NEMF-mediated CAT-tailing defines distinct branches of translocation-associated quality control.

Ribosome stalling during co-translational translocation at the endoplasmic reticulum (ER) causes translocon clogging and impairs ER protein biogenesis. Mammalian cells resolve translocon clogging vial a poorly characterized translocation-associated quality control (TAQC) process. Here, we combine genome-wide CRISPR screen with live cell imaging to dissect the molecular linchpin of TAQC. We show that substrates translated from mRNAs bearing a ribosome stalling poly(A) sequence are degraded by lysosomes and the proteasome, while substrates encoded by non-stop mRNAs are degraded by an unconventional ER-associated degradation (ERAD) mechanism involving ER-to-Golgi trafficking and KDEL-dependent substrate retrieval. The triaging diversity appears to result from the heterogeneity of NEMF-mediated CATylation, because a systematic characterization of representative CAT-tail mimetics establishes an AT-rich tail as a "degron" for ERAD, whereas an AG-rich tail can direct a secretory protein to the lysosome. Our study reveals an unexpected protein sorting function for CAT-tailing that safeguards ER protein biogenesis.

Proximity labeling expansion microscopy (PL-ExM) evaluates interactome labeling techniques.

Understanding protein-protein interactions (PPIs) through proximity labeling has revolutionized our comprehension of cellular mechanisms and pathology. Various proximity labeling techniques, such as HRP, APEX, BioID, TurboID, and µMap, have been widely used to biotinylate PPIs or organelles for proteomic profiling. However, the variability in labeling precision and efficiency of these techniques often results in limited reproducibility in proteomic detection. We address this persistent challenge by introducing proximity labeling expansion microscopy (PL-ExM), a super-resolution imaging technique that combines expansion microscopy with proximity labeling techniques. PL-ExM enabled up to 17 nm resolution with microscopes widely available, providing visual comparison of the labeling precision, efficiency, and false positives of different proximity labeling methods. Our mass spectrometry proteomic results confirmed that PL-ExM imaging is reliable in guiding the selection of proximity labeling techniques and interpreting the proteomic results with new spatial information.

Trioxane-based MS-cleavable Cross-linking Mass Spectrometry for Profiling Multimeric Interactions of Cellular Networks.

Cross-linking mass spectrometry (XL-MS) is a powerful technology for mapping protein-protein interactions (PPIs) at the systems-level. By covalently connecting pairs of proximal residues, cross-linking reagents provide distance restraints to infer protein conformations and interaction interfaces. While binary cross-links have been remarkably informative, multimeric cross-links can offer enhanced spatial resolution to facilitate the characterization of dynamic and heterogeneous protein complexes. However, the identification of multimeric cross-links remains extremely challenging due to fragmentation complexity and the vast expansion of database search space. Here, we present a novel trioxane-based MS-cleavable homotrifunctional cross-linker TSTO, which can target three proximal lysine residues simultaneously. Owing to its unique structure and MS-cleavability, TSTO enables fast and unambiguous identification of cross-linked peptides using LC-MS n analysis. Importantly, we have demonstrated that the TSTO-based XL-MS platform is effective for mapping PPIs of protein complexes and cellular networks. The trimeric interactions captured by TSTO have uncovered new structural details that cannot be easily revealed by existing reagents, allowing in-depth description of PPIs to facilitate structural modeling. This development not only advances XL-MS technologies for global PPI profiling from living cells, but also offers a new direction for creating multifunctional MS-cleavable cross-linkers to further push structural systems biology forward in the future.

Huntingtin contains an ubiquitin-binding domain and regulates lysosomal targeting of mitochondrial and RNA-binding proteins.

Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.

Cadmium binding by the F-box domain induces p97-mediated SCF complex disassembly to activate stress response programs.

The F-box domain is a highly conserved structural motif that defines the largest class of ubiquitin ligases, Skp1/Cullin1/F-box protein (SCF) complexes. The only known function of the F-box motif is to form the protein interaction surface with Skp1. Here we show that the F-box domain can function as an environmental sensor. We demonstrate that the F-box domain of Met30 is a cadmium sensor that blocks the activity of the SCFMet30 ubiquitin ligase during cadmium stress. Several highly conserved cysteine residues within the Met30 F-box contribute to binding of cadmium with a KD of 8 µM. Binding induces a conformational change that allows for Met30 autoubiquitylation, which in turn leads to recruitment of the segregase Cdc48/p97/VCP followed by active SCFMet30 disassembly. The resulting inactivation of SCFMet30 protects cells from cadmium stress. Our results show that F-box domains participate in regulation of SCF ligases beyond formation of the Skp1 binding interface.

Mono-UFMylation promotes misfolding-associated secretion of α-synuclein.

Stressed cells secret misfolded proteins lacking signaling sequence via an unconventional protein secretion (UcPS) pathway, but how misfolded proteins are targeted selectively in UcPS is unclear. Here, we report that misfolded UcPS clients are subject to modification by a ubiquitin-like protein named ubiquitin-fold modifier 1 (UFM1). Using α-synuclein (α-Syn) as a UcPS model, we show that mutating the UFMylation sites in α-Syn or genetic inhibition of the UFMylation system mitigates α-Syn secretion, whereas overexpression of UFBP1, a component of the endoplasmic reticulum-associated UFMylation ligase complex, augments α-Syn secretion in mammalian cells and in model organisms. UFM1 itself is cosecreted with α-Syn, and the serum UFM1 level correlates with that of α-Syn. Because UFM1 can be directly recognized by ubiquitin specific peptidase 19 (USP19), a previously established UcPS stimulator known to associate with several chaperoning activities, UFMylation might facilitate substrate engagement by USP19, allowing stringent and regulated selection of misfolded proteins for secretion and proteotoxic stress alleviation.

DSBSO-Based XL-MS Analysis of Breast Cancer PDX Tissues to Delineate Protein Interaction Network in Clinical Samples.

Protein-protein interactions (PPIs) are fundamental to understanding biological systems as protein complexes are the active molecular modules critical for carrying out cellular functions. Dysfunctional PPIs have been associated with various diseases including cancer. Systems-wide PPI analysis not only sheds light on pathological mechanisms, but also represents a paradigm in identifying potential therapeutic targets. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for defining endogenous PPIs of cellular networks. While proteome-wide studies have been performed in cell lysates, intact cells and tissues, applications of XL-MS in clinical samples have not been reported. In this study, we adopted a DSBSO-based in vivo XL-MS platform to map interaction landscapes from two breast cancer patient-derived xenograft (PDX) models. As a result, we have generated a PDX interaction network comprising 2,557 human proteins and identified interactions unique to breast cancer subtypes. Interestingly, most of the observed differences in PPIs correlated well with protein abundance changes determined by TMT-based proteome quantitation. Collectively, this work has demonstrated the feasibility of XL-MS analysis in clinical samples, and established an analytical workflow for tissue cross-linking that can be generalized for mapping PPIs from patient samples in the future to dissect disease-relevant cellular networks.

Proximity Labeling Expansion Microscopy (PL-ExM) resolves structure of the interactome.

Elucidating the spatial relationships within the protein interactome is pivotal to understanding the organization and regulation of protein-protein interactions. However, capturing the 3D architecture of the interactome presents a dual challenge: precise interactome labeling and super-resolution imaging. To bridge this gap, we present the Proximity Labeling Expansion Microscopy (PL-ExM). This innovation combines proximity labeling (PL) to spatially biotinylate interacting proteins with expansion microscopy (ExM) to increase imaging resolution by physically enlarging cells. PL-ExM unveils intricate details of the 3D interactome's spatial layout in cells using standard microscopes, including confocal and Airyscan. Multiplexing PL-ExM imaging was achieved by pairing the PL with immunofluorescence staining. These multicolor images directly visualize how interactome structures position specific proteins in the protein-protein interaction network. Furthermore, PL-ExM stands out as an assessment method to gauge the labeling radius and efficiency of different PL techniques. The accuracy of PL-ExM is validated by our proteomic results from PL mass spectrometry. Thus, PL-ExM is an accessible solution for 3D mapping of the interactome structure and an accurate tool to access PL quality.

Structural surfaceomics reveals an AML-specific conformation of integrin ß2 as a CAR T cellular therapy target.

Safely expanding indications for cellular therapies has been challenging given a lack of highly cancer-specific surface markers. Here we explore the hypothesis that tumor cells express cancer-specific surface protein conformations that are invisible to standard target discovery pipelines evaluating gene or protein expression, and these conformations can be identified and immunotherapeutically targeted. We term this strategy integrating cross-linking mass spectrometry with glycoprotein surface capture 'structural surfaceomics'. As a proof of principle, we apply this technology to acute myeloid leukemia (AML), a hematologic malignancy with dismal outcomes and no known optimal immunotherapy target. We identify the activated conformation of integrin ß2 as a structurally defined, widely expressed AML-specific target. We develop and characterize recombinant antibodies to this protein conformation and show that chimeric antigen receptor T cells eliminate AML cells and patient-derived xenografts without notable toxicity toward normal hematopoietic cells. Our findings validate an AML conformation-specific target antigen and demonstrate a tool kit for applying these strategies more broadly.

The Hippo pathway noncanonically drives autophagy and cell survival in response to energy stress.

The Hippo pathway is known for its crucial involvement in development, regeneration, organ size control, and cancer. While energy stress is known to activate the Hippo pathway and inhibit its effector YAP, the precise role of the Hippo pathway in energy stress response remains unclear. Here, we report a YAP-independent function of the Hippo pathway in facilitating autophagy and cell survival in response to energy stress, a process mediated by its upstream components MAP4K2 and STRIPAK. Mechanistically, energy stress disrupts the MAP4K2-STRIPAK association, leading to the activation of MAP4K2. Subsequently, MAP4K2 phosphorylates ATG8-family member LC3, thereby facilitating autophagic flux. MAP4K2 is highly expressed in head and neck cancer, and its mediated autophagy is required for head and neck tumor growth in mice. Altogether, our study unveils a noncanonical role of the Hippo pathway in energy stress response, shedding light on this key growth-related pathway in tissue homeostasis and cancer.

New advances in cross-linking mass spectrometry toward structural systems biology.

Elucidating protein-protein interaction (PPI) networks and their structural features within cells is central to understanding fundamental biology and associations of cell phenotypes with human pathologies. Owing to technological advancements during the last decade, cross-linking mass spectrometry (XL-MS) has become an enabling technology for delineating interaction landscapes of proteomes as they exist in living systems. XL-MS is unique due to its capability to simultaneously capture PPIs from native environments and uncover interaction contacts though identification of cross-linked peptides, thereby permitting the determination of both identity and connectivity of PPIs in cells. In combination with high resolution structural tools such as cryo-electron microscopy and AI-assisted prediction, XL-MS has contributed significantly to elucidating architectures of large protein assemblies. This review highlights the latest developments in XL-MS technologies and their applications in proteome-wide analysis to advance structural systems biology.

Structural basis of gRNA stabilization and mRNA recognition in trypanosomal RNA editing.

In Trypanosoma brucei, the editosome, composed of RNA-editing substrate-binding complex (RESC) and RNA-editing catalytic complex (RECC), orchestrates guide RNA (gRNA)-programmed editing to recode cryptic mitochondrial transcripts into messenger RNAs (mRNAs). The mechanism of information transfer from gRNA to mRNA is unclear owing to a lack of high-resolution structures for these complexes. With cryo-electron microscopy and functional studies, we have captured gRNA-stabilizing RESC-A and gRNA-mRNA-binding RESC-B and RESC-C particles. RESC-A sequesters gRNA termini, thus promoting hairpin formation and blocking mRNA access. The conversion of RESC-A into RESC-B or -C unfolds gRNA and allows mRNA selection. The ensuing gRNA-mRNA duplex protrudes from RESC-B, likely exposing editing sites to RECC-catalyzed cleavage, uridine insertion or deletion, and ligation. Our work reveals a remodeling event facilitating gRNA-mRNA hybridization and assembly of a macromolecular substrate for the editosome's catalytic modality.

Adolescent exposure to low-dose THC disrupts energy balance and adipose organ homeostasis in adulthood.

One of cannabis' most iconic effects is the stimulation of hedonic high-calorie eating-the "munchies"-yet habitual cannabis users are, on average, leaner than non-users. We asked whether this phenotype might result from lasting changes in energy balance established during adolescence, when use of the drug often begins. We found that daily low-dose administration of cannabis' intoxicating constituent, Δ9-tetrahydrocannabinol (THC), to adolescent male mice causes an adult metabolic phenotype characterized by reduced fat mass, increased lean mass and utilization of fat as fuel, partial resistance to diet-induced obesity and dyslipidemia, enhanced thermogenesis, and impaired cold- and ß-adrenergic receptor-stimulated lipolysis. Further analyses revealed that this phenotype is associated with molecular anomalies in the adipose organ, including ectopic overexpression of muscle-associated proteins and heightened anabolic processing. Thus, adolescent exposure to THC may promote an enduring "pseudo-lean" state that superficially resembles healthy leanness but might in fact be rooted in adipose organ dysfunction.

Exploring an Alternative Cysteine-Reactive Chemistry to Enable Proteome-Wide PPI Analysis by Cross-Linking Mass Spectrometry.

The development of MS-cleavable cross-linking mass spectrometry (XL-MS) has enabled the effective capture and identification of endogenous protein-protein interactions (PPIs) and their residue contacts at the global scale without cell engineering. So far, only lysine-reactive cross-linkers have been successfully applied for proteome-wide PPI profiling. However, lysine cross-linkers alone cannot uncover the complete PPI map in cells. Previously, we have developed a maleimide-based cysteine-reactive MS-cleavable cross-linker (bismaleimide sulfoxide (BMSO)) that is effective for mapping PPIs of protein complexes to yield interaction contacts complementary to lysine-reactive reagents. While successful, the hydrolysis and limited selectivity of maleimides at physiological pH make their applications in proteome-wide XL-MS challenging. To enable global PPI mapping, we have explored an alternative cysteine-labeling chemistry and thus designed and synthesized a sulfoxide-containing MS-cleavable haloacetamide-based cross-linker, Dibromoacetamide sulfoxide (DBrASO). Our results have demonstrated that DBrASO cross-linked peptides display the same fragmentation characteristics as other sulfoxide-containing MS-cleavable cross-linkers, permitting their unambiguous identification by MSn. In combination with a newly developed two-dimensional peptide fractionation method, we have successfully performed DBrASO-based XL-MS analysis of HEK293 cell lysates and demonstrated its capability to complement lysine-reactive reagents and expand PPI coverage at the systems-level.

Discovery, structure, and function of filamentous 3-methylcrotonyl-CoA carboxylase.

3-methylcrotonyl-CoA carboxylase (MCC) is a biotin-dependent mitochondrial enzyme necessary for leucine catabolism in most organisms. While the crystal structure of recombinant bacterial MCC has been characterized, the structure and potential polymerization of native MCC remain elusive. Here, we discovered that native MCC from Leishmania tarentolae (LtMCC) forms filaments, and determined the structures of different filament regions at 3.4, 3.9, and 7.3 Å resolution using cryoEM. α6ß6 LtMCCs assemble in a twisted-stacks architecture, manifesting as supramolecular rods up to 400 nm. Filamentous LtMCCs bind biotin non-covalently and lack coenzyme A. Filaments elongate by stacking α6ß6 LtMCCs onto the exterior α-trimer of the terminal LtMCC. This stacking immobilizes the biotin carboxylase domains, sequestering the enzyme in an inactive state. Our results support a new model for LtMCC catalysis, termed the dual-swinging-domains model, and cast new light on the function of polymerization in the carboxylase superfamily and beyond.

Discovery of compounds that reactivate p53 mutants in vitro and in vivo.

The tumor suppressor p53 is the most frequently mutated protein in human cancer. The majority of these mutations are missense mutations in the DNA binding domain of p53. Restoring p53 tumor suppressor function could have a major impact on the therapy for a wide range of cancers. Here we report a virtual screening approach that identified several small molecules with p53 reactivation activities. The UCI-LC0023 compound series was studied in detail and was shown to bind p53, induce a conformational change in mutant p53, restore the ability of p53 hotspot mutants to associate with chromatin, reestablish sequence-specific DNA binding of a p53 mutant in a reconstituted in vitro system, induce p53-dependent transcription programs, and prevent progression of tumors carrying mutant p53, but not p53null or p53WT alleles. Our study demonstrates feasibility of a computation-guided approach to identify small molecule corrector drugs for p53 hotspot mutations.

Structure and conformational dynamics of Clostridioides difficile toxin A.

Clostridioides difficile toxin A and B (TcdA and TcdB) are two major virulence factors responsible for diseases associated with C. difficile infection (CDI). Here, we report the 3.18-Å resolution crystal structure of a TcdA fragment (residues L843-T2481), which advances our understanding of the complete structure of TcdA holotoxin. Our structural analysis, together with complementary single molecule FRET and limited proteolysis studies, reveal that TcdA adopts a dynamic structure and its CROPs domain can sample a spectrum of open and closed conformations in a pH-dependent manner. Furthermore, a small globular subdomain (SGS) and the CROPs protect the pore-forming region of TcdA in the closed state at neutral pH, which could contribute to modulating the pH-dependent pore formation of TcdA. A rationally designed TcdA mutation that trapped the CROPs in the closed conformation showed drastically reduced cytotoxicity. Taken together, these studies shed new lights into the conformational dynamics of TcdA and its roles in TcdA intoxication.

Two-Dimensional Fractionation Method for Proteome-Wide Cross-Linking Mass Spectrometry Analysis.

Cross-linking mass spectrometry (XL-MS) is an emergent technology for studying protein-protein interactions (PPIs) and elucidating architectures of protein complexes. The development of various MS-cleavable cross-linkers has facilitated the identification of cross-linked peptides, enabling XL-MS studies at the systems level. However, the scope and depth of cellular networks revealed by current XL-MS technologies remain limited. Due to the inherently broad dynamic range and complexity of proteomes, interference from highly abundant proteins impedes the identification of low-abundance cross-linked peptides in complex samples. Thus, peptide enrichment prior to MS analysis is necessary to enhance cross-link identification for proteome-wide studies. Although chromatographic techniques including size exclusion (SEC) and strong cation exchange (SCX) have been successful in isolating cross-linked peptides, new fractionation methods are still needed to further improve the depth of PPI mapping. Here, we present a two-dimensional (2D) separation strategy by integrating peptide SEC with tip-based high pH reverse-phase (HpHt) fractionation to expand the coverage of proteome-wide XL-MS analyses. Combined with the MS-cleavable cross-linker DSSO, we have successfully mapped in vitro PPIs from HEK293 cell lysates with improved identification of cross-linked peptides compared to existing approaches. The method developed here is effective and can be generalized for cross-linking studies of complex samples.

Developing a Targeted Quantitative Strategy for Sulfoxide-Containing MS-Cleavable Cross-Linked Peptides to Probe Conformational Dynamics of Protein Complexes.

In recent years, cross-linking mass spectrometry (XL-MS) has made enormous strides as a technology for probing protein-protein interactions (PPIs) and elucidating architectures of multisubunit assemblies. To define conformational and interaction dynamics of protein complexes under different physiological conditions, various quantitative cross-linking mass spectrometry (QXL-MS) strategies based on stable isotope labeling have been developed. These QXL-MS approaches have effectively allowed comparative analysis of cross-links to determine their relative abundance changes at global scales. Although successful, it remains challenging to consistently obtain quantitative measurements on low-abundant cross-links. Therefore, targeted QXL-MS is needed to enable MS "Western" analysis of cross-links to enhance sensitivity and reliability in quantitation. To this end, we have established a robust parallel reaction monitoring (PRM)-based targeted QXL-MS platform using sulfoxide-containing MS-cleavable cross-linker disuccinimidyl sulfoxide (DSSO), permitting label-free comparative analysis of selected cross-links across multiple samples. In addition, we have applied this methodology to study phosphorylation-dependent conformational dynamics of the human 26S proteasome. The PRM-based targeted QXL-MS analytical platform described here is applicable for all sulfoxide-containing MS-cleavable cross-linkers and can be directly adopted for comparative studies of protein-protein interactions in various cellular contexts.

The Hippo pathway kinases LATS1 and LATS2 attenuate cellular responses to heavy metals through phosphorylating MTF1.

Heavy metals are both integral parts of cells and environmental toxicants, and their deregulation is associated with severe cellular dysfunction and various diseases. Here we show that the Hippo pathway plays a critical role in regulating heavy metal homeostasis. Hippo signalling deficiency promotes the transcription of heavy metal response genes and protects cells from heavy metal-induced toxicity, a process independent of its classic downstream effectors YAP and TAZ. Mechanistically, the Hippo pathway kinase LATS phosphorylates and inhibits MTF1, an essential transcription factor in the heavy metal response, resulting in the loss of heavy metal response gene transcription and cellular protection. Moreover, LATS activity is inhibited following heavy metal treatment, where accumulated zinc directly binds and inhibits LATS. Together, our study reveals an interplay between the Hippo pathway and heavy metals, providing insights into this growth-related pathway in tissue homeostasis and stress response.