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Results for “proximity proteomics”

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PubMed2006

Proteomic analysis of the adaptive response of rat renal proximal tubules to metabolic acidosis.

Proximal tubules were isolated from control and acidotic rats by collagenase digestion and Percoll density gradient centrifugation. Western blot analysis indicated that the tubules were approximately 95% pure. The samples were analyzed by two-dimensional difference gel electrophoresis (DIGE) and DeCyder software was used to quantify the temporal changes in proteins that exhibit enhanced or reduced expression. The mass-to-charge ratios and the amino acid sequences of the recovered tryptic peptides were determined by MALDI-TOF/TOF mass spectrometry and the proteins were identified using Mascot software. This analysis confirmed the well-characterized adaptive responses in glutaminase (GA), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase (PEPCK). This approach also identified 17 previously unrecognized proteins that are increased with ratios of 1.5 to 5.6 and 16 proteins that are decreased with ratios of 0.67 to 0.03 when tubules from 7-day acidotic vs. control rats were compared. Some of these changes were confirmed by Western blot analysis. Temporal studies identified proteins that were induced either with rapid kinetics similar to PEPCK or with more gradual profiles similar to GA and GDH. All of the mRNAs that encode the latter proteins contain an AU sequence that is homologous to the pH response element found in GA mRNA. Thus selective mRNA stabilization may be a predominant mechanism by which protein expression is increased in response to acidosis.

Acidosis↗

PubMed2002

Acute and chronic effect of dietary phosphorus restriction on protein expression in young rat renal proximal tubules.

Renal proximal tubules play a vital role in phosphorus (P) homeostasis. It is well known that dietary P restriction up-regulates the activities of 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1-OHase), an enzyme that is involved in activation of vitamin D and thereby maintaining P balance. However, the mechanism involved in such regulation is not known. In the present study, we aim to identify proteins that might be involved in the renal adaptation to dietary P restriction using a proteomic approach. Renal proximal tubules were harvested from young rats fed either normal P diet or low P diet (LPD) for 1 to 7 days. Western blotting analysis of 1-OHase and signaling proteins in insulin-like growth factor I axis indicated an increase in expression of these proteins upon dietary P restriction. Using two-dimensional electrophoresis, we found that LPD reduced the total number of protein species expressed in renal proximal tubules. Differentially expressed proteins were analyzed and located using the software Melanie III, and their identities were found using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Our results showed that beta-actin, gamma-actin, major urinary protein, phosphatidylinositol transfer protein beta isoform, and G1/S-specific cyclin D3 are up-regulated and nonspecific lipid transfer protein is down-regulated by LPD.

Actins↗

PubMed2003

A correlation between a proteomic evaluation and conventional measurements in the assessment of renal proximal tubular toxicity.

4-Aminophenol (4-AP), D-serine, and cisplatin are established rodent nephrotoxins that damage proximal tubules within the renal cortex. Using high throughput 2D gel proteomics to profile protein changes in the plasma of compound-treated animals, we identified several markers of kidney toxicity. Male F344 and Alpk rats were treated with increasing doses of 4-AP, D-serine, or cisplatin, and plasma samples were collected over time. Control groups received saline or nontoxic isomers, L-serine, and transplatin. Plasma proteins that displayed dose- and temporal-dependent regulation in each study were further characterized by mass spectrometry to elucidate the protein identity. Several isoforms of the rat-specific T-kininogen protein were identified in each study. T-kininogen was elevated in the plasma of 4-AP-, D-serine-, and cisplatin-treated animals at early time points, returning to baseline levels 3 weeks after treatment. The protein was not elevated in the plasma of control animals or those treated with nontoxic compounds. We propose that T-kininogen may be required to counteract apoptosis in proximal tubular cells in order to minimize tissue damage following a toxic insult. In addition, T-kininogen may be required to stimulate localized inflammation to aid tissue repair. We also identified several isoforms of the inter-alpha inhibitor H4P heavy chain in the 4-AP and D-serine studies. In each case, the protein expression levels in the blood samples paralleled the extent of kidney toxicity, highlighting the correlation between protein alterations and clinical chemistry endpoints. A further set of proteins correlating with kidney damage was found to be a component of the complement cascade and other blood clotting factors, indicating a contribution of the immune system to the observed toxicity. These observations underscore the value of proteomics in identifying new biomarkers and in the elucidation of mechanisms of toxicity.

4-Aminopyridine↗

PubMed2026

From stem cells to somites: Revealing genetic and exogenous factors of human embryogenesis.

Stem-cell-based human embryo models offer an ethically tractable platform for studying early human development. This study employs somitoids, three-dimensional models of human somitogenesis, to investigate how transcriptional programs and culture conditions influence somite formation and segmentation. We show that pre-differentiation culture medium impacts the developmental potential of induced pluripotent stem cells (iPSCs), with StemFit medium and Matrigel embedding outperforming mTeSR Plus medium in generating robust somite-like structures. Strikingly, these differences arise despite only subtle changes in transcriptomic and time-resolved proteomic profiles. P300-based proximity labeling also reveals a largely overlapping set of chromatin-associated regulators across iPSC conditions. In somitoids, enhancer-associated profiling highlights factors linked to somitogenesis, including MESP2 and TBX6. Knockout of three identified regulators, BPTF, RBPJ, and CITED2, demonstrate their essential roles in somite formation. Together, these findings highlight how culture conditions and enhancer-associated networks influence early human development and demonstrate somitoids as a scalable system for functional genomics.

Humans↗

PubMed2007

Proximity ligation: a specific and versatile tool for the proteomic era.

Knowledge about the total human genome sequence now provides opportunities to study its myriad gene products. However, the presence of alternative splicing, post-translational modifications, and innumerable protein-protein interactions among proteins occurring at widely different concentrations, all combine to place extreme demands on the specificity and sensitivity of assays. The choice of method also depends on matters such as whether proteins will be analyzed in body fluids and lysates, or localized inside single cells. In this review we discuss commonly used detection methods and compare these to the recently-developed proximity ligation technique.

Animals↗

PubMed2004

High-resolution proteomic mapping in the vertebrate central nervous system: close proximity of connexin35 to NMDA glutamate receptor clusters and co-localization of connexin36 with immunoreactivity for zonula occludens protein-1 (ZO-1).

Combined confocal microscopy and freeze-fracture replica immunogold labeling (FRIL) were used to examine the connexin identity at electrical synapses in goldfish brain and rat retina, and to test for "co-localization" vs. "close proximity" of connexins to other functionally interacting proteins in synapses of goldfish and mouse brain and rat retina. In goldfish brain, confocal microscopy revealed immunofluorescence for connexin35 (Cx35) and NMDA-R1 (NR1) glutamate receptor protein in Mauthner Cell/Club Ending synapses. By FRIL double labeling, NR1 glutamate receptors were found in clusters of intramembrane particles in the postsynaptic membrane extraplasmic leaflets, and these distinctive postsynaptic densities were in close proximity (0.1-0.3 microm) to neuronal gap junctions labeled for Cx35, which is the fish ortholog of connexin36 (Cx36) found at neuronal gap junctions in mammals. Immunogold labeling for Cx36 in adult rat retina revealed abundant gap junctions, including several previously unrecognized morphological types. As in goldfish hindbrain, immunogold double labeling revealed NR1-containing postsynaptic densities localized near Cx36-labeled gap junction in rat inferior olive. Confocal immunofluorescence microscopy revealed widespread co-localization of Cx36 and ZO-1, particularly in the reticular thalamic nucleus and amygdala of mouse brain. By FRIL, ZO-1 immunoreactivity was co-localized with Cx36 at individual gap junction plaques in rat retinal neurons. As cytoplasmic accessory proteins, ZO-1 and possibly related members of the membrane-associated guanylate kinase (MAGUK) family represent scaffolding proteins that may bind to and regulate the activity of many neuronal gap junctions. These data document the power of combining immunofluorescence confocal microscopy with FRIL ultrastructural imaging and immunogold labeling to determine the relative proximities of proteins that are involved in short- vs. intermediate-range molecular interactions in the complex membrane appositions at synapses between neurons.

Animals↗

PubMed2026

Machine learning-assisted plasma PEA proteomics enables differential diagnosis of melancholic depression and bipolar disorder.

Differentiating bipolar disorder (BD) from major depressive disorder (MDD) remains a critical unmet need in psychiatry due to overlapping clinical presentations and the absence of reliable biological markers. In this study, we assessed the capacity of multivariate machine learning models to accurately differentiate BD from MDD with melancholic features using plasma proteomic profiles obtained via Proximity Extension Assay (PEA) technology. A total of 67 participants were included (23 BD, 20 MDD, and 24 HC), and plasma protein expression was assessed using the Olink Target 96 Neurology panel. Differential proteomic analysis revealed distinct disorder-specific expression patterns, identifying 21 differentially expressed proteins in BD versus MDD, 18 in BD versus healthy controls, and 7 in MDD versus healthy controls. Using a stepwise feature reduction strategy, machine learning models were trained on three feature sets comprising all proteins, the top 20 most informative proteins, and the top 5 most beneficial proteins, and evaluated across BD-MDD, BD-HC, and MDD-HC classification tasks using five algorithms. For BD-MDD discrimination, the Random Forest model achieved the highest performance when trained on the top 5 protein set (LXN, HAGH, MATN3, PLXNB1, and CTSC), yielding an AUC of 0.905, with similarly strong performance observed using the top 20 protein set. Feature importance analysis highlighted proteins involved in neurodevelopmental processes, immune regulation, and extracellular matrix organization. Overall, these findings demonstrate that integrating plasma proteomics with machine learning enables robust differentiation between BD and MDD with melancholic features, supporting the development of scalable and biologically informed diagnostic tools for precision psychiatry.

Bipolar disorder↗

PubMed2004

The urinary proteome in Fanconi syndrome implies specificity in the reabsorption of proteins by renal proximal tubule cells.

Polypeptides present in the glomerular filtrate are almost completely reabsorbed in the first segment of the proximal tubule by receptor-mediated endocytosis; in renal Fanconi syndrome (FS), there is failure to reabsorb many of these polypeptides. We have compared the urinary proteomes in patients with Dent's disease (due to a CLC5 mutation), a form of FS, with normal subjects using three different proteomic methods. No differences in the levels of several plasma proteins were detected when standardized to total protein amounts. In contrast, several vitamin and prosthetic group carrier proteins were found in higher amounts in Dent's urine (with respect to total protein). Similarly, complement components, apolipoproteins, and some cytokines represented a larger proportion of the Dent's urinary proteome, suggesting that such proteins are reabsorbed more efficiently than other classes of proteins. Conversely, proteins of renal origin were found in proportionately higher amounts in normal urine. Thus the uptake of filtered vitamins, which are normally bound to their respective carrier proteins to prevent urinary losses, seems a key function of the proximal tubule; in addition, this nephron segment may also play a critical role in reabsorbing potentially cytotoxic polypeptides of plasma origin, preventing them from acting at more distal nephron sites.

Adolescent↗

PubMed2004

Cytokine detection by antibody-based proximity ligation.

Efficient and precise detection techniques, along with extensive repertoires of specific binding reagents, will be needed to meet the challenges of proteome analyses. The recently established proximity ligation mechanism enables sensitive high-capacity protein measurements by converting the detection of specific proteins to the analysis of DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins and to form amplifiable tag sequences by ligation when brought in proximity. In our previous report, both the ligatable arms and the protein binders were DNA molecules. We now generalize the method by providing simple and convenient protocols to convert any polyclonal antibodies or matched pair of monoclonal antibodies to proximity probe sets through the attachment of oligonucleotide sequences. Sufficient reagent for >100,000 proximity ligation assays can be prepared from 1 microg of antibody. The technique is applied to measure cytokines in a homogenous test format with femtomolar detection sensitivities in 1-microl samples, and we exemplify its utility in situations when only minute sample amounts are available.

Antibodies↗

PubMed2026

Serum Olink Proteomics Reveals Novel Biomarkers for Early Diagnosis of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a highly prevalent malignant tumor in China, and early diagnosis critically affects the prognosis. Current imaging and pathological biopsy techniques have limitations, including high invasiveness and limited accessibility, while the insufficient sensitivity of serum biomarkers (such as AFP) restricts their use in early screening. In this study, using the Olink proteomics platform based on the proximity extension assay (PEA), we screened for hepatocellular carcinoma-related differentially expressed proteins (DEPs) and constructed a multiprotein diagnostic model. In the discovery cohort, we included 15 patients with newly diagnosed HCCs and 16 healthy controls. DEPs were identified using Olink, and their diagnostic performance was analyzed to identify the candidate biomarkers. In an independent validation cohort, including 116 HCC patients (50 early stage, 66 late stage) and 83 healthy controls, we further validated the expression levels and diagnostic performance of identified proteins─C1QA and GFER. The C1QA and GFER expression levels were significantly higher in the serum of patients with early and late HCC stages compared to healthy controls. By constructing a multiprotein diagnostic model, we identified C1QA, GFER, and AFP as the optimal diagnostic combination, demonstrating a combined diagnostic AUC of 0.92 and 0.99 for early-stage and advanced-stage HCC, respectively.

Humans↗

PubMed2005

A new murine model of giant proximal axonopathy.

The aromatic gamma-diketone 1,2-diacetylbenzene (1,2-DAB), the putative active metabolite of the organic solvent 1,2-diethylbenzene, forms blue-colored polymeric protein adducts and induces the formation of amyotrophic lateral sclerosis (ALS)-like giant, intraspinal neurofilamentous axonal swellings in Sprague Dawley rats. The pathogenetic mechanism of this neuropathy has yet to be understood. We assessed whether these pathological changes are also seen in the C57BL/6 mouse, the animal of choice for toxicogenomic studies. Mice were treated intraperitoneally with 30, 35, 50, or 70 mg/kg 1,2-DAB or its inactive isomer 1,3-DAB per day (or on alternate days) for up to 43 days. Animals treated with 30 or 35 mg/kg per day 1,2-DAB, but not with 1,3-DAB, developed muscle spasms and progressive weakness, most prominently in hind limbs. Light microscopy revealed swollen axons in spinal anterior horns and proximal ventral roots, and to a lesser extent in dorsal root ganglia of 1,2-DAB-treated animals. Ultrastructural examination of swollen axons revealed clumps of maloriented 10-nm neurofilaments. Sciatic nerves showed clustering of axonal microtubules and other organelles. These findings are qualitatively comparable to those reported in rats treated with 1,2-DAB and represent a suitable phenotype with which to explore molecular mechanisms of proximal, giant neurofilamentous axonopathy using proteomic and genomic technologies.

Acetophenones↗

PubMed2026

Dual proximity-based interactome mapping of FKBP51 and FKBP52 uncovers shared metabolic networks.

The 51 kDa FK506-binding protein (FKBP51) has been studied for its involvement in regulating multiple biological systems, particularly as a regulator of steroid hormone receptors, but roles in metabolism, pain response, cell survival, protein turnover, autophagy, immune response, and insulin signaling have also been described. Genetic variants of FKBP51 are associated with various stress-related mental disorders. While recent research has clarified aspects of these processes, the complete range of FKBP51 interactions remains undetermined. FKBP52, a closely related homolog, also affects similar pathways. Recent studies have identified new protein partners for FKBP51 and FKBP52, suggesting an even broader interactome with transient associations. To further characterize interactions, TurboID-based proximity labeling was performed in HeLa cells. Proteomic analysis confirmed known FKBP51 and FKBP52 interactions, while also identifying additional shared and unique binding partners with strong enrichment in metabolic pathways, amino acid biosynthesis, and carbon metabolism. Although FKBP51 and FKBP52 proximal proteins were primarily cytosolic, FKBP51 showed additional associations with exosomal proteins while FKBP52 engaged with additional nuclear proteins. These findings highlight the overlapping roles in metabolic signaling and differentiate pathway-specific partners.

Tacrolimus Binding Proteins↗

PubMed2026

Tissue-layer-resolved proteome landscape of Crohn's disease strictures highlights potential drivers of fibrosis progression.

The chronic inflammation of Crohn's disease frequently leads to fibrosis and muscular hypertrophy of the intestinal wall. This often culminates in strictures, a serious condition lacking directed therapy. Severe pathological changes occur in the submucosa and muscularis propria intestinal wall layers of strictures, yet stricture-associated proteome changes in these layers is unexplored. We perform unbiased proteomics on submucosa and muscularis propria microdissected from transmural sections of strictured and nonstrictured ileum. Proteome changes in strictured submucosa reflected a transition from homeostasis to tissue remodeling, inflammation, and smooth muscle changes. Top submucosal features included reduced vascular components and lipid metabolism proteins accompanied by increased proteins with immune-, ECM-, or stress-related functions, including CTHRC1, TNC, IL-16, MZB1, and TXNDC5. In parallel, predominant changes in strictured muscularis propria included increased ECM (POSTN) and immune (mast cell CPA3) proteins alongside decreased proteins with lipid metabolic, mitochondrial, or key muscle functions. Finally, trends of differentially expressed proteins along nonstrictured submucosa suggest progressive profibrotic tissue remodeling and muscle expansion as proximity to strictures increases. The comprehensive proteome map presented here offers tissue-layer-resolved insight into the stricture microenvironment and potential drivers of fibrotic disease, providing a valuable resource to fuel biomarker and therapeutic target research.

Humans↗

PubMed2026

Resolving cellular signaling in space and time: From organelle proteomics to spatial phosphoproteomics.

Cellular signaling is inherently organized in space and time, requiring coordinated control of protein localization, molecular interactions, and enzymatic activity across subcellular compartments. Recent advances in chemical biology, protein engineering, and quantitative proteomics have made it possible to interrogate these dimensions in an integrated manner. Here, we highlight emerging strategies to resolve signaling organization across three interconnected dimensions: organelle-resolved proteome mapping to define spatial context, proximity labeling to capture local protein interaction networks, and spatially resolved phosphoproteomics to quantify signaling outputs. Developments in proximity labeling, including split, conditionally activated and light-gated enzymes, enable temporally controlled, context-dependent profiling of transient protein assemblies in living cells. Advances in high-throughput and low-input phosphoproteomics, together with improved computational frameworks for kinase activity inference and subcellular enrichment strategies, are enabling spatially resolved measurement of signaling activity. Together, these approaches are shifting the field from static localization maps toward dynamic models of signaling networks.

Proteomics↗

PubMed2026

Calcium-Sensing Receptor Activation Disrupts Phosphatidylserine Asymmetry and Promotes Calcium Oxalate Crystal-Induced Epithelial Injury in Renal Tubular Epithelial Cells.

BACKGROUND: Calcium oxalate (CaOx) crystal retention on the renal tubular epithelium is a key step in urolithiasis. Phosphatidylserine (PS) exposure may facilitate crystal-cell adhesion, but the upstream signaling mechanisms and the relative contributions of impaired inward PS flipping versus outward PS redistribution remain unclear. MATERIALS AND METHODS: Global proteomic profiling using 2-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (2-DE/MALDI-TOF/TOF) in an immortalized human proximal tubular epithelial cell line (HK-2) cells exposed to calcium oxalate monohydrate (COM) identified upregulation of the calcium-sensing receptor (CaSR). HK-2 cells were treated with COM with or without the CaSR antagonist NPS2390 or the CaSR agonist gadolinium chloride (GdCl3). Bidirectional PS transport was assessed using an N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-labeled phosphatidylserine (NBD-PS) fluorescence-quenching assay, and surface PS exposure was measured by annexin V binding. Aminophospholipid translocase (APLT) expression, APLT-dependent inward PS transport, crystal adhesion, oxidative stress, and apoptosis-related signaling were evaluated. RESULTS: COM increased CaSR expression, enhanced surface PS exposure, and promoted crystal adhesion with concurrent oxidative stress and apoptosis-related signaling. COM induced a CaSR-sensitive defect in APLT-dependent inward PS flipping: NPS2390 partially restored inward PS transport and APLT expression, whereas GdCl3 exacerbated these changes. In contrast, COM-enhanced outward PS redistribution and externalization was largely unaffected by CaSR modulation, indicating relative CaSR insensitivity of the outward process. Consistently, CaSR activation aggravated, while CaSR inhibition attenuated, crystal adhesion and injury-related readouts. CONCLUSIONS: COM was associated with enhanced crystal-cell adhesion, CaSR activation, and a CaSR-sensitive impairment of APLT-dependent inward PS flipping, whereas enhanced outward PS redistribution appeared largely CaSR-insensitive. Pharmacologic inhibition of CaSR attenuated epithelial injury and crystal retention-related readouts, suggesting that CaSR may represent a potential therapeutic target.

Receptors, Calcium-Sensing↗

PubMed2025

Spatial Proteomics Using BiFCPL Identifies Regulators of DMV Formation Involved in Coronavirus Replication.

β-Coronaviruses hijack host factors to remodel host endo-membranes to form double membrane vesicles (DMVs), which act as central hubs for the replication of viral genomes. Understanding the molecular mechanism underlying DMV formation is critical for developing effective antiviral strategies and has garnered significant attention. However, the host factors involved in DMV formation remain scanty. Here, we employed a bimolecular fluorescence complementation-based proximity labeling (BiFCPL) strategy to investigate the proteome of DMVs generated by co-expression of SARS-CoV-2 NSP3 and NSP4. Our analysis identified 62 proteins with high confidence, among which five proteins were localized in the endoplasmic reticulum (ER), and were further confirmed to be recruited to DMVs through interactions with NSP3/NSP4. Moreover, we demonstrated that the absence of GRAMD1B or TEX2 resulted in the formation of enlarged DMVs induced by either NSP3/NSP4 or coronaviruses infection, and impaired coronaviruses replication as well. Collectively, our study concerning host-virus interactions sheds light on novel host factors involved in DMV formation.

Virus Replication↗

PubMed2006

Convergence of the proteomic pattern in cancer.

MOTIVATION: On the histological level the differentiation of normal epithelial tissues is well known. The phenomenon of dedifferentiation, which occurs as cells develop towards malignancy is also well described. To identify an epithelial tumor-specific proteomic profile as well as to measure the proximities between we used data from tumor tissue and adjacent normal tissue microdissected from head and neck and colon cancer samples which were analyzed using ProteinChip technology and performed a bioinformatic meta-analysis on the resulting four complex datasets. RESULTS: All four groups could be identified based on their proteomic signatures and the tumor tissues were found to be more similar to one another than to the normal epithelial tissue from which they progressed. This study shows at the proteomic level that changes in the histological features of tumors as compared to the tissues from which they arise are reflected in the convergence of proteomic pattern during the development to cancer.

Cell Differentiation↗

PubMed2006

Do distantly related parasites rely on the same proximate factors to alter the behaviour of their hosts?

Phylogenetically unrelated parasites often increase the chances of their transmission by inducing similar phenotypic changes in their hosts. However, it is not known whether these convergent strategies rely on the same biochemical precursors. In this paper, we explored such aspects by studying two gammarid species (Gammarus insensibilis and Gammarus pulex; Crustacea: Amphipoda: Gammaridae) serving as intermediate hosts in the life cycle of two distantly related parasites: the trematode, Microphallus papillorobustus and the acanthocephalan, Polymorphus minutus. Both these parasite species are known to manipulate the behaviour of their amphipod hosts, bringing them towards the water surface, where they are preferentially eaten by aquatic birds (definitive hosts). By studying and comparing the brains of infected G. insensibilis and G. pulex with proteomics tools, we have elucidated some of the proximate causes involved in the parasite-induced alterations of host behaviour for each system. Protein identifications suggest that altered physiological compartments in hosts can be similar (e.g. immunoneural connexions) or different (e.g. vision process), and hence specific to the host-parasite association considered. Moreover, proteins required to alter the same physiological compartment can be specific or conversely common in both systems, illustrating in the latter case a molecular convergence in the proximate mechanisms of manipulation.

Acanthocephala↗