搜索结果：Molecular & cellular proteomics : MCP

Pathogenic variants in the CACNA1F gene are linked to congenital stationary night blindness type 2 though their specific molecular effects remain elusive. This study examines the retinal impact of two variants: a truncation (RX) and a gain-of-function (IT) to explore variant-specific retinal proteome changes. Electroretinography showed that RX primarily affects rod pathways, while IT disrupts both rod and cone signaling, consistent with morphological findings. Comprehensive quantitative proteomic analysis using mass spectrometry identified approximately 4000 proteins across wild-type control and mutant retinas, including also low-abundant membrane proteins. IT retinas exhibited widespread proteomic remodeling suggesting broad cellular responses and also compensatory molecular adaptations. In contrast, RX retinas displayed a more restricted profile. Similar to IT retinas, we found reduced Cav1.4 protein levels but without transcriptional downregulation in RX, alongside selective changes in synaptic proteins such as Erc1, Lrfn2, vGlut1, and Rab3a. These findings suggest selective molecular changes in synaptic organization and calcium-related pathways in RX retinas, offering insights into the mechanisms of Cav1.4 dysfunction in retinal disease. Deep proteomic analysis demonstrates how retinal cells reorganize their molecular architecture in response to calcium channel defects and highlights the utility of comprehensive proteomics to characterize adaptive cellular responses to genetic perturbations in retinal synaptic organization.

Extracellular vesicles (EVs) are central to intercellular communication and have gained attention as rich sources of molecular information in cancer research, but their molecular composition remains incompletely characterized. Protein glycosylation is a frequent post-translational modification; however, most EV studies focus on proteomics, whereas mapping glycosylation changes of proteins is still under-represented. To address this gap, we analyzed the proteomic, N-glycoproteomic, and chondroitin sulfate/dermatan sulfate (CS/DS) glycosaminoglycan (GAG) profiles of small EVs (sEVs) derived from A549 lung adenocarcinoma and BEAS-2B nontumorigenic epithelial cells. Principal component analysis and hierarchical clustering revealed that all three profiles strongly reflect sEV origin. Comparative proteomic analysis showed enrichment of proteins associated with cell cycle regulation, DNA repair, metabolism, and protein synthesis in A549 sEVs, whereas immune-related processes were enriched in BEAS-2B sEVs. Five differentially expressed CS proteoglycans were identified, highlighting the value of complementary GAG-level analysis. N-glycoproteomics revealed a shift from oligomannose to complex N-glycans in A549 sEVs. Prominent glycoproteins with multiple glycosylation sites included versican, galectin-3-binding protein, and laminins. CS/DS content increased 3.4-fold in A549 sEVs, whereas the ratio of the two monosulfated disaccharides changed twofold. These findings demonstrate the utility of N-glycoproteomics and GAG profiling for sensitively characterizing molecular differences between sEVs derived from different cell culture models, thereby providing a foundation for future EV biomarker studies.

Extracellular vesicles (EVs) have gained increasing attention with their intriguing biological functions and their molecular cargoes serving as potential biomarkers for various diseases, including cancers. A relatively lower abundance of EV proteins compared to cellular counterparts necessitates sensitive and accurate quantitative proteomic strategies. Multiplexed proteomics combined with data-independent acquisition (mDIA) has shown promise for improving sensitivity and quantification over traditional DDA and label-free methods. Despite this, mDIA pipelines that utilize various types of spectral libraries and search software suites have not been thoroughly evaluated with EV proteome samples. In this study, we aim to establish a robust mDIA pipeline based on dimethyl labeling for quantitative proteomics of EVs. EVs were isolated using the extracellular vesicle total recovery and purification (EVtrap) technique and processed directly through an on-bead one-pot sample preparation workflow to obtain digested peptides. We evaluated different mDIA pipelines, including library-free and library-based DIA on the timsTOF HT platform. Results showed that library-based DIA, with project-specific spectral libraries generated from StageTip-based fractionation, outperformed other pipelines in protein identification and quantification. We demonstrated for the first time EV proteome landscape changes caused by the IDH1 mutation and inhibitor treatment in intrahepatic cholangiocarcinoma, highlighting the utility of mDIA in EV-based biomarker discovery.

The spatial organization of the cellular proteome is vital for cellular physiology, as protein localization is closely linked to post-translational modifications, subcellular trafficking, and protein-protein interactions. Systematic profiling of these spatial features can greatly enhance our understanding of protein functions. Recent advances in enzyme-mediated proximity labeling (PL) techniques, such as TurboID and APEX2, have improved our ability to map subcellular proteomes in living cells. This review discusses emerging trends in PL methods, which now offer subcellular precision with multi-dimensional protein features, including post-translational modifications, trafficking, turnover, and interaction with other biomolecules. Additionally, new techniques such as photoactivatable PL (optoPL) and antibody-targeted PL (immunoPL) provide enhanced spatiotemporal control and allow for detailed subcellular proteome mapping without genetic manipulation.

Hydroxychloroquine (HCQ) and chloroquine have been utilized as antimalarial drugs for decades. Recently, these compounds were reported to inhibit various viruses utilizing the endosomal entry pathway. However, their direct molecular targets in host cells remain elusive. In this study, we developed a clickable photo-crosslinking probe in combination with proteomic approaches to identified cathepsin L (CTSL) as the binding target of HCQ. Extensive biochemical and in silico analyses were conducted to validate the HCQ-CTSL interactions. HCQ significantly inhibited the protease activity of CTSL and suppressed CTSL-dependent coronavirus entry in cells that support endosomal entry pathway. These findings not only reveal the underlying mechanism of how HCQ inhibits endosomal viral entry but also guide the rational use of HCQ against other emerging infectious agents.

Tumor-derived small extracellular vesicles (EVs) play a crucial role in modulating immune responses and shaping the tumor microenvironment; however, their functional impact on airway immunity in NSCLC remains largely unexplored. This study represents the first attempt to investigate the immunomodulatory and tumor-promoting effects of NSCLC-derived EVs in a human 3D bronchial airway model, which closely mimics the human lung microenvironment. EVs were isolated from the plasma and bronchoalveolar lavage fluid (BALF) of NSCLC patients and analyzed via nanoparticle tracking analysis (NTA) and high-resolution imaging flow cytometry. The lymphocyte compositions of the matched blood and BALF samples were profiled. To assess the functional effects of EVs, we employed a pioneering in vitro 3D airway coculture model that combines primary human airway epithelial cells and alveolar macrophages at the air‒liquid interface. Proteomic analysis of EV-treated cells and their secretome was performed to identify key molecular pathways underlying EV-driven immunomodulation. Surprisingly, no significant molecular differences were detected between EVs from cancerous (cBALF) and opposite (oBALF) lung compartments, despite a localized increase in regulatory T cells (Tregs) in the cBALF, suggesting regional immunosuppression. Plasma-derived EVs exhibited highly diverse, patient-specific molecular signatures but were not directly correlated with clinical or immune parameters. Functional studies of EVs with high and low surface molecular cargo in a 3D airway model revealed that both EV subgroups promoted monocyte/macrophage recruitment, angiogenesis, and epithelial-to-mesenchymal transition (EMT) via MCP-1 secretion and induced an immunosuppressive airway microenvironment, enhancing IL-10 production and shifting macrophages toward a tumor-promoting M2 phenotype. Proteomic analysis revealed distinct differentially expressed protein (DEP) profiles across epithelial and macrophage populations, ultimately resulting in protumorigenic and immunosuppressive outcomes. Notably, functional enrichment analysis of macrophages revealed that EV-driven M2 polarization occurred through the suppression of EGFR activity, a previously underrecognized mechanism that links EV-mediated immune suppression to lung cancer progression. This study provides the first functional evidence that NSCLC-derived EVs drive immune suppression and tumor-supportive changes in a human 3D airway model, closely mimicking in vivo lung conditions. The identification of EGFR suppression as a driver of macrophage polarization underscores the need to consider macrophage-specific EGFR regulation in anti-EGFR therapies to prevent unintended protumorigenic effects. These findings pave the way for future studies exploring EV cargo, such as miRNAs, as potential therapeutic targets in NSCLC.

Large macromolecular assemblies are integral to most cellular processes, making their identification and structural characterization an important strategy for advancing our understanding of protein functions. In this pilot study, we investigated large multiprotein assemblies from the cytoplasm of the slime mold Dictyostelium discoideum using shotgun electron microscopy, the combined application of mass spectrometry-based proteomics and cryo-EM to heterogenous mixtures of proteins. With its similarities in cell structure and behavior to mammalian cells, D. discoideum has long served as an invaluable model organism, particularly in the study of immune cell chemotaxis, phagocytosis, bacterial infection, and other processes. We subjected D. discoideum soluble protein complexes to two-step fractionation, performing size-exclusion chromatography followed by mixed-bed ion-exchange chromatography. Isolated fractions containing a subset of high molecular weight-scale protein assemblies were subsequently analyzed using mass spectrometry to identify the proteins and cryo-EM to characterize their structures. Mass spectrometry analysis revealed 179 unique proteins in the isolated fractions, then single-particle cryo-EM analysis generated distinct 2D projections of several visually distinctive protein assemblies, from which we successfully identified and reconstructed three major protein complexes: the 20S proteasome, the dihydrolipoyllysine-residue succinyltransferase (Odo2) of the mitochondrial 2-oxoglutarate dehydrogenase complex, and polyketide synthase 16 (Pks16), thought to be the primary fatty acid synthase of D. discoideum. Based on the Pks16 structure, the first of the 40 D. discoideum PKSs to be experimentally determined, models for the full set of D. discoideum PKSs were constructed with help from AlphaFold 3. Comparative analysis enabled structural characterization of their reaction chambers. Shotgun EM thus provides a view of proteins in their native or near-native biological conformations and scaling up this approach offers an effective route to characterize new structures of multiprotein assemblies directly from complex samples.

O-linked β-N-acetylglucosamine (O-GlcNAc) modification (i.e., O-GlcNAcylation) on proteins is an essential modification in physiology and pathology. Although O-GlcNAcylation is functionally critical, its analysis has been challenging. Despite the existence of a number of methods developed in the past years, which one(s) might have the best performance is largely unclear. To that end, we conducted a rigorous comparison of several cleavable bioorthogonal biotin-alkyne probes which showed promise for sensitive O-GlcNAc proteomics. In brief, we developed chemoenzymatic labeling/click chemistry-based analytical workflows for O-GlcNAc proteomics by utilizing four cleavable bioorthogonal probes, including photocleavabe-biotin-alkyne (PC-biotin-alkyne), dialkoxydiphenylsilane-biotin-alkyne (DADPS-biotin-alkyne); 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-biotin-alkyne (Dde-biotin-alkyne), and diazobenzene-biotin-alkyne (Diazo-biotin-alkyne). The analytical performance of these probes was evaluated with synthetic O-GlcNAc peptides and then benchmarked by using mouse brain lysates for O-GlcNAc proteomics. Besides providing valuable technical insights into O-GlcNAc proteomics methods, our work yielded an unprecedented O-GlcNAc proteome depth in the mouse brain. In total, 2906 O-GlcNAc sites were unambiguously assigned on 878 proteins. Among them, 1611 sites were newly identified, including 138 O-GlcNAcylated tyrosine residues. Our work will help guide the selection/development of O-GlcNAc proteomics methods for future studies, provide an invaluable resource for functional elucidation of protein O-GlcNAcylation in brain biology, and yield critical insights into tyrosine O-GlcNAcylation.

Tumor stiffening plays a pivotal role in cancer progression. Increased tumor stiffness, resulting from interactions between cancer cells and their surrounding microenvironment, alters the tumor's mechanical properties and significantly impacts cancer growth and metastasis, the primary cause of cancer-related death. Despite the importance of tumor stiffness, systematic studies exploring its effect on protein dysregulation are limited. In this study, focused on colorectal cancer, we show by in-depth proteomics that matrix stiffness significantly alters the expression of secreted proteins, while intracellular protein levels remain largely unaffected. Functional assays reveal that the changes observed by proteomics in the secretome, driven by matrix stiffness, enhance cell migration, angiogenesis, and matrix remodeling, which collectively would contribute to a more aggressive cancer phenotype in a real scenario. Our findings emphasize the critical role of matrix stiffness in driving colorectal cancer progression through changes in the secretome, offering valuable insights for the development of biomechanical cancer therapies.

Peripartum cardiomyopathy (PPCM) is a rare form of acute heart failure that develops in women toward the end of pregnancy or early postpartum. No effective, specific treatment for PPCM is available and heart transplantation or mechanical circulatory support may be required in severe cases where drug treatment for heart failure is insufficient. The mechanisms through which the disease progresses are not well understood, and despite similar clinical characteristics to dilated cardiomyopathy of other etiologies (nonperipartum cardiomyopathy; NPCM) it is not known how the molecular remodeling differs between these groups. We aimed to provide insight into the human PPCM heart using unbiased methodologies, and to use changes occurring within the heart tissue to facilitate biomarker discovery. We obtained heart tissue from female patients with end-stage disease receiving either heart transplantation or left ventricular assist device implantation, or from organ donors without heart disease as a control group. We performed deep proteomics, single nucleus transcriptomics and spatial transcriptomics, providing a comprehensive map of the molecular phenotype in advanced PPCM compared to both control and NPCM hearts. Consistent with similarities in the clinical phenotypes of PPCM and NPCM, we observed regulation of canonical markers of end-stage heart failure in both PPCM and NPCM hearts in comparison to controls. Among the changes specific to PPCM and that were consistently observed across multiple data types and cohorts was an upregulation of chymase and carboxypeptidase A3, consistent with mast cell proliferation/activation. Analysis of the proteome of peripheral blood serum from a larger cohort of patients with PPCM and controls showed that chymase was strongly predictive of cardiomyopathy in peripartum women. PPCM heart tissue is characterized by increased mast cell proteins chymase and carboxypeptidase A3. Chymase may have clinical utility as a biomarker for the diagnosis of cardiomyopathy in peripartum women.

Acute kidney injury (AKI), characterized by a rapid decline in renal function, has high mortality rates and frequently progresses to chronic kidney disease (CKD). A major contributor to AKI is ischemia-reperfusion injury (IRI). However, the global molecular changes underlying the AKI-to-CKD transition post-IRI remain to be fully elucidated. Using 4D label-free proteomic and phosphoproteomic analyses in a murine unilateral IRI model at 1 h, 1 day, 3 days, 7 days, and 28 days post injury, we systematically identified dysregulated proteins, phosphoproteins, and signaling pathways involved in the progression from AKI to CKD. Critically, these analyses consistently revealed the enrichment and sustained activation of NF-κB signaling, a key pathway driving inflammatory and fibrotic responses, across multiple time points. In addition, we identified significant impairment of fatty acid β-oxidation. Notably, our omics analysis specifically identified the dedicator of cytokinesis (Dock) protein family, with Dock2 emerging as a prime candidate due to its known immune regulatory functions. Dock2 expression showed significant upregulation post-IRI and was found predominantly localized to injured tubular epithelial cells. Functional validation demonstrated that Dock2 knockdown attenuated proinflammatory responses in tubular epithelial cells by inhibiting IKKβ-mediated NF-κB activation in vitro. Consistently, pharmacological inhibition of Dock2 by CPYPP ameliorated renal tubular injury, inflammation, and fibrosis in vivo. To our knowledge, this is the first study to reveal the role and mechanism of Dock2 in the AKI-to-CKD progression post-IRI. In conclusion, our findings delineate molecular mechanisms underpinning the transition from AKI to CKD and nominate Dock2 as a promising therapeutic target for mitigating this process.

Extracellular vesicles (EVs), including exosomes and microvesicles, act as transmitters of various biological signals through cell-cell communication. Although EVs derived from immune response cells have been partially studied, the characteristics of EVs mediated by NLR family pyrin domain-containing 3 (NLRP3) inflammasome activation remain unclear. Here, we characterize inflammatory EVs, termed infosomes, derived from NLRP3 inflammasome-activated macrophages, which play a role in inducing inflammation. Proteomic analysis revealed that EV production was increased in macrophages with activated NLRP3 inflammasomes and that these EVs were enriched with marker proteins involved in metabolism, membrane structure, and cytoskeletal organization. Furthermore, significantly increased proteins were associated with signaling pathways and biological processes related to immune response, phagocytosis, endocytosis, and neurodegenerative diseases. Crucially, these alterations in EV secretion and molecular composition were dependent on NLRP3 and its subsequent inflammasome activity. Functionally, these infosomes were shown to amplify the expression of inflammatory factors in both macrophages and endothelial cells. These findings provide insights into the biological roles of infosomes, suggesting that EVs generated and loaded by NLRP3 inflammasome activation act as key biological mediators that disseminate and amplify inflammatory responses through cell-cell communication. This highlights their potential as novel biomarkers and therapeutic targets for inflammatory diseases.

Platycodin D (PD), a major bioactive saponin isolated from the traditional Chinese medicine (TCM) Platycodon grandiflorus, has shown promising therapeutic potential against non-small cell lung cancer (NSCLC). However, the functional mechanisms of PD in NSCLC progression remains unclear. This study aimed to explore the pharmacological mechanism of PD against NSCLC. Thermal proteome profiling (TPP) approach, molecular docking, cellular thermal shift assay (CETSA) and peptide-centric local stability assay (PELSA) were employed to identify the potential binding target of PD. Subsequent Western Blot and immunoprecipitation-Western Blot (IP-WB) experiments were conducted to investigate the downstream signaling pathways of the target. Furthermore, proteomic and ubiquitinomic profiling of PD-treated cells were performed to investigate its functions on global. Replication factor C subunit 4 (RFC4) was identified as a potential binding target of PD by TPP and their binding sites were further exposed by PELSA. PD-RFC4 complex promotes the degradation of Notch1 and Notch3 by reducing nuclear entry of their domains. Compared with control treatment, the differentially expressed proteins induced by PD were found to be primarily involved in ferroptosis, ubiquitination, platinum drug resistance and ribosome-related processes. The ubiquitin proteome analysis revealed that proteins associated with the Notch pathway underwent ubiquitin modifications. PD binds to RFC4 and inhibits its activity, leading to downregulation of the Notch signaling pathway, ultimately triggering cancer cell apoptosis. PD is a natural product with potential therapeutic value for NSCLC.

The involvement of the oral microbiome (OM) in the pathophysiology of Alzheimer's disease and vascular dementia has been recognized epidemiologically, but the molecular mechanisms remain elusive. In this study, we uncovered the presence of OM-derived proteins (OMdPs) in brain extracellular vesicles (bEVs) from post-mortem Alzheimer's disease and vascular dementia subjects using unbiased metaproteomics. OMdP circulation in blood EVs was also confirmed in an independent cohort. Our findings also reveal that specific OMdPs are present in bEVs, with their levels varying with disease progression. Peptidome-wide correlation analyses further explored their exchange dynamics and composition within bEVs. In addition, we validated the ability of OM-derived EVs to cross the blood-brain barrier using a blood-brain barrier-on-a-chip model, confirming a potential route for bacterial-derived molecules to reach the central nervous system. Bioinformatics-driven interaction analyses indicated that OMdPs engage with key neuropathological proteins, including amyloid-beta and tau, suggesting a novel mechanism linking dysbiotic OM to dementia. These results provide new insights into the role of the OM in neurodegeneration and highlight OMdPs as potential biomarkers and therapeutic targets.

The precise structure of glycosaminoglycans is critical for their bioactivity and the development of glycopharmaceuticals. Herein, cellular and animal experiments were conducted to assess the differences in the activities of heparin (HP) and heparan sulfate (HS) against liver cancer and drug-induced liver injury. Label-free quantitative proteomics, bioinformatics, biolayer interferometry, and immunohistochemical analyses were used to determine key proteins with differential expression. As a result, HP demonstrated superior antiliver cancer activity compared with HS, whereas HS exhibited strong potential in resisting acetaminophen-induced liver injury. DIRAS family GTPase 2 (DIRAS2) was identified as a key HS-binding protein that was strongly associated with cell proliferation, and its expression levels in cells and tissues showed opposite trends following HP and HS administration. HP significantly reduced the abundance of DIRAS2 in the tumor tissue, thereby inhibiting tumor cell proliferation, whereas HS promoted proliferation by increasing DIRAS2 expression. Cluster sequencing revealed that consecutive GlcNS6S-IdoA2S domains in HP and IdoA2S-GlcNS6S, GlcA-GlcNS6S, and IdoA-GlcNAc domains in HS were required for affinity binding within the decasaccharide region. Molecular docking suggested that differences in the binding modes of HP and HS chains to DIRAS2 underlie their functional diversity. These findings indicate that HP and HS oligosaccharides with well-defined structures may serve as potential therapeutic agents for liver-related diseases.

Essential tremor (ET) stands as one of the most prevalent movement disorders originating from cerebellar dysfunction. However, effective treatment remains limited, largely due to a poor understanding of its molecular pathology. The harmaline-induced tremor in mice is a well-established model for ET research, though its mechanisms remain unclear. This study aimed to get insight into the molecular intricacies underlying cerebellar dysfunction in this model. Combining LC-MS/MS and RNA-Seq approach, we delved into the cerebellar alterations in harmaline-induced tremor in mouse. Multi-omics profiling identified 5194 correlated coding molecules, among which 19 were significantly dysregulated. Further KEGG enrichment analysis identified cerebellar serotonin transporter (SERT) as the key molecule in harmaline-induced tremor. We validated the upregulation of SERT in the cerebellar cortex following harmaline induction, particularly within Purkinje cells, and demonstrated that pharmacological inhibition or genetical knockdown of SERT significantly attenuated tremor severity and neuronal hyperexcitability. Further mechanistic studies revealed that harmaline-induced SERT upregulation leads to depleted serotonin levels in the cerebellum, contributing to tremor pathogenesis. In general, our study unveils crucial insights that could pave the way for molecular target identification and effective therapeutic interventions for ET.

Intracellular signaling pathways form networks through which information is transmitted, often in the form of kinase-mediated phosphorylation events, to interpret extracellular signals and elicit appropriate cellular responses. Yet, even isogenic cells in a homogenous environment show heterogeneity in their intracellular "cell-state", as well as their response to extracellular signals. Here, we aimed to better understand this relation between these phenomena by investigating how information flows through the EGF-receptor centered network upon targeted drug treatment, and how this is affected by cell-to-cell-state differences. Using single-cell ID-seq, we profiled the cell-state and signaling activity in primary human epidermal stem cells by measuring 69 (phospho-)proteins upon inhibition of the Erk/MAPK (p90RSK) and Akt/mTOR (p70S6K) routes downstream of the EGF pathway. We found that the effects of drug treatment propagated from the EGF-signaling pathway to other connected parts of the cellular signaling network, indicating altered signaling flow. We identified nine distinct cell-states that show pervasive state-dependent drug-responses for many (phospho-)proteins. Computational modeling of the signaling network using single-cell Comparative Network Reconstruction showed that many interactions between phospho-proteins (i.e. network wiring) were quantitatively different between cell-states. Furthermore, (phospho-)proteins with a cell-state dependent drug response, were more likely to be involved in interactions that showed a cell-state dependent strength. Overall, our results indicate that drug treatment response and signaling interactions between proteins are closely related and modulated by cell-state.

In orthopaedic foot and ankle procedures, bone debris generated during osteotomies is typically discarded. However, this autograft bone debris has good handling properties and can be amenable for use as a stimulus to bone healing at the site of the osteotomy. In the present study, discarded bone debris was harvested intraoperatively during minimally invasive chevron Akin (MICA) hallux valgus corrections, isolated Akin osteotomies of the proximal phalanx of the great toe, cheilectomies, and calcaneal osteotomies from 9 participants. Multiplex protein arrays of 40 cytokines and 41 growth factors identified 76 immunomodulatory and reparative proteins within the bone debris. Fifteen key growth factors and cytokines (VEGF, PDGF-BB, M-CSF, EGF-R, HGF, ICAM1, GCSF, TIMP-2, sTNFRII, MCP-1, GM-CSF, IL-6sR, IL-10, MCP-2, RANTES) were prominent, suggesting that bone debris proteins may have potential effects on immunomodulation and bone regeneration including cytokine-cytokine receptor interaction, interleukins, MAPK, PI3K-Akt, Wnt, BMP, and TGFβ signaling pathways.Mesenchymal stem/stromal cells (MSCs) were isolated from bone debris of the osteotomies. MSCs expressed genes involved in bone and cartilage formation, homeostasis, angiogenesis, and immunomodulation. Fourteen genes were associated with maintaining cell stemness, whereas seventeen genes were linked to osteochondral development and spatial organization. Additionally, the study identified eight genes promoting angiogenesis and ten genes regulating immune responses in the mesenchymal stem cell environment. This repurposed surgical waste contains a concentrated array of growth factors, antiinflammatory mediators, and viable MSC that might enhance bone healing when reintroduced to surgical sites. The results could serve as a foundation for repurposing previously discarded bone debris as autologous bone grafts for reimplantation in minimally invasive orthopaedic procedures to potentially enhance bone tissue healing. To confirm clinical relevance, further well-controlled trials are required to establish whether these findings improve bone healing rates and related patient-reported outcomes.

Usher syndrome is the leading cause of inherited deaf-blindness, with type 2 (Usher syndrome type 2, USH2) being the most common form. USH2A, ADGRV1, and WHRN are the three known USH2 causative genes, which are also linked to isolated retinal degeneration and hearing loss. These genes encode usherin, ADGRV1, and whirlin, respectively, collectively called USH2 proteins. These proteins form a multiprotein complex (USH2 complex) at the periciliary membrane in retinal photoreceptors and at the stereociliary ankle link in inner ear hair cells. The molecular function of the USH2 complex and its disease mechanisms are poorly understood. Currently, there is no cure for diseases caused by mutations in the three USH2 genes. In this study, we employed multiple affinity purification methods combined with mass spectrometry to systematically identify the interaction partners of USH2 proteins in the retina. The ADGRV1 intracellular bait pulled down proteins involved in actin-based cell projections, the chaperone-containing TCP-1 complex, and the Bardet-Biedl syndrome complex. The extracellular domains of ADGRV1 and usherin pulled down proteins related to peptidase regulation, collagen biosynthesis and modification, and elastic fiber formation. The EAR/EPTP repeats of ADGRV1 specifically pulled down TGFβ signaling proteins. Further immunoprecipitation experiments identified, with high confidence, Gαi and Gαq as ADGRV1-interacting proteins, and retinal degeneration and ciliary proteins as interaction partners of USH2 proteins. We also demonstrated that the usherin extracellular domains interact with each other and with ADGRV1. Overall, these findings suggest that the USH2 complex connects the extracellular matrix (ECM) to the intracellular actin network, signals through Gαi and Gαq, and participates in ECM remodeling, TGFβ signaling, cell adhesion, and ciliary function in photoreceptors.