMSD of less than 1.5 Å to the design models. In contrast to library screening approaches for which determining binding mode can be a major bottleneck, the binding modes of RFpeptides-generated macrocycles are known by design, which should greatly facilitate downstream optimization. RFpeptides thus provides a powerful framework for rapid and custom design of macrocyclic peptides for diagnostic and therapeutic applications.
    DOI:  https://doi.org/10.1101/2024.11.18.622547
  38. Science. 2024 Nov 29. 386(6725): eado8476
    Molecular basis of mRNA delivery to the bacterial ribosome.
    Michael W Webster, Adrien Chauvier, Huma Rahil, Andrea Graziadei, Kristine Charles, Nataliya Miropolskaya, Maria Takacs, Charlotte Saint-André, Juri Rappsilber, Nils G Walter, Albert Weixlbaumer.
      Protein synthesis begins with the formation of a ribosome-messenger RNA (mRNA) complex. In bacteria, the small ribosomal subunit (30S) is recruited to many mRNAs through base pairing with the Shine-Dalgarno (SD) sequence and RNA binding by ribosomal protein bS1. Translation can initiate on nascent mRNAs, and RNA polymerase (RNAP) can promote the recruitment of the pioneering 30S. Here, we examined 30S recruitment to nascent mRNAs using cryo-electron microscopy, single-molecule fluorescence colocalization, and in-cell cross-linking mass spectrometry. We show that bS1 delivers the mRNA to the ribosome for SD duplex formation and 30S activation. Additionally, bS1 and RNAP stimulate translation initiation. Our work provides a mechanistic framework for how the SD duplex, ribosomal proteins, and RNAP cooperate in 30S recruitment to mRNAs and establish transcription-translation coupling.
    DOI:  https://doi.org/10.1126/science.ado8476
  39. bioRxiv. 2024 Nov 12. pii: 2024.11.12.623272. [Epub ahead of print]
    Stress Changes the Bacterial Biomolecular Condensate Material State and Shifts Function from mRNA Decay to Storage.
    Luis A Ortiz-Rodríguez, Hadi Yassine, Vidhyadhar Nandana, Christopher A Azaldegui, Jiayu Cheng, Jared M Schrader, Julie S Biteen.
      Bacterial ribonucleoprotein bodies (BR-bodies) are dynamic biomolecular condensates that play a pivotal role in RNA metabolism. We investigated how BR-bodies significantly influence mRNA fate by transitioning between liquid- and solid-like states in response to stress. With a combination of single-molecule and bulk fluorescence microscopy, biochemical assays, and quantitative analyses, we determine that BR-bodies promote efficient mRNA decay in a liquid-like condensate during exponential growth. On the other hand, BR-bodies are repurposed from sites of mRNA decay to reservoirs for mRNA storage under stress, a functional change that is enabled by their transition to a more rigid state, marked by reduced internal dynamics, increased molecular density, and prolonged residence time of ribonuclease E. Furthermore, we manipulated ATP levels and translation rates and conclude that the accumulation of ribosome-depleted mRNA is a key factor driving these material state transitions, and that condensate maturation further contributes to this process. Upon nutrient replenishment, stationary-phase BR-bodies disassemble, releasing stored mRNAs for rapid translation, demonstrating that BR-body function is governed by a reversible mechanism for resource management. These findings reveal adaptive strategies by which bacteria regulate RNA metabolism through condensate-mediated control of mRNA decay and storage.
    DOI:  https://doi.org/10.1101/2024.11.12.623272
  40. Nat Commun. 2024 Nov 29. 15(1): 10388
    Thioredoxin 1 moonlights as a chaperone for an interbacterial ADP-ribosyltransferase toxin.
    Baptiste Dumont, Laurent Terradot, Eric Cascales, Laurence Van Melderen, Dukas Jurėnas.
      Formation and breakage of disulfide bridges strongly impacts folding and activity of proteins. Thioredoxin 1 (TrxA) is a small, conserved enzyme that reduces disulfide bonds in the bacterial cytosol. In this study, we provide an example of the emergence of a chaperone role for TrxA, which is independent of redox catalysis. We show that the activity of the secreted bacterial ADP-ribosyltransferase (ART) toxin TreX, which does not contain any cysteines, is dependent on TrxA. TreX binds to the reduced form of TrxA via its carboxy-terminal extension to form a soluble and active complex. Structural studies revealed that TreX-like toxins are homologous to Scabin-like ART toxins which possess cysteine residues and form disulfide bridges at the position that superimposes the TrxA binding site in TreX. Our study therefore suggests that thioredoxin 1 evolved alternative functions by maintaining the interaction with cysteine-free substrates.
    DOI:  https://doi.org/10.1038/s41467-024-54892-w
  41. Nat Commun. 2024 Nov 25. 15(1): 10193
    Structural basis for Retriever-SNX17 assembly and endosomal sorting.
    Amika Singla, Daniel J Boesch, Ho Yee Joyce Fung, Chigozie Ngoka, Avery S Enriquez, Ran Song, Daniel A Kramer, Yan Han, Esther Banarer, Andrew Lemoff, Puneet Juneja, Daniel D Billadeau, Xiaochen Bai, Zhe Chen, Emre E Turer, Ezra Burstein, Baoyu Chen.
      During endosomal recycling, Sorting Nexin 17 (SNX17) facilitates the transport of numerous membrane cargo proteins by tethering them to the Retriever complex. Despite its importance, the mechanisms underlying this interaction have remained elusive. Here, we provide biochemical, structural, cellular, and proteomic analyses of the SNX17-Retriever interaction. Our data reveal that SNX17 adopts an autoinhibited conformation in the basal state, with its FERM domain sequestering its C-terminal tail. The binding of cargo proteins to the FERM domain displaces the C-terminal tail through direct competition. The released tail engages with Retriever by binding to a highly conserved interface between its VPS35L and VPS26C subunits, as revealed by cryogenic electron microscopy (cryo-EM). Disrupting this interface impairs the Retriever-SNX17 interaction, subsequently affecting the recycling of SNX17-dependent cargoes and altering the composition of the plasma membrane proteome. Intriguingly, the SNX17-binding pocket on Retriever can be utilized by other ligands containing a consensus acidic C-terminal tail motif. Together, our findings uncover a mechanism underlying endosomal trafficking of critical cargo proteins and reveal how Retriever can potentially engage with other regulatory factors or be exploited by pathogens.
    DOI:  https://doi.org/10.1038/s41467-024-54583-6
  42. Nat Commun. 2024 Nov 25. 15(1): 10223
    Interformer: an interaction-aware model for protein-ligand docking and affinity prediction.
    Houtim Lai, Longyue Wang, Ruiyuan Qian, Junhong Huang, Peng Zhou, Geyan Ye, Fandi Wu, Fang Wu, Xiangxiang Zeng, Wei Liu.
      In recent years, the application of deep learning models to protein-ligand docking and affinity prediction, both vital for structure-based drug design, has garnered increasing interest. However, many of these models overlook the intricate modeling of interactions between ligand and protein atoms in the complex, consequently limiting their capacity for generalization and interpretability. In this work, we propose Interformer, a unified model built upon the Graph-Transformer architecture. The proposed model is designed to capture non-covalent interactions utilizing an interaction-aware mixture density network. Additionally, we introduce a negative sampling strategy, facilitating an effective correction of interaction distribution for affinity prediction. Experimental results on widely used and our in-house datasets demonstrate the effectiveness and universality of the proposed approach. Extensive analyses confirm our claim that our approach improves performance by accurately modeling specific protein-ligand interactions. Encouragingly, our approach advances docking tasks state-of-the-art (SOTA) performance.
    DOI:  https://doi.org/10.1038/s41467-024-54440-6
  43. Nat Methods. 2024 Nov 27.
    SurfDock is a surface-informed diffusion generative model for reliable and accurate protein-ligand complex prediction.
    Duanhua Cao, Mingan Chen, Runze Zhang, Zhaokun Wang, Manlin Huang, Jie Yu, Xinyu Jiang, Zhehuan Fan, Wei Zhang, Hao Zhou, Xutong Li, Zunyun Fu, Sulin Zhang, Mingyue Zheng.
      Accurately predicting protein-ligand interactions is crucial for understanding cellular processes. We introduce SurfDock, a deep-learning method that addresses this challenge by integrating protein sequence, three-dimensional structural graphs and surface-level features into an equivariant architecture. SurfDock employs a generative diffusion model on a non-Euclidean manifold, optimizing molecular translations, rotations and torsions to generate reliable binding poses. Our extensive evaluations across various benchmarks demonstrate SurfDock's superiority over existing methods in docking success rates and adherence to physical constraints. It also exhibits remarkable generalizability to unseen proteins and predicted apo structures, while achieving state-of-the-art performance in virtual screening tasks. In a real-world application, SurfDock identified seven novel hit molecules in a virtual screening project targeting aldehyde dehydrogenase 1B1, a key enzyme in cellular metabolism. This showcases SurfDock's ability to elucidate molecular mechanisms underlying cellular processes. These results highlight SurfDock's potential as a transformative tool in structural biology, offering enhanced accuracy, physical plausibility and practical applicability in understanding protein-ligand interactions.
    DOI:  https://doi.org/10.1038/s41592-024-02516-y
  44. Arch Biochem Biophys. 2024 Nov 25. pii: S0003-9861(24)00349-7. [Epub ahead of print] 110227
    Reveal the autophagic degradation of glutaredoxin Grx4 in Schizosaccharomyces pombe.
    Rong Li, Ying Huang.
      Glutaredoxins (Grxs) are small, heat-stable proteins that serve as multi-functional glutathione-dependent thiol transferases. Recent studies have elucidated their role in regulating cellular iron and copper homeostasis. To further elucidate their functions, we employed a combination of bioinformatics and experimental analyses. In S. pombe, five Grxs have been identified. Our study utilized multiple sequence alignment and conserved domain prediction, revealing that Grx4 and its homologs possess a glutaredoxin domain (GRX domain) at the C-terminal and a thioredoxin-like domain (TRX domain) exclusively at the N-terminal. The functional roles of the GRX domain and TRX domain were investigated by constructing strains that express a truncated Grx4 under the regulation of either a constitutive cam1 promoter or its native promoter. Our findings indicated that two Atg8 interacting motifs (AIM), FLKI and FQEI, located within the TRX domain of Grx4, are sufficient to induce autophagic degradation under nitrogen- or iron-starvation conditions, respectively. This represents a significant advancement in understanding TRX domain function within Grxs for the first time. Moreover, the altered expression level of Pcl1 in Δatg5 or Δatg8 strains under iron starvation suggests that autophagy is essential for maintaining iron homeostasis. Further investigations revealed that Grx4 is required for cellular survival and endoplasmic reticulum autophagy (ER-phagy) during DTT treatment, implying a potential correlation between Grxs and the endoplasmic reticulum (ER). Additionally, the loss of Grx4 disrupted nuclear integrity during ER stress, highlighting the versatility and importance of further investigations into the functions of Grx4.
    Keywords:  Autophagy; ER stress; Glutaredoxins; S. pombe; iron homeostasis
    DOI:  https://doi.org/10.1016/j.abb.2024.110227
  45. Proc Natl Acad Sci U S A. 2024 Dec 03. 121(49): e2409139121
    Molecular insights into the interaction between a disordered protein and a folded RNA.
    Rishav Mitra, Emery T Usher, Selin Dedeoğlu, Matthew J Crotteau, Olivia A Fraser, Neela H Yennawar, Varun V Gadkari, Brandon T Ruotolo, Alex S Holehouse, Loïc Salmon, Scott A Showalter, James C A Bardwell.
      Intrinsically disordered protein regions (IDRs) are well established as contributors to intermolecular interactions and the formation of biomolecular condensates. In particular, RNA-binding proteins (RBPs) often harbor IDRs in addition to folded RNA-binding domains that contribute to RBP function. To understand the dynamic interactions of an IDR-RNA complex, we characterized the RNA-binding features of a small (68 residues), positively charged IDR-containing protein, Small ERDK-Rich Factor (SERF). At high concentrations, SERF and RNA undergo charge-driven associative phase separation to form a protein- and RNA-rich dense phase. A key advantage of this model system is that this threshold for demixing is sufficiently high that we could use solution-state biophysical methods to interrogate the stoichiometric complexes of SERF with RNA in the one-phase regime. Herein, we describe our comprehensive characterization of SERF alone and in complex with a small fragment of the HIV-1 Trans-Activation Response (TAR) RNA with complementary biophysical methods and molecular simulations. We find that this binding event is not accompanied by the acquisition of structure by either molecule; however, we see evidence for a modest global compaction of the SERF ensemble when bound to RNA. This behavior likely reflects attenuated charge repulsion within SERF via binding to the polyanionic RNA and provides a rationale for the higher-order assembly of SERF in the context of RNA. We envision that the SERF-RNA system will lower the barrier to accessing the details that support IDR-RNA interactions and likewise deepen our understanding of the role of IDR-RNA contacts in complex formation and liquid-liquid phase separation.
    Keywords:  RNA binding proteins; disordered proteins; molecular condensates
    DOI:  https://doi.org/10.1073/pnas.2409139121
  46. Cell Rep. 2024 Nov 23. pii: S2211-1247(24)01299-3. [Epub ahead of print]43(12): 114948
    IFN-γ-dependent regulation of intestinal epithelial homeostasis by NKT cells.
    Marta Lebrusant-Fernandez, Tom Ap Rees, Rebeca Jimeno, Nikolaos Angelis, Joseph C Ng, Franca Fraternali, Vivian S W Li, Patricia Barral.
      Intestinal homeostasis is maintained through the combined functions of epithelial and immune cells that collaborate to preserve the integrity of the intestinal barrier. However, the mechanisms by which immune cell populations regulate intestinal epithelial cell (IEC) homeostasis remain unclear. Here, we use a multi-omics approach to study the immune-epithelial crosstalk and identify CD1d-restricted natural killer T (NKT) cells as key regulators of IEC biology. We find that NKT cells are abundant in the proximal small intestine and show hallmarks of activation at steady state. Subsequently, NKT cells regulate the survival and the transcriptional and cellular composition landscapes of IECs in intestinal organoids, through interferon-γ (IFN-γ) and interleukin-4 secretion. In vivo, lack of NKT cells results in an increase in IEC turnover, while NKT cell activation leads to IFN-γ-dependent epithelial apoptosis. Our findings propose NKT cells as potent producers of cytokines that contribute to the regulation of IEC homeostasis.
    Keywords:  CP: Immunology; IFN-γ; NKT cell; intestinal epithelial cell; intestinal organoids
    DOI:  https://doi.org/10.1016/j.celrep.2024.114948
  47. bioRxiv. 2024 Nov 25. pii: 2024.11.21.623966. [Epub ahead of print]
    Structural diversity and oligomerization of bacterial ubiquitin-like proteins.
    Minheng Gong, Qiaozhen Ye, Yajie Gu, Lydia R Chambers, Andrey A Bobkov, Neal K Arakawa, Mariusz Matyszewski, Kevin D Corbett.
      Bacteria possess a variety of operons with homology to eukaryotic ubiquitination pathways that encode predicted E1, E2, E3, deubiquitinase, and ubiquitin-like proteins. Some of these pathways have recently been shown to function in anti-bacteriophage immunity, but the biological functions of others remain unknown. Here, we show that ubiquitin-like proteins in two bacterial operon families show surprising architectural diversity, possessing one to three β-grasp domains preceded by diverse N-terminal domains. We find that a large group of bacterial ubiquitin-like proteins possess three β-grasp domains and form homodimers and helical filaments mediated by conserved Ca 2+ ion binding sites. Our findings highlight a distinctive mode of self-assembly for ubiquitin-like proteins, and suggest that Ca 2+ -mediated ubiquitin-like protein filament assembly and/or disassembly enables cells to sense and respond to stress conditions that alter intracellular metal ion concentration.
    DOI:  https://doi.org/10.1101/2024.11.21.623966
  48. Nature. 2024 Nov 27.
    Stereochemistry in the disorder-order continuum of protein interactions.
    Estella A Newcombe, Amanda D Due, Andrea Sottini, Steffie Elkjær, Frederik Friis Theisen, Catarina B Fernandes, Lasse Staby, Elise Delaforge, Christian R O Bartling, Inna Brakti, Katrine Bugge, Benjamin Schuler, Karen Skriver, Johan G Olsen, Birthe B Kragelund.
      Intrinsically disordered proteins can bind via the formation of highly disordered protein complexes without the formation of three-dimensional structure1. Most naturally occurring proteins are levorotatory (L)-that is, made up only of L-amino acids-imprinting molecular structure and communication with stereochemistry2. By contrast, their mirror-image dextrorotatory (D)-amino acids are rare in nature. Whether disordered protein complexes are truly independent of chiral constraints is not clear. Here, to investigate the chiral constraints of disordered protein-protein interactions, we chose as representative examples a set of five interacting protein pairs covering the disorder-order continuum. By observing the natural ligands and their stereochemical mirror images in free and bound states, we found that chirality was inconsequential in a fully disordered complex. However, if the interaction relied on the ligand undergoing extensive coupled folding and binding, correct stereochemistry was essential. Between these extremes, binding could be observed for the D-ligand with a strength that correlated with disorder in the final complex. These findings have important implications for our understanding of the molecular processes that lead to complex formation, the use of D-peptides in drug discovery and the chemistry of protein evolution of the first living entities on Earth.
    DOI:  https://doi.org/10.1038/s41586-024-08271-6
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