bims-proteo Biomed News
on Proteostasis
Issue of 2024‒11‒17
forty-two papers selected by
Eric Chevet, INSERM



  1. Elife. 2024 Nov 12. pii: RP89606. [Epub ahead of print]12
      Degrons are minimal protein features that are sufficient to target proteins for degradation. In most cases, degrons allow recognition by components of the cytosolic ubiquitin proteasome system. Currently, all of the identified degrons only function within the cytosol. Using Saccharomyces cerevisiae, we identified the first short linear sequences that function as degrons from the endoplasmic reticulum (ER) lumen. We show that when these degrons are transferred to proteins, they facilitate proteasomal degradation through the endoplasmic reticulum associated degradation (ERAD) system. These degrons enable degradation of both luminal and integral membrane ER proteins, expanding the types of proteins that can be targeted for degradation in budding yeast and mammalian tissue culture. This discovery provides a framework to target proteins for degradation from the previously unreachable ER lumen and builds toward therapeutic approaches that exploit the highly conserved ERAD system.
    Keywords:  ERAD; S. cerevisiae; cell biology; degrons; endoplasmic reticulum associated degradation; human; protein degradation
    DOI:  https://doi.org/10.7554/eLife.89606
  2. J Cell Sci. 2024 Nov 08. pii: jcs.262333. [Epub ahead of print]
      Cleavage of transmembrane segments on target proteins by the aspartyl intramembrane protease signal peptide peptidase (SPP) has been linked to immunity, viral infection and protein quality control. How SPP recognizes its various substrates and specifies their fate remains elusive. Here we identified the lanosterol demethylase CYP51A1 as an SPP substrate and show that SPP-catalyzed cleavage triggers CYP51A1 clearance by ER-associated degradation (ERAD). We observe that SPP targets only a fraction of CYP51A1 molecules and identified an amphipathic helix in the N-terminus as a key determinant for SPP recognition. SPP recognition is remarkably specific to CYP51A1 molecules with the amphipathic helix aberrantly inserted in the membrane with a type II orientation. Thus, our data are consistent with a role for SPP in topology surveillance, triggering the clearance of certain, potentially non-functional conformers.
    Keywords:  Endoplasmic reticulum; Endoplasmic reticulum-associated protein degradation (ERAD); Intramembrane proteolysis; Protein quality control; Signal peptide peptidase (SPP); Ubiquitin ligase
    DOI:  https://doi.org/10.1242/jcs.262333
  3. J Cell Biol. 2024 Dec 02. pii: e202407123. [Epub ahead of print]223(12):
      Most eukaryotic genes encode polypeptides that are either obligate members of hetero-stoichiometric complexes or clients of organelle-targeting pathways. Proteins in these classes can be released from the ribosome as "orphans"-newly synthesized proteins not associated with their stoichiometric binding partner(s) and/or not targeted to their destination organelle. Here we integrate recent findings suggesting that although cells selectively degrade orphan proteins under homeostatic conditions, they can preserve them in chaperone-regulated biomolecular condensates during stress. These orphan protein condensates activate the heat shock response (HSR) and represent subcellular sites where the chaperones induced by the HSR execute their functions. Reversible condensation of orphan proteins may broadly safeguard labile precursors during stress.
    DOI:  https://doi.org/10.1083/jcb.202407123
  4. J Cell Biol. 2025 Jan 06. pii: e202406103. [Epub ahead of print]224(1):
      Protein secretion is an essential process that drives cell growth and communication. Enrichment of soluble secretory proteins into ER-derived transport carriers occurs via transmembrane cargo receptors that connect lumenal cargo to the cytosolic COPII coat. Here, we find that the cargo receptor, SURF4, recruits different SEC24 cargo adaptor paralogs of the COPII coat to export different cargoes. The secreted protease, PCSK9, requires both SURF4 and a co-receptor, TMED10, for export via SEC24A. In contrast, secretion of Cab45 and NUCB1 requires SEC24C/D. We further show that ER export signals of Cab45 and NUCB1 bind co-translationally to SURF4 via a lumenal pocket, contrasting prevailing models of receptor engagement only upon protein folding/maturation. Bioinformatics analyses suggest that strong SURF4-binding motifs are features of proteases, receptor-binding ligands, and Ca2+-binding proteins. We propose that certain classes of proteins are fast-tracked for rapid export to protect the health of the ER lumen.
    DOI:  https://doi.org/10.1083/jcb.202406103
  5. Science. 2024 Nov 08. 386(6722): 667-672
      One-fifth of human proteins are N-glycosylated in the endoplasmic reticulum (ER) by two oligosaccharyltransferases, OST-A and OST-B. Contrary to the prevailing view of N-glycosylation as a housekeeping function, we identified an ER pathway that modulates the activity of OST-A. Genetic analyses linked OST-A to HSP90B1, an ER chaperone for membrane receptors, and CCDC134, an ER luminal protein. During its translocation into the ER, an N-terminal peptide in HSP90B1 templates the assembly of a translocon complex containing CCDC134 and OST-A that protects HSP90B1 during folding, preventing its hyperglycosylation and degradation. Disruption of this pathway impairs WNT and IGF1R signaling and causes the bone developmental disorder osteogenesis imperfecta. Thus, N-glycosylation can be regulated by specificity factors in the ER to control cell surface receptor signaling and tissue development.
    DOI:  https://doi.org/10.1126/science.adp7201
  6. Cell Chem Biol. 2024 Nov 07. pii: S2451-9456(24)00442-2. [Epub ahead of print]
      Targeted protein degradation (TPD) has emerged as a powerful strategy to selectively eliminate cellular proteins using small-molecule degraders, offering therapeutic promise for targeting proteins that are otherwise undruggable. However, a remaining challenge is to unambiguously identify primary TPD targets that are distinct from secondary downstream effects in the proteome. Here we introduce an approach for selective analysis of protein degradation by mass spectrometry (DegMS) at proteomic scale, which derives its specificity from the exclusion of confounding effects of altered transcription and translation induced by target depletion. We show that the approach efficiently operates at the timescale of TPD (hours) and we demonstrate its utility by analyzing the cyclin K degraders dCeMM2 and dCeMM4, which induce widespread transcriptional downregulation, and the GSPT1 degrader CC-885, an inhibitor of protein translation. Additionally, we apply DegMS to characterize a previously uncharacterized degrader, and identify the zinc-finger protein FIZ1 as a degraded target.
    Keywords:  PROTAC; click chemistry; molecular glue; protein degradation; proteomics; stable isotope labeling; targeted protein degradation
    DOI:  https://doi.org/10.1016/j.chembiol.2024.10.007
  7. Dev Cell. 2024 Nov 01. pii: S1534-5807(24)00627-0. [Epub ahead of print]
      The cellular proteome determines the functional state of cells and is often skewed to direct pathological conditions. Autophagy shapes cellular proteomes primarily through lysosomal degradation of either damaged or unnecessary proteins. Here, we show that autophagy directs the senescence-specific translatome to fuel inflammation by coupling selective protein degradation with alternative splicing. RNA splicing is significantly altered during senescence, some of which surprisingly depend on autophagy, including exon 5 skipping of the translation regulator EIF4H. Systematic translatome profiling indicates that this event is key to the translational bias toward inflammation in senescence. Autophagy promotes these changes by selectively degrading the splicing regulator splicing factor proline and glutamine rich (SFPQ) via the autophagy receptor NBR1. These autophagy-centric inflammatory controls appear to be conserved during human tissue aging and cancer. Our work highlights the role of autophagy in the on-demand functional remodeling of cellular proteomes as well as the crosstalk between autophagy, alternative splicing, and inflammatory translation.
    Keywords:  RNA homeostasis; aging; alternative splicing; autophagy; cancer; cellular senescence; inflammation; protein translation; selective autophagy
    DOI:  https://doi.org/10.1016/j.devcel.2024.10.008
  8. Nat Commun. 2024 Nov 15. 15(1): 9899
      Specificity of the ubiquitin-proteasome system depends on E3 ligase-substrate interactions. Many such pairings depend on E3 ligases binding to peptide-like sequences - termed N- or C-degrons - at the termini of substrates. However, our knowledge of structural features distinguishing closely related C-degron substrate-E3 pairings is limited. Here, by systematically comparing ubiquitylation activities towards a suite of common model substrates, and defining interactions by biochemistry, crystallography, and cryo-EM, we reveal principles of C-degron recognition across the KLHDCX family of Cullin-RING ligases (CRLs). First, a motif common across these E3 ligases anchors a substrate's C-terminus. However, distinct locations of this C-terminus anchor motif in different blades of the KLHDC2, KLHDC3, and KLHDC10 β-propellers establishes distinct relative positioning and molecular environments for substrate C-termini. Second, our structural data show KLHDC3 has a pre-formed pocket establishing preference for an Arg or Gln preceding a C-terminal Gly, whereas conformational malleability contributes to KLHDC10's recognition of varying features adjacent to substrate C-termini. Finally, additional non-consensus interactions, mediated by C-degron binding grooves and/or by distal propeller surfaces and substrate globular domains, can substantially impact substrate binding and ubiquitylatability. Overall, the data reveal combinatorial mechanisms determining specificity and plasticity of substrate recognition by KLDCX-family C-degron E3 ligases.
    DOI:  https://doi.org/10.1038/s41467-024-54126-z
  9. Cell Chem Biol. 2024 Nov 07. pii: S2451-9456(24)00440-9. [Epub ahead of print]
      Chemical proteomics enables the global analysis of small molecule-protein interactions in native biological systems and has emerged as a versatile approach for ligand discovery. The range of small molecules explored by chemical proteomics has, however, remained limited. Here, we describe a diversity-oriented synthesis (DOS)-inspired library of stereochemically defined compounds bearing diazirine and alkyne units for UV light-induced covalent modification and click chemistry enrichment of interacting proteins, respectively. We find that these "photo-stereoprobes" interact in a stereoselective manner with hundreds of proteins from various structural and functional classes in human cells and demonstrate that these interactions can form the basis for high-throughput screening-compatible NanoBRET assays. Integrated phenotypic screening and chemical proteomics identified photo-stereoprobes that modulate autophagy by engaging the mitochondrial serine protease CLPP. Our findings show the utility of DOS-inspired photo-stereoprobes for expanding the ligandable proteome, furnishing target engagement assays, and facilitating the discovery and characterization of bioactive compounds in phenotypic screens.
    Keywords:  NanoBRET; autophagy; chemical proteomics; diazirine; diversity-oriented synthesis; ligands; phenotypic screening; photoreactive; probes; proteomics; stereochemistry
    DOI:  https://doi.org/10.1016/j.chembiol.2024.10.005
  10. Nat Struct Mol Biol. 2024 Nov 07.
      Proteins traverse the eukaryotic secretory pathway through membrane trafficking between organelles. The coat protein complex II (COPII) mediates the anterograde transport of newly synthesized proteins from the endoplasmic reticulum, engaging cargoes with a wide range of size and biophysical properties. The native architecture of the COPII coat and how cargo might influence COPII carrier morphology remain poorly understood. Here we reconstituted COPII-coated membrane carriers using purified Saccharomyces cerevisiae proteins and cell-derived microsomes as a native membrane source. Using cryo-electron tomography with subtomogram averaging, we demonstrate that the COPII coat binds cargo and forms largely spherical vesicles from native membranes. We reveal the architecture of the inner and outer coat layers and shed light on how spherical carriers are formed. Our results provide insights into the architecture and regulation of the COPII coat and advance our current understanding of how membrane curvature is generated.
    DOI:  https://doi.org/10.1038/s41594-024-01413-4
  11. Trends Cell Biol. 2024 Nov 14. pii: S0962-8924(24)00209-5. [Epub ahead of print]
      Aging trajectories vary among individuals, characterized by progressive functional decline, often leading to disease states. One of the central hallmarks of aging is the deterioration of proteostasis, where the function of the endoplasmic reticulum (ER) is dramatically affected. ER stress is monitored and adjusted by the unfolded protein response (UPR); a signaling pathway that mediates adaptive processes to restore proteostasis. Studies in multiple model organisms (yeast, worms, flies, and mice) in addition to human tissue indicates that adaptive UPR signaling contributes to healthy aging. Strategies to improve ER proteostasis using small molecules and gene therapy reduce the decline of organ function during normal aging in mammals. This article reviews recent advances in understanding the significance of the ER proteostasis network to normal aging and its relationship with other hallmarks of aging such as senescence.
    Keywords:  aging; endoplasmic reticulum stress; proteostasis; senescence; unfolded protein response
    DOI:  https://doi.org/10.1016/j.tcb.2024.10.003
  12. J Cell Biol. 2024 Dec 02. pii: e202404094. [Epub ahead of print]223(12):
      Efficient import of nuclear-encoded proteins into mitochondria is crucial for proper mitochondrial function. The conserved translation factor eIF5A binds ribosomes, alleviating stalling at polyproline-encoding sequences. eIF5A impacts mitochondrial function across species, though the precise molecular mechanism is unclear. We found that eIF5A depletion in yeast reduces the translation and levels of the TCA cycle and oxidative phosphorylation proteins. Loss of eIF5A causes mitoprotein precursors to accumulate in the cytosol and triggers a mitochondrial import stress response. We identify an essential polyproline protein as a direct target of eIF5A: the mitochondrial inner membrane protein and translocase component Tim50. Thus, eIF5A controls mitochondrial protein import by alleviating ribosome stalling along Tim50 mRNA at the mitochondrial surface. Removal of polyprolines from Tim50 partially rescues the mitochondrial import stress response and translation of oxidative phosphorylation genes. Overall, our findings elucidate how eIF5A impacts the mitochondrial function by promoting efficient translation and reducing ribosome stalling of co-translationally imported proteins, thereby positively impacting the mitochondrial import process.
    DOI:  https://doi.org/10.1083/jcb.202404094
  13. J Biol Chem. 2024 Nov 08. pii: S0021-9258(24)02479-7. [Epub ahead of print] 107977
      Oxidants produced through endogenous metabolism or encountered in the environment react directly with reactive sites in biological macromolecules. Many proteins, in particular, are susceptible to oxidative damage, which can lead their altered structure and function. Such structural and functional changes trigger a cascade of events that influence key components of the proteostasis network. Here, we highlight recent advances in our understanding of how cells respond to the challenges of protein folding and metabolic alterations that occur during oxidative stress. Immediately after an oxidative insult, cells selectively block the translation of most new proteins and shift molecular chaperones from a folding to a holding role to prevent wholesale protein aggregation. At the same time, adaptive responses in gene expression are induced, allowing for increased expression of antioxidant enzymes, enzymes that carry out reduction of oxidized proteins, and molecular chaperones, all of which serve to mitigate oxidative damage and rebalance proteostasis. Likewise, concomitant activation of protein clearance mechanisms, namely proteasomal degradation and particular autophagic pathways, promotes degradation of irreparably damaged proteins. As oxidative stress is associated with inflammation, aging, and numerous age-related disorders, the molecular events described herein are therefore major determinants of health and disease.
    Keywords:  chaperone; foldase; heat shock protein; holdase; oxidation; oxidative stress; post-translational modification; protein degradation; proteostasis; redox regulation; thiol modification; transcriptional response; translation repression
    DOI:  https://doi.org/10.1016/j.jbc.2024.107977
  14. Curr Biol. 2024 Nov 10. pii: S0960-9822(24)01457-X. [Epub ahead of print]
      Maintenance of lysosomal integrity is essential for cell viability. Upon injury, lysosomes may be targeted for degradation via a selective form of autophagy known as lysophagy. The engulfment of a damaged lysosome by an autophagosome is mediated by the recruitment of adaptor proteins, including SQSTM1/p62. p62 promotes lysophagy via the formation of phase-separated condensates in a mechanism that is regulated by the heat shock protein HSP27. Here, we demonstrate a direct interaction between HSP27 and p62. We used structural modeling to predict the binding interface between HSP27 and p62 and identify several disease-associated mutations that map to this interface. We used proteomics to identify post-translational modifications of HSP27 that regulate HSP27 recruitment to stressed lysosomes, finding robust phosphorylation at several serine residues. Next, we characterized the upstream signaling mechanism leading to HSP27 phosphorylation and found that p38 mitogen-activated protein kinase (MAPK) and its effector kinase MAP kinase-activated protein kinase 2 (MK2) are activated upon lysosomal damage by the kinase mTOR and the production of intracellular reactive oxygen species (ROS). Increased ROS activates p38 MAPK, which in turn allows MK2-dependent phosphorylation of HSP27. Depletion of HSP27 or the inhibition of HSP27 phosphorylation alters the dynamics of p62 condensates on stressed lysosomes, significantly inhibiting p62-dependent lysophagy. Thus, we define a novel lysosomal quality control mechanism in which lysosomal injury triggers a p38 MAPK/MK2 signaling cascade promoting p62-dependent lysophagy. Further, this signaling cascade is activated by many cellular stressors, including oxidative and heat stress, suggesting that other forms of selective autophagy may be regulated by p38 MAPK/MK2/HSP27.
    Keywords:  HSP27; SQSTM1/p62; lysophagy; oligomers; p38 MAPK; phase separation; phosphorylation
    DOI:  https://doi.org/10.1016/j.cub.2024.10.061
  15. Science. 2024 Nov 15. 386(6723): 768-776
      RNA splicing enables the functional adaptation of cells to changing contexts. Impaired splicing has been associated with diseases, including retinitis pigmentosa, but the underlying molecular mechanisms and cellular responses remain poorly understood. In this work, we report that deficiency of ubiquitin-specific protease 39 (USP39) in human cell lines, zebrafish larvae, and mice led to impaired spliceosome assembly and a cytotoxic splicing profile characterized by the use of cryptic 5' splice sites. Disruptive cryptic variants evaded messenger RNA (mRNA) surveillance pathways and were translated into misfolded proteins, which caused proteotoxic aggregates, endoplasmic reticulum (ER) stress, and, ultimately, cell death. The detrimental consequence of splicing-induced proteotoxicity could be mitigated by up-regulating the ubiquitin-proteasome system and selective autophagy. Our findings provide insight into the molecular pathogenesis of spliceosome-associated diseases.
    DOI:  https://doi.org/10.1126/science.adi5295
  16. Nat Commun. 2024 Nov 12. 15(1): 9797
      Increased activity of the heat shock factor, HSF-1, suppresses proteotoxicity and enhances longevity. However, the precise mechanisms by which HSF-1 promotes lifespan are unclear. Using an RNAi screen, we identify ubiquilin-1 (ubql-1) as an essential mediator of lifespan extension in worms overexpressing hsf-1. We find that hsf-1 overexpression leads to transcriptional downregulation of all components of the CDC-48-UFD-1-NPL-4 complex, which is central to both endoplasmic reticulum and mitochondria associated protein degradation, and that this is complemented by UBQL-1-dependent turnover of NPL-4.1. As a consequence, mitochondrial network dynamics are altered, leading to increased lifespan. Together, our data establish that HSF-1 mediates lifespan extension through mitochondrial network adaptations that occur in response to down-tuning of components associated with organellar protein degradation pathways.
    DOI:  https://doi.org/10.1038/s41467-024-54136-x
  17. Mol Cell. 2024 Nov 07. pii: S1097-2765(24)00864-5. [Epub ahead of print]
      PARP enzymes transfer ADP-ribose from NAD+ onto proteins as a covalent modification that regulates multiple aspects of cell biology. Here, we identify an undiscovered catalytic activity for human PARP1: de novo generation of free PAR molecules that are not attached to proteins. Free PAR production arises when a molecule of NAD+ or ADP-ribose docks in the PARP1 acceptor site and attaches to an NAD+ molecule bound to the donor site, releasing nicotinamide and initiating ADP-ribose chains that emanate from NAD+/ADP-ribose rather than protein. Free PAR is also produced by human PARP2 and the PARP enzyme Tankyrase. We demonstrate that free PAR in cells is generated mostly by PARP1 de novo synthesis activity rather than by PAR-degrading enzymes PAR glycohydrolase (PARG), ARH3, and TARG1 releasing PAR from protein. The coincident production of free PAR and protein-linked modifications alters models for PAR signaling and broadens the scope of PARP enzyme signaling capacity.
    Keywords:  ADP-ribose; ARH3; HPF1; PARG; PARP1; PARP2; Parthanatos; TARG1; Tankyrase; poly(ADP-ribose)
    DOI:  https://doi.org/10.1016/j.molcel.2024.10.024
  18. EMBO J. 2024 Nov 12.
      Ubiquitin-conjugating enzymes (E2) play a crucial role in the attachment of ubiquitin to proteins. Together with ubiquitin ligases (E3), they catalyze the transfer of ubiquitin (Ub) onto lysines with high chemoselectivity. A subfamily of E2s, including yeast Ubc6 and human Ube2J2, also mediates noncanonical modification of serines, but the structural determinants for this chemical versatility remain unknown. Using a combination of X-ray crystallography, molecular dynamics (MD) simulations, and reconstitution approaches, we have uncovered a two-layered mechanism that underlies this unique reactivity. A rearrangement of the Ubc6/Ube2J2 active site enhances the reactivity of the E2-Ub thioester, facilitating attack by weaker nucleophiles. Moreover, a conserved histidine in Ubc6/Ube2J2 activates a substrate serine by general base catalysis. Binding of RING-type E3 ligases further increases the serine selectivity inherent to Ubc6/Ube2J2, via an allosteric mechanism that requires specific positioning of the ubiquitin tail at the E2 active site. Our results elucidate how subtle structural modifications to the highly conserved E2 fold yield distinct enzymatic activity.
    Keywords:  ER-associated Protein Degradation; Noncanonical Ubiquitination; Posttranslational Modification; RING E3 Ligase; Ubiquitin-conjugating Enzyme
    DOI:  https://doi.org/10.1038/s44318-024-00301-3
  19. EMBO J. 2024 Nov 12.
      Lysosomal damage induces stress granule (SG) formation. However, the importance of SGs in determining cell fate and the precise mechanisms that mediate SG formation in response to lysosomal damage remain unclear. Here, we describe a novel calcium-dependent pathway controlling SG formation, which promotes cell survival during lysosomal damage. Mechanistically, the calcium-activated protein ALIX transduces lysosomal damage signals to SG formation by controlling eIF2α phosphorylation after sensing calcium leakage. ALIX enhances eIF2α phosphorylation by promoting the association between PKR and its activator PACT, with galectin-3 inhibiting this interaction; these regulatory events occur on damaged lysosomes. We further find that SG formation plays a crucial role in promoting cell survival upon lysosomal damage caused by factors such as SARS-CoV-2ORF3a, adenovirus, malarial pigment, proteopathic tau, or environmental hazards. Collectively, these data provide insights into the mechanism of SG formation upon lysosomal damage and implicate it in diseases associated with damaged lysosomes and SGs.
    Keywords:  ALG2-ALIX; Calcium-dependent Pathway; Lysosomal Damage; PACT-PKR-eIF2α; Stress Granules
    DOI:  https://doi.org/10.1038/s44318-024-00292-1
  20. mBio. 2024 Nov 13. e0232224
      In bacteria, if a ribosome translates an mRNA lacking a stop codon it becomes stalled at the 3' end of the message. These ribosomes must be rescued by trans-translation or the alternative rescue factors (ArfA or ArfB). However, mounting evidence suggests that the ribosome quality control (RQC) pathway may also rescue non-stop ribosomes. Here, we surveyed the conservation of ribosome rescue pathways in >15,000 bacterial genomes. We found that trans-translation is conserved in >97% of bacterial genomes, while the other rescue pathways are restricted to particular phyla. We did not detect the gene encoding RqcH, the major mediator of RQC, in Proteobacteria (Pseudomonadota). In all Proteobacteria investigated to date, trans-translation is essential in the absence of the Arf proteins. Therefore, we tested whether expression of RQC components from Bacillus subtilis could rescue viability in the absence of trans-translation and ArfA in Escherichia coli. We found that the RQC pathway indeed functions in E. coli and rescues the well-documented synthetic lethal phenotype of ∆ssrA∆arfA. Moreover, we show that the RQC pathway in B. subtilis is essential in the absence of trans-translation and ArfA, further supporting a role for the RQC pathway in the rescue of non-stop ribosomes. Finally, we report a strong co-occurrence between RqcH and the ribosome splitting factor MutS2, but present experimental evidence that there are likely additional ribosome splitting factors beyond MutS2 in B. subtilis. Altogether, our work supports a role for RQC in non-stop ribosome rescue and provides a broad survey of ribosome rescue pathways in diverse bacteria.IMPORTANCE: In bacteria, it is estimated that 2%-4% of all translation reactions terminate with the ribosome stalled on a damaged mRNA lacking a stop codon. Mechanisms that rescue these ribosomes are essential for viability. We determined the functional overlap between the ribosome quality control pathway and the classical non-stop rescue systems [alternative rescue factor (ArfA) and trans-translation] in a representative Firmicute and Proteobacterium, phyla that are evolutionarily distinct. Furthermore, we used a bioinformatics approach to examine the conservation and overlap of various ribosome rescue systems in >15,000 species throughout the bacterial domain. These results provide key insights into ribosome rescue in diverse phyla.
    Keywords:  RNA binding proteins; bacteria; molecular genetics; ribosomes; trans-translation; translation
    DOI:  https://doi.org/10.1128/mbio.02322-24
  21. Chembiochem. 2024 Nov 07. e202400617
      Cells utilize ubiquitin as a posttranslational protein modifier to convey various signals such as proteasomal degradation. The disfunction of ubiquitylation or following proteasomal degradation can give rise to the accumulation and aggregation of improperly ubquitylated proteins, which is known to be a general causation of many neurodegenerative diseases. Thus, the characterization of substrate peptide sequences of E3 ligases is crucial in biological and pharmaceutical sciences. In this study, we developed a novel high-throughput screening system for substrate peptide sequences of E3 ligases using a cDNA display method, which enables covalent conjugation between peptide sequences and their corresponding cDNA sequences. First, we focused on the MDM2 E3 ligase and its known peptide substrate as a model to establish the screening method, and confirmed that cDNA display method was compatible with in vitro ubiquitylation. Then, we demonstrated identification of MDM2 substrate sequences from random libraries to identify a novel motif (VKFTGGQLA). Bioinformatics analysis of the hit sequences was performed to gain insight about endogenous substrate proteins.
    Keywords:  E3 ligase; cDNA display; degron; high-throughput screening; ubiquitylation
    DOI:  https://doi.org/10.1002/cbic.202400617
  22. Nucleus. 2024 Dec;15(1): 2423660
      The Nuclear envelope (NE) is frequently challenged by mechanical stimuli involving cells passing through a tight space and such stress is known as "NE stress." Various factors that cooperate to repair the NE have been identified, including endosomal sorting complex required for transport-III (ESCRT-III). Recently, vacuolar protein sorting 4 homolog B (VPS4B) has been reported to modulate the recycling of ESCRT-III during NE repair, but the regulatory mechanism remains unclear. Our previous study revealed that U251MG cells, derived from the glioblastoma (GBM), exhibited nuclear deformation followed by DNA damage upon mechanical NE stress while these phenotypes were not observed in U87MG, another GBM-derived cell line. Here, we found that VPS4B expression was lower in U251MG than in U87MG. Our functional analysis demonstrated that insufficient VPS4B triggers an inadequate response to NE stress and that VPS4B regulates the dynamics of ESCRT-III, uncovering the mechanism underlying the NE stress response in GBM.
    Keywords:  ESCRT-III; VPS4B; glioblastoma; nuclear envelope; nuclear envelope stress
    DOI:  https://doi.org/10.1080/19491034.2024.2423660
  23. Proc Natl Acad Sci U S A. 2024 Nov 19. 121(47): e2403330121
      The concept of targeted protein degradation is at the forefront of modern drug discovery, which aims to eliminate disease-causing proteins using specific molecules. In this paper, we explored the idea to design protein degraders based on the section of ligands that cause protein destabilization, hence that facilitate the cellular breakdown of the target. Our studies present covalent agents targeting Pin1, a cis-trans prolyl isomerase that plays a crucial role in tumorigenesis. Our design strategy entailed iterative optimizations of agents for potency and Pin1 destabilization in vitro. Biophysical and cellular studies suggest that the agents may act like molecular crowbars, displacing protein-stabilizing interactions that open the structure for recognition by the proteasome degradation machinery. This approach resulted in a series of potent and effective Pin1 degraders with potential applications in target validation and in therapeutic development. We propose that our design strategy can identify molecular degraders without engineering bifunctional agents that artificially create interactions between a disease-causing protein and a ubiquitin ligase.
    Keywords:  PIN1; drug discovery; molecular crowbars; protein degradation
    DOI:  https://doi.org/10.1073/pnas.2403330121
  24. Nat Commun. 2024 Nov 08. 15(1): 9681
      AAA+ proteolytic machines unfold proteins before degrading them. Here, we present cryoEM structures of ClpXP-substrate complexes that reveal a postulated but heretofore unseen intermediate in substrate unfolding/degradation. A ClpX hexamer draws natively folded substrates tightly against its axial channel via interactions with a fused C-terminal degron tail and ClpX-RKH loops that flexibly conform to the globular substrate. The specific ClpX-substrate contacts observed vary depending on the substrate degron and affinity tags, helping to explain ClpXP's ability to unfold/degrade a wide array of different cellular substrates. Some ClpX contacts with native substrates are enabled by upward movement of the seam subunit in the AAA+ spiral, a motion coupled to a rearrangement of contacts between the ClpX unfoldase and ClpP peptidase. Our structures additionally highlight ClpX's ability to translocate a diverse array of substrate topologies, including the co-translocation of two polypeptide chains.
    DOI:  https://doi.org/10.1038/s41467-024-53681-9
  25. Bioorg Chem. 2024 Nov 04. pii: S0045-2068(24)00848-4. [Epub ahead of print]153 107943
      Under hypoxic conditions in tumor cells, HIF-1α is unable to bind to VHL E3 ligase due to the blocked hydroxylation reaction, resulting in impaired degradation and intracellular accumulation. Mounting evidences show a close association between HIF-1α overexpression and drug resistance, treatment failure, and increased mortality. To address HIF-1α overexpression, we innovatively introduced an E3 ligase ligand to the HIF-1α inhibitor IDF-11774 using the PROTACs strategy, aiming to reactivate the degradative pathway impeded under hypoxia, and thereby achieve the degradation of HIF-1α protein under hypoxia. Western blotting analyses demonstrated that most of our designed PROTACs effectively degraded HIF-1α. Among these, compounds C3 and V2 exhibited excellent anti-proliferation activity on MDA-MB-231 cells with IC50 values of 48.98 μM and 7.54 μM, respectively. Both compounds induced protein degradation in a concentration-dependent manner, achieving degradation rates up to 80 %. Additionally, this degradation was inhibited by the proteasome inhibitor MG132. As a part of the ongoing effort to develop HIF-1 inhibitors, targeting the degradation of HIF-1α may offer an effective treatment strategy against solid tumors.
    Keywords:  E3 ligase; HIF-1α; IDF-11774; PROTACs; Reactivated degradation
    DOI:  https://doi.org/10.1016/j.bioorg.2024.107943
  26. FEBS Lett. 2024 Nov 14.
      HUWE1, a HECT E3 ligase, is critical for processes like protein degradation and tumor development. Contrary to previous findings which suggested minimal non-covalent interactions between the HUWE1 HECT domain and ubiquitin, we identified a non-covalent interaction between the HUWE1 HECT N-lobe and ubiquitin using NMR spectroscopy, revealing a conserved ubiquitin-binding mode shared across HECT E3 ligases. Molecular dynamics simulations not only confirmed the stability of this interaction but also uncovered conformational changes in key residues, which likely influence binding affinity. Additionally, we highlighted the roles of both conserved and unique residues in ubiquitin binding. These findings advance our understanding of the interactions between the HUWE1 HECT domain and ubiquitin, and highlight potential targets for therapeutic intervention in the ubiquitin-proteasome pathway.
    Keywords:  HECT N‐lobe; HUWE1; NMR; molecular dynamics simulations; ubiquitin
    DOI:  https://doi.org/10.1002/1873-3468.15050
  27. Cell. 2024 Nov 14. pii: S0092-8674(24)01207-8. [Epub ahead of print]187(23): 6784-6784.e1
      Targeted protein degradation strategies leverage endogenous cellular degradation machinery to selectively eliminate a protein of interest. Emerging technologies are opening avenues in drug discovery and functional characterization of intracellular, membrane, and extracellular proteins. To view this SnapShot, open or download the PDF.
    DOI:  https://doi.org/10.1016/j.cell.2024.10.025
  28. Proc Natl Acad Sci U S A. 2024 Nov 19. 121(47): e2402818121
      Axonemal dynein, the macromolecular machine that powers ciliary motility, assembles in the cytosol with the help of dynein axonemal assembly factors (DNAAFs). These DNAAFs localize in cytosolic foci thought to form via liquid-liquid phase separation. However, the functional significance of DNAAF foci formation and how the production and assembly of multiple components are so efficiently coordinated, at such enormous scale, remain unclear. Here, we unveil an axonemal dynein production and assembly hub enriched with translating heavy chains (HCs) and DNAAFs. We show that mRNAs encoding interacting HCs of outer dynein arms colocalize in cytosolic foci, along with nascent HCs. The formation of these mRNA foci and their colocalization relies on HC translation. We observe that a previously identified DNAAF assembly, containing the DNAAF Lrrc6 and cochaperones Ruvbl1 and Ruvbl2, colocalizes with these HC foci, and is also dependent on HC translation. We additionally show that Ruvbl1 is required for the recruitment of Lrrc6 into the HC foci and that both proteins function cotranslationally. We propose that these DNAAF foci are anchored by stable interactions between translating HCs, ribosomes, and encoding mRNAs, followed by cotranslational molecular condensation of cochaperones and assembly factors, providing a potential mechanism that coordinates HC translation, folding, and assembly at scale.
    Keywords:  Ruvbl1/Ruvbl2; cilia; co-translational assembly; dynein axonemal assembly factors; primary ciliary dyskinesia
    DOI:  https://doi.org/10.1073/pnas.2402818121
  29. Sci Rep. 2024 11 12. 14(1): 27658
      Living systems rely on coordinated molecular interactions, especially those related to gene expression and protein activity. The Unfolded Protein Response is a crucial mechanism in eukaryotic cells, activated when unfolded proteins exceed a critical threshold. It maintains cell homeostasis by enhancing protein folding, initiating quality control, and activating degradation pathways when damage is irreversible. This response functions as a dynamic signaling network, with proteins as nodes and their interactions as edges. We analyze these protein-protein networks across different organisms to understand their intricate intra-cellular interactions and behaviors. In this work, analyzing twelve organisms, we assess how fundamental measures in network theory can individuate seed proteins and specific pathways across organisms. We employ network robustness to evaluate and compare the strength of the investigated protein-protein interaction networks, and the structural controllability of complex networks to find and compare the sets of driver nodes necessary to control the overall networks. We find that network measures are related to phylogenetics, and advanced network methods can identify main pathways of significance in the complete Unfolded Protein Response mechanism.
    Keywords:  Complex networks; Endoplasmic reticulum stress; Network analysis; Protein-protein interactions
    DOI:  https://doi.org/10.1038/s41598-024-79086-8
  30. Mol Cell. 2024 Nov 05. pii: S1097-2765(24)00862-1. [Epub ahead of print]
      Lipophagy is a ubiquitous mechanism for degradation of lipid droplets (LDs) in lysosomes. Autophagy receptors selectively target organelles for lysosomal degradation. The selective receptor for lipophagy remains elusive. Using mouse liver phosphoproteomics and human liver transcriptomics, we identify vacuolar-protein-sorting-associated protein 4A (VPS4A), a member of a large family AAA+ ATPases, as a selective receptor for lipophagy. We show that phosphorylation of VPS4A on Ser95,97 and its localization to LDs in response to fasting drives lipophagy. Imaging/three-dimensional (3D) reconstruction and biochemical analyses reveal the concomitant degradation of VPS4A and LDs in lysosomes in an autophagy-gene-7-sensitive manner. Either silencing VPS4A or targeting VPS4AS95,S97 phosphorylation or VPS4A binding to LDs or LC3 blocks lipophagy without affecting other forms of selective autophagy. Finally, VPS4A levels and markers of lipophagy are markedly reduced in human steatotic livers-revealing a fundamental role of VPS4A as the lipophagy receptor in mice and humans.
    Keywords:  MASLD; VPS4A; autophagy; human; lipid droplet; lipophagy; liver; lysosome; phosphorylation; receptor
    DOI:  https://doi.org/10.1016/j.molcel.2024.10.022
  31. Elife. 2024 Nov 07. pii: RP93232. [Epub ahead of print]12
      Plants distribute many nutrients to chloroplasts during leaf development and maturation. When leaves senesce or experience sugar starvation, the autophagy machinery degrades chloroplast proteins to facilitate efficient nutrient reuse. Here, we report on the intracellular dynamics of an autophagy pathway responsible for piecemeal degradation of chloroplast components. Through live-cell monitoring of chloroplast morphology, we observed the formation of chloroplast budding structures in sugar-starved leaves. These buds were then released and incorporated into the vacuolar lumen as an autophagic cargo termed a Rubisco-containing body. The budding structures did not accumulate in mutants of core autophagy machinery, suggesting that autophagosome creation is required for forming chloroplast buds. Simultaneous tracking of chloroplast morphology and autophagosome development revealed that the isolation membranes of autophagosomes interact closely with part of the chloroplast surface before forming chloroplast buds. Chloroplasts then protrude at the site associated with the isolation membranes, which divide synchronously with autophagosome maturation. This autophagy-related division does not require DYNAMIN-RELATED PROTEIN 5B, which constitutes the division ring for chloroplast proliferation in growing leaves. An unidentified division machinery may thus fragment chloroplasts for degradation in coordination with the development of the chloroplast-associated isolation membrane.
    Keywords:  A. thaliana; autophagy; cell biology; chloroplast; plant; plant biology
    DOI:  https://doi.org/10.7554/eLife.93232
  32. Chemistry. 2024 Nov 14. e202403676
      The ubiquitous molecular chaperone Heat shock protein 90 (Hsp90) is pivotal in many cellular processes through folding of client proteins under stressed and normal conditions. Despite intensive research on its function as a chaperone, the influence of posttranslational modifications on Hsp90 (the 'chaperone code'), and its interactions with co-chaperones and client proteins, still remains to be elucidated. The C-terminal domain (CTD) of Hsp90 is essential for formation of the active homodimer state of Hsp90 and contains recognition sites for co-chaperones and client proteins. Here we used expressed protein selenoester ligation to introduce site-selective phosphorylations in the Hsp90 CTD, while preserving the native amino acid sequence. The two phosphorylations do not affect the overall secondary structure, but in combination, slightly decrease the thermal stability of the CTD. The Hsp90 CTD functions as a chaperone in decreasing aggregation of model client proteins, but the C-terminal phosphorylations do not significantly alter the anti-aggregation activity for these clients. The optimization of expressed protein selenoester ligation to carry out several steps in one pot provides an efficient route to access site-specifically modified Hsp90 CTD variants, allowing the generation of Hsp90 variants with site-specific PTMs to decipher the chaperone code.
    Keywords:  Chaperones; Expressed Protein Selenoester Ligation; Hsp90; Posttranslational modifications; Protein synthesis
    DOI:  https://doi.org/10.1002/chem.202403676
  33. EMBO J. 2024 Nov 12.
      Terminal oligopyrimidine motif-containing mRNAs (TOPs) encode all ribosomal proteins in mammals and are regulated to tune ribosome synthesis to cell state. Previous studies have implicated LARP1 in 40S- or 80S-ribosome complexes that are thought to repress and stabilize TOPs. However, a molecular understanding of how LARP1 and TOPs interact with these ribosome complexes is lacking. Here, we show that LARP1 directly binds non-translating ribosomal subunits. Cryo-EM structures reveal a previously uncharacterized domain of LARP1 bound to and occluding the mRNA channel of the 40S subunit. Increased availability of free ribosomal subunits downstream of various stresses promote 60S joining at the same interface to form LARP1-80S complexes. Simultaneously, LARP1 engages the TOP via its previously characterized La/PAM2 and DM15 domains. Contrary to expectations, ribosome binding within these complexes is not required for LARP1-mediated TOP repression or stabilization, two canonical LARP1 functions. Together, this work provides molecular insight into how LARP1 directly binds ribosomal subunits and challenges existing models describing the function of repressed LARP1-40S/80S-TOP complexes.
    Keywords:  Cryo-EM; LARP1; Ribosome; TOP mRNA; Translation
    DOI:  https://doi.org/10.1038/s44318-024-00294-z
  34. ACS Chem Biol. 2024 Nov 12.
      Inositol-requiring enzyme 1α (IRE1α) is a sensor of endoplasmic reticulum (ER) stress and drives ER stress response pathways. Activated IRE1α exhibits RNase activity and cleaves mRNA encoding X-box binding protein 1, a transcription factor that induces the expression of genes that maintain ER proteostasis for cell survival. Previously, we showed that IRE1α undergoes S-nitrosylation, a post-translational modification induced by nitric oxide (NO), resulting in reduced RNase activity. Therefore, S-nitrosylation of IRE1α compromises the response to ER stress, making cells more vulnerable. We conducted virtual screening and cell-based validation experiments to identify compounds that inhibit the S-nitrosylation of IRE1α by targeting nitrosylated cysteine residues. We ultimately identified a compound (1ACTA) that selectively inhibits the S-nitrosylation of IRE1α and prevents the NO-induced reduction of RNase activity. Furthermore, 1ACTA reduces the rate of NO-induced cell death. Our research identified S-nitrosylation as a novel target for drug development for IRE1α and provides a suitable screening strategy.
    DOI:  https://doi.org/10.1021/acschembio.4c00403
  35. Nat Commun. 2024 Nov 07. 15(1): 9629
      Structure-based machine learning algorithms have been utilized to predict the properties of protein-protein interaction (PPI) complexes, such as binding affinity, which is critical for understanding biological mechanisms and disease treatments. While most existing algorithms represent PPI complex graph structures at the atom-scale or residue-scale, these representations can be computationally expensive or may not sufficiently integrate finer chemical-plausible interaction details for improving predictions. Here, we introduce MCGLPPI, a geometric representation learning framework that combines graph neural networks (GNNs) with MARTINI molecular coarse-grained (CG) models to predict PPI overall properties accurately and efficiently. Extensive experiments on three types of downstream PPI property prediction tasks demonstrate that at the CG-scale, MCGLPPI achieves competitive performance compared with the counterparts at the atom- and residue-scale, but with only a third of computational resource consumption. Furthermore, CG-scale pre-training on protein domain-domain interaction structures enhances its predictive capabilities for PPI tasks. MCGLPPI offers an effective and efficient solution for PPI overall property predictions, serving as a promising tool for the large-scale analysis of biomolecular interactions.
    DOI:  https://doi.org/10.1038/s41467-024-53583-w
  36. FASEB J. 2024 Nov 15. 38(21): e70162
      Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 and PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), which are required for the regulation of the renal tubular diameter. Loss of polycystin function results in cyst formation. Atypical forms of ADPKD are caused by mutations in genes encoding endoplasmic reticulum (ER)-resident proteins through mechanisms that are not well understood. Here, we investigate the function of DNAJB11, an ER co-chaperone associated with atypical ADPKD. We generated mouse models with constitutive and conditional Dnajb11 inactivation and Dnajb11-deficient renal epithelial cells to investigate the mechanism underlying autosomal dominant inheritance, the specific cell types driving cyst formation, and molecular mechanisms underlying DNAJB11-dependent polycystic kidney disease. We show that biallelic loss of Dnajb11 causes cystic kidney disease and fibrosis, mirroring human disease characteristics. In contrast to classical ADPKD, cysts predominantly originate from proximal tubules. Cyst formation begins in utero and the timing of Dnajb11 inactivation strongly influences disease severity. Furthermore, we identify impaired PC1 cleavage as a potential mechanism underlying DNAJB11-dependent cyst formation. Proteomic analysis of Dnajb11- and Pkd1-deficient cells reveals common and distinct pathways and dysregulated proteins, providing a foundation to better understand phenotypic differences between different forms of ADPKD.
    DOI:  https://doi.org/10.1096/fj.202401763R
  37. Bio Protoc. 2024 Oct 20. 14(20): e5091
      Regulation of gene expression in response to various biological processes, including extracellular stimulation and environmental adaptation, requires nascent mRNA synthesis and translation. Simultaneous analysis of the coordinated regulation of dynamic mRNA synthesis and translation using the same experiment remains a major challenge in the field. Here, we describe a step-by-step protocol for the simultaneous measurement of transcription of nascent mRNA and its translation at the gene level during the acute unfolded protein response (UPR) in HEK293 cells by combining 4-thiouridine metabolic mRNA labeling with translational ribosome affinity purification (TRAP) using a monoclonal antibody against evolutionarily conserved ribosomal P-stalk proteins (P-TRAP). Since P-TRAP captures full-length RNAs bound to ribosomes, it is compatible with 3' mRNA-seq, which analyzes the uridine-rich 3' UTRs of polyadenylated RNAs, allowing robust quantification of T>C conversions. Our nascent P-TRAP (nP-TRAP) method, in which P-TRAP is combined with metabolic mRNA labeling, can serve as a simple and powerful tool to analyze the coordinated regulation of transcription and translation of individual genes in cultured cells. Key features • Simple and retriable analysis of nascent mRNA synthesis and its translation in cultured cells • Combination of 4-thiouridine metabolic RNA labeling with translating ribosome affinity purification (TRAP) • Ribosomal P-stalk-mediated TRAP (P-TRAP) allows single-step and efficient purification of non-tagged ribosomes and translated mRNAs.
    Keywords:  4-thiouridine; Deep sequencing; Metabolic RNA labeling; TRAP-seq; Transcription; Translating ribosome affinity purification; Translation
    DOI:  https://doi.org/10.21769/BioProtoc.5091
  38. Sci Adv. 2024 Nov 15. 10(46): eadp5689
      N6-methyladenosine (m6A)-modified mRNAs and their cytoplasmic reader YTHDFs are colocalized with stress granules (SGs) under stress conditions, but the interplay between m6A modification and SG stability remains unclear. Here, we presented a spatiotemporal m6A imaging system (SMIS) that can monitor the m6A modification and the translation of mRNAs with high specificity and sensitivity in a single live cell. SMIS showed that m6A-modified reporter mRNAs dynamically enriched into SGs under arsenite stress and gradually partitioned into the cytosol as SG disassembled. SMIS revealed that knockdown of YTHDF2 contributed to SG disassembly, resulting in the fast redistribution of mRNAs from SGs and rapid recovery of stalled translation. The mechanism is that YTHDF2 can regulate SG stability through the interaction with G3BP1 in m6A-modified RNA-dependent manner. Our results suggest a mechanism for the interplay between m6A modification and SG through YTHDF2 regulation.
    DOI:  https://doi.org/10.1126/sciadv.adp5689
  39. J Cell Biol. 2024 Dec 02. pii: e202408173. [Epub ahead of print]223(12):
      We identify BEACH domain-containing proteins (BDCPs) as novel membrane coat proteins involved in the sorting of transmembrane proteins (TMPs) on the trans-Golgi network and tubular sorting endosomes. The seven typical mammalian BDCPs share a predicted alpha-solenoid-beta propeller structure, suggesting they have a protocoatomer origin and function. We map the subcellular localization of seven BDCPs based on their dynamic colocalization with RAB and ARF small GTPases and identify five typical BDCPs on subdomains of dynamic tubular-vesicular compartments on the intersection of endocytic recycling and post-Golgi secretory pathways. We demonstrate that BDCPs interact directly with the cytosolic tails of selected TMPs and identify a subset of TMPs, whose trafficking to the plasma membrane is affected in cells lacking BDCP. We propose that the competitive binding of BDCPs and clathrin coat adaptors to the cytosolic tails of TMPs, followed by their clustering to distinct subdomains of secretory/recycling tubules function as a mechanism for sorting of TMPs in pleomorphic tubular-vesicular compartments that lack a clathrin coat.
    DOI:  https://doi.org/10.1083/jcb.202408173
  40. Cell Syst. 2024 Nov 06. pii: S2405-4712(24)00301-6. [Epub ahead of print]
      Secreted proteins play crucial roles in paracrine and endocrine signaling; however, identifying ligand-receptor interactions remains challenging. Here, we benchmarked AlphaFold2 (AF2) as a screening approach to identify extracellular ligands to single-pass transmembrane receptors. Key to the approach is the optimization of AF2 input and output for screening ligands against receptors to predict the most probable ligand-receptor interactions. The predictions were performed on ligand-receptor pairs not used for AF2 training. We demonstrate high discriminatory power and a success rate of close to 90% for known ligand-receptor pairs and 50% for a diverse set of experimentally validated interactions. Further, we show that screen accuracy does not correlate linearly with prediction of ligand-receptor interaction. These results demonstrate a proof of concept of a rapid and accurate screening platform to predict high-confidence cell-surface receptors for a diverse set of ligands by structural binding prediction, with potentially wide applicability for the understanding of cell-cell communication.
    Keywords:  AlphaFold; binding prediction; deorphanization; ligand-receptor interactions
    DOI:  https://doi.org/10.1016/j.cels.2024.10.004
  41. Cell. 2024 Nov 14. pii: S0092-8674(24)01253-4. [Epub ahead of print]187(23): 6501-6517
      The identification of individual protein-protein interactions (PPIs) began more than 40 years ago, using protein affinity chromatography and antibody co-immunoprecipitation. As new technologies emerged, analysis of PPIs increased to a genome-wide scale with the introduction of intracellular tagging methods, affinity purification (AP) followed by mass spectrometry (MS), and co-fractionation MS (CF-MS). Now, combining the resulting catalogs of interactions with complementary methods, including crosslinking MS (XL-MS) and cryogenic electron microscopy (cryo-EM), helps distinguish direct interactions from indirect ones within the same or between different protein complexes. These powerful approaches and the promise of artificial intelligence applications like AlphaFold herald a future where PPIs and protein complexes, including energy-driven protein machines, will be understood in exquisite detail, unlocking new insights in the contexts of both basic biology and disease.
    DOI:  https://doi.org/10.1016/j.cell.2024.10.038
  42. Elife. 2024 Nov 11. pii: RP95595. [Epub ahead of print]13
      In response to an ever-increasing demand of new small molecules therapeutics, numerous chemical and genetic tools have been developed to interrogate compound mechanism of action. Owing to its ability to approximate compound-dependent changes in thermal stability, the proteome-wide thermal shift assay has emerged as a powerful tool in this arsenal. The most recent iterations have drastically improved the overall efficiency of these assays, providing an opportunity to screen compounds at a previously unprecedented rate. Taking advantage of this advance, we quantified more than one million thermal stability measurements in response to multiple classes of therapeutic and tool compounds (96 compounds in living cells and 70 compounds in lysates). When interrogating the dataset as a whole, approximately 80% of compounds (with quantifiable targets) caused a significant change in the thermal stability of an annotated target. There was also a wealth of evidence portending off-target engagement despite the extensive use of the compounds in the laboratory and/or clinic. Finally, the combined application of cell- and lysate-based assays, aided in the classification of primary (direct ligand binding) and secondary (indirect) changes in thermal stability. Overall, this study highlights the value of these assays in the drug development process by affording an unbiased and reliable assessment of compound mechanism of action.
    Keywords:  biochemistry; chemical biology; chemoproteomics; human; human cell lines; kinase inhibitors; proteomics; thermal proteome profiling
    DOI:  https://doi.org/10.7554/eLife.95595