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Results for “proximity ligation assay”

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At least 19 recordsLinked to original sources
PubMed2006

A sensitive proximity ligation assay for active PSA.

Prostate-specific antigen (PSA) is a widely used marker for prostate cancer. The utility of PSA tests is limited by their inability to differentiate prostate cancer from non-malignant conditions such as benign prostatic hyperplasia and prostatitis. In circulation, PSA occurs in various complexed and free forms, and specific determination of some of these can be used to improve the diagnostic accuracy of PSA tests. We have previously identified peptides that specifically bind to enzymatically active PSA and using such a peptide we have developed an immunopeptidometric assay for this form of PSA. However, the sensitivity of that assay is too low to measure active PSA at clinically important levels. Recently a novel sensitive immunoassay for analysis of proteins, termed the proximity ligation assay, has been established. Here we describe a sensitive implementation of the proximity ligation assay, which utilizes a PSA-binding peptide and antibody as probes to detect active PSA. The assay has a sensitivity of 0.07 microg/l, which is approximately ten-fold lower than that of our previous assay. It does not cross-react with inactive proPSA or the highly similar kallikrein hK2. Our results show that a highly sensitive immunopeptidometric assay can be developed using proximity ligation. This principle should facilitate establishment of specific assays for active forms of other proteases.

Antibody Specificity↗
PubMed2005

Proximity ligation assays with peptide conjugate 'burrs' for the sensitive detection of spores.

The proximity ligation assay (PLA) has previously been used for the sensitive and specific detection of single proteins. In order to adapt PLA methods for the detection of cell surfaces, we have generated multivalent peptide-oligonucleotide-phycoerythrin conjugates ('burrs') that can bind adjacent to one another on a cell surface and be ligated together to form unique amplicons. Real-time PCR detection of burr ligation events specifically identified as few as 100 Bacillus anthracis, 10 Bacillus subtilis and 1 Bacillus cereus spore. Burrs should prove to be generally useful for detecting and mapping interactions and distances between cell surface proteins.

Bacillus anthracis↗
PubMed2006

Sensitive protein detection via triple-binder proximity ligation assays.

The detection of weakly expressed proteins and protein complexes in biological samples represents a fundamental challenge. We have developed a new proximity-ligation strategy named 3PLA that uses three recognition events for the highly specific and sensitive detection of as little as a hundred molecules of the vascular endothelial growth factor (VEGF), the biomarkers troponin I, and prostate-specific antigen (PSA) alone or in complex with an inhibitor--demonstrating the versatility of 3PLA.

Antibodies↗
PubMed2006

Assays for cytokines using aptamers.

Aptamers are short nucleic acid sequences that are used as ligands to bind their targets with high affinity. They are generated via the combinatorial chemistry procedure systematic evolution of ligands by exponential enrichment (SELEX). Aptamers have shown much promise towards detection of a variety of protein targets, including cytokines. Specifically, for the determination of cytokines and growth factors, several assays making use of aptamers have been developed, including aptamer-based enzyme-linked immunosorbent assays, antibody-linked oligonucleotide assay, fluorescence (anisotropy and resonance energy transfer) assays, and proximity ligation assays. In this article, the concept of aptamer selection using SELEX and the assay formats using aptamers for the detection of cytokines are discussed.

Animals↗
PubMed2002

Protein detection using proximity-dependent DNA ligation assays.

The advent of in vitro DNA amplification has enabled rapid acquisition of genomic information. We present here an analogous technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe. The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein. This proximity ligation assay detects zeptomole (40 x 10(-21) mol) amounts of the cytokine platelet-derived growth factor (PDGF) without washes or separations, and the mechanism can be generalized to other forms of protein analysis.

Animals↗
PubMed2026

Participation of the purinergic P2X7 receptor in molecular complexes in the nucleus of human chondrocytes.

In addition to the purinergic receptor P2X7R's known activity as a sensor of damage-associated molecular patterns (DAMPs), evidences support its role in maintaining tissue homeostasis. Its presence in cellular compartments other than its usual transmembrane localization suggests its involvement in specific signaling pathways. This study aimed to analyze P2X7R in the nucleus of human chondrocytes and search for potential interacting partners. Through co-immunoprecipitation and proximity ligation assay we discovered that, independent of extracellular ATP levels, P2X7R is abundantly present in both the nuclear membrane and in the nucleoplasm, where it is found in close proximity to lamin A/C (a component of the nuclear lamina), emerin (a protein involved in the assembly and disassembly of the nuclear envelope), and SUN2 (an inner nuclear membrane protein that facilitates the transmission of mechanical forces). Furthermore, chromatin immunoprecipitation revealed the participation of P2X7R in molecular complexes located in the promoter of specific genes including Sox9, TRPS1, FOXO3a, integrin β2 and connective tissue growth factor. Overall, this evidence reveals for the first time novel partners of P2X7R that place it in an intricate network that influences nuclear structure, mechanosensitivity, chromatin organization, and gene expression. Specifically, on the one hand, a close association between P2X7R and nuclear proteins participating in the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex (lamin A/C, emerin, and SUN2) places it among the factors involved in mechanosignaling and the maintenance of nuclear integrity; on the other, its recruitment to specific gene promoters suggests that it may act as a transcription regulator.

Humans↗
PubMed2025

Hexokinase 2 is an RNA-binding protein that regulates mRNA translation independently of glycolysis and induces melanoma cell proliferation.

Although metabolic benefits of glycolysis have been extensively described in tumor cells, the extra-metabolic functions linked to this energetic pathway in tumor growth and cell proliferation have not been clearly established yet. Recently, some key glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase 2, were reported to regulate mRNA translation. Translational control of gene expression is considered as a critical effector in cancer biology, representing a highly promising area of research. Here, we report that Hexokinase 2 (HK2), a glucose kinase that catalyzes the first step of glycolysis at the outer mitochondrial membrane (OMM), is an RNA-binding protein (RBP) that regulates mRNA translation in melanoma cell lines. Polysome profiling experiments followed by RNA sequencing indicate that the translational regulation exerted by HK2 is partly independent of the metabolic status or the glycolytic pathway. We found that HK2 specifically regulates translation of the mRNA encoding SOX10, a transcription factor implicated in the regulation of tumor initiation, maintenance, and progression in melanoma. RNA-protein interaction assays, including CrossLinking ImmunoPrecipitation (CLIP), indicate that HK2 is an RBP whose interaction with RNA is independent of its enzymatic activity, its ability to bind glucose or its association with the OMM. HK2 directly interacts with the 5' untranslated region (5'UTR) of the SOX10 mRNA through a stem-loop RNA secondary structure. Using RNA-protein proximity ligation assays and a fluorescence-based ribosome-bound mRNA mapping method, we found that high glucose conditions, which promote the release of HK2 from the OMM, induce an increase in HK2-SOX10 mRNA interaction and SOX10 mRNA translation in the cytoplasm. We further showed that HK2-dependent SOX10 mRNA translation is involved in melanoma cell proliferation and colony formation. Collectively, our data highlight a nonmetabolic function of HK2 acting as an RBP and translation regulator.

Hexokinase↗
PubMed2026

A Spatiotemporal Atlas of the Androgen Receptor Proximal Interactome.

Androgen receptor-interacting proteins (AR-IPs) number close to 1,000, yet their organization across subcellular space and time remains uncharted. Proximity labeling identifies direct partners and neighboring proteins, thereby expanding AR-IPs to AR-proximal interacting proteins (AR-PIPs). Using proximity labeling quantitative mass spectrometry (PL-qMS), we construct a spatiotemporal atlas of the cytosolic, microsomal, and nuclear compartments in LNCaP prostate tumor cells. PL-qMS recovered 82.2% of the known AR-interactome in extranuclear compartments and 84.2% in the nucleus, identifying 4,751 AR-PIPs that remodel across an androgen time course. The retromer formed an androgen-sensitive AR-proximal interaction network (AR-PIN) verified by proximity ligation assays (PLAs). Moreover, partial VPS26A disruption attenuated androgen-regulated transcription and mislocalized the AR coactivator TMF1, defining a retromer-AR-TMF1 axis. In the nucleus, AR-PINs recover 100% of the Launonen 2021 ChIP-SICAP chromatome and reveal a PLA-verified translation-to-transcription handoff involving eIF4G and 4E-BP1. This spatiotemporal atlas provides a proximal framework for probing AR function in cells.

Journal Article↗
PubMed2026

Characterization of a Ku-binding motif in the C-terminal region of RAG2.

We applied an unsupervised interactome analysis with the RAG2 C-terminal region (R2CT) in v-abl pro-B cells undergoing V(D)J recombination. Mass-spectrometry analyses showed that Ku70 and Ku80 were among the top 10 hits. To further strengthen these observations, we performed Proximity Ligation Assay (PLA) and characterize the existence of a GFP-R2CT-Ku complex formation in cellulo. The interaction of several partners with Ku70/80 (Ku) through Ku-binding motifs (KBMs) in their sequences governs their enrolment in NHEJ repair complexes. Through sequence analysis, we identified a KBM within R2CT (R-KBM, amino acids 589-527). We confirmed by calorimetry a specific micromolar interaction between this RAG2 region and Ku70/80/DNA complex. The RAG2 motif KBM can be subdivided in two conserved parts that have no interaction individually. AlphaFold2 prediction coupled with molecular dynamic simulations indicate that the C-terminal part of the RAG2 motif interacts with Ku80 on the same site than the NHEJ factor XLF. These in silico analyses indicated that the N-terminal part of the RAG2 motif interacts with DNA adjacent to Ku with a major role of the K503 residue in agreement with disruption of the interaction observed with the K503E mutant. This study further extends the large ensemble of proteins recruited at DSBs by KBM motifs and substantiates the model of a tight coupling between DNA breakage and repair during V(D)J recombination, mediated by the Ku-RAG2 C-terminus interaction.

Ku Autoantigen↗
PubMed2026

Direct targeting of ORAI1 by ginsenoside Rg3 modulates calcium signaling and senescence-associated AMPK-NRF2 activation.

BACKGROUND: 20(S)-ginsenoside Rg3 (Rg3(S)), a major saponin derived from red ginseng, exhibits diverse biological activities, including antioxidant and anti-senescence effects. However, the direct molecular targets through which Rg3(S) regulates calcium signaling and its role at membrane contact sites during cellular senescence remain largely unknown. METHODS: Plasma membrane (PM)-endoplasmic reticulum (ER) contacts and related protein interactions were analyzed using proximity ligation assays and co-immunoprecipitation. Direct binding of Rg3(S) to ORAI1 was validated using cellular thermal shift assays and microscale thermophoresis. Molecular docking simulations followed by site-directed mutagenesis were used to define critical binding residues. Cytosolic calcium levels and cellular senescence were assessed using calcium imaging and senescence-associated β-galactosidase staining. RESULTS: Rg3(S) increased cytosolic calcium levels independently of ER calcium depletion and was accompanied by a reduction in PM-ER contacts. Rg3(S) directly bound to ORAI1 in a dose-dependent manner, identifying ORAI1 as a previously unrecognized molecular target of ginsenoside Rg3. Molecular docking revealed LYS204 and ILE229 within the extracellular loop of ORAI1 as key residues maintaining this interaction. Mutation of these residues abolished Rg3(S)-induced calcium influx, leading to impaired activation of the AMPK-NRF2 pathway and attenuation of the anti-senescence effect of Rg3(S). CONCLUSION: These findings identify ORAI1 as a key molecular mediator of ginsenoside Rg3(S)-induced calcium signaling linked to cellular senescence. By modulating PM-ER contact sites and cytosolic calcium dynamics, Rg3(S) attenuates senescence, providing new mechanistic insight into the anti-aging potential of ginseng-derived compounds beyond autophagy-centered pathways.

Calcium signaling↗
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

The cellular protein TIAR mediates rapid initiation of West Nile virus genome RNA synthesis.

During the intracellular replication cycle of West Nile virus (WNV), genome RNA synthesis is initially inefficient but increases exponentially as viral replication complexes are sequestered in invaginations in the endoplasmic reticulum. In this study, we investigated the functional role of the cellular protein TIAR (T-cell intracellular antigen-related protein) in the transcription of WNV genome RNA. Close colocalization of cytoplasmic TIAR with viral double-stranded RNA was detected by a proximity ligation assay in WNV-infected cells. TIAR binds specifically to the WNV 3'(-) SL but not to the complementary WNV 5'(+) SL in in vitro RNA binding assays. Only the 3' end of the WNV minus-strand RNA was enriched by immunoprecipitation of infected cell lysates with anti-TIAR antibody. Stable overexpression of TIAR in clonal A549 cells increased the ratio of intracellular viral plus-strand to minus-strand RNA in a dose-dependent manner. TIAR contains three RNA recognition motifs (RRMs). Biophysical data indicated that only RRM2 directly contacts RNA and that up to three TIAR molecules can bind cooperatively to the WNV 3'(-) SL RNA. These data provide additional evidence that TIAR functions as a proviral host factor facilitating exponential amplification of WNV genome production in infected cells.IMPORTANCEWest Nile virus (WNV) is a mosquito-borne orthoflavivirus associated with increasing global human disease incidence. The molecular mechanisms underlying viral replication are not fully understood. In early stages of infection, viral genome transcription is inefficient; however, in late stages, viral genome transcription increases exponentially. T-cell intracellular antigen-related (TIAR) protein is a cellular protein that has been shown to interact with the 3' end of the WNV negative-sense antigenomic RNA. We obtained data showing colocalization of cellular TIAR with viral replication complexes in infected cells and an increased ratio of intracellular genomic to antigenomic viral RNA in TIAR-overexpressing cells, and confirmed preferential binding of TIAR to the 3' end of the WNV antigenome both in vitro and in infected cell extracts. We also demonstrated that multiple TIAR proteins can bind cooperatively to the WNV 3'(-) stem-loop RNA. These data provide supporting evidence for a model of TIAR-mediated rapid initiation of nascent genome RNA synthesis in infected cells.

TIAR↗
PubMed2025

Proviral functions of HMGB1 in HAdV-C5 replication compartments.

UNLABELLED: Human adenoviruses (HAdVs) induce significant reorganization of the nuclear environment, leading to the formation of virus-induced subnuclear structures known as replication compartments (RCs). Within these RCs, viral genome replication, gene expression, and modulation of cellular antiviral responses are tightly coordinated, making them valuable models for studying virus-host interactions. In a recent study, we analyzed the protein composition of HAdV type 5 (HAdV-C5) RCs isolated from infected primary cells at different time points during infection using quantitative proteomics. We identified several chromatin modifiers, including the high-mobility group box 1 protein (HMGB1) as components associated with RCs and demonstrated that HMGB1 can be relocalized to RCs from different HAdV species, thereby modulating viral replication in a species-specific manner. In the present work, using click-chemistry and proximity ligation assays, we discovered that HMGB1 localizes to sites of DNA replication within RCs and that its interaction with DBP in RCs is dependent on both DNA replication and RC assembly. HMGB1-knockdown experiments demonstrated that HMGB1 is required for efficient viral gene expression. However, despite its proviral role in viral replication, we found that HMGB1 levels decreased in late stages of infection due to transcriptional downregulation. Furthermore, by overexpressing HMGB1, we showed that this regulation of HMGB1 levels during infection is critical for optimal HAdV-C5 replication. These results highlight the complex regulatory relationship between HMGB1 and HAdV-C5 infection. IMPORTANCE: In an extensive proteomics analysis, we found that HMGB1, an important cellular chromatin protein, was enriched in adenovirus replication compartments. In this study, we aimed to better understand the role of HMGB1 in the infection process of a human DNA virus, HAdV-C5. We tested different virus types, including some with specific gene deletions and mutations. Our results showed that during infection, HMGB1 levels decreased because the virus suppressed its production. Despite this, even at lower levels, HMGB1 still helped the virus replicate by interacting with key viral proteins and DNA at sites where the virus is actively replicating. Overall, our findings highlight how HMGB1 plays a crucial role in facilitating efficient virus replication, making it an important factor in the infection process.

HMGB1 Protein↗
PubMed2026

A Family Exhibiting Autosomal Dominant Inheritance of Multiple Acyl-Coenzyme A (CoA) Dehydrogenase Deficiency (MADD) Disease.

Multiple acyl-CoA dehydrogenase deficiency (MADD) is considered an autosomal recessive disorder; yet, recent findings suggest up to 10% of cases may result from heterozygous electron transfer flavoprotein dehydrogenase (ETFDH) variants exhibiting dominant or dominant-like effects. Here, a novel heterozygous ETFDH variant (c.1798A>C, p.Asn600His) was identified within a three-generation family. The grandfather presented with muscular weakness at age 35, and the father developed similar symptoms at 19 following a tonsillectomy. Both were diagnosed with MADD based on muscle biopsies revealing neutral lipid accumulation and acylcarnitine profiles and responded fully to riboflavin therapy (150 mg/day). The two siblings, aged 8 and 10, carry the same mutation and show increased acyl-carnitine levels but remain asymptomatic due to early riboflavin treatment. Skin fibroblasts from affected individuals were immortalized and subjected to normal and reduced riboflavin levels. Gene expression analysis demonstrated unchanged ETFDH RNA but reduced protein levels in mutant cells, particularly under low riboflavin. Structural modelling suggested the Asn600His substitution destabilizes the protein, diminishing its mitochondrial function. Proximity ligation assays indicated a decreased interaction with mitochondrial complex III, while oxygen consumption via fatty acid oxidation was impaired, especially at reduced riboflavin. The novel ETFDH variant found in this family gives a possible dominant pattern of inheritance for MADD, where a single mutant allele impairs the mitochondrial metabolism, particularly under riboflavin-deficient conditions, and highlights the importance of early riboflavin supplementation in preventing clinical symptoms.

Humans↗
PubMed2006

Detection of proteins based on amino acid sequences by multiple aptamers against tripeptides.

A number of different ligands have been tested in the course of the development of protein array technology. The most extensively studied example of protein ligands has been based on antibody-antigen interaction. Other examples include protein-protein, protein-nucleic acid, and protein-small molecule interactions. All these ligands can recognize and specifically bind to protein epitopes. In this study, we have developed a novel technology using DNA-based aptamers to detect proteins based on their amino acid sequences. Mouse cathepsin D was used for the proof of principle experiment. Four tripeptides, Leu-Ala-Ser, Asp-Gly-Ile, Gly-Glu-Leu, and Lys-Ala-Ile, were selected based on the published amino acid sequence of mouse cathepsin D. DNA aptamers against the tripeptides were isolated using the systematic evolution of ligands of exponential enrichment method. We have demonstrated that the aptamers specifically interacted with mouse cathepsin D using the structure-switch method. We further performed a proximity-dependent ligation assay to demonstrate that multiple aptamers could specifically detect the protein from cell extracts. In principle, one library containing 8000 aptamers should be enough to detect almost all proteins in the whole proteome in all organisms. This technology could be applied to generate a new generation of protein arrays.

Amino Acid Sequence↗
PubMed2026

Spatial proximity sequencing maps developmental dynamics in the germinal center.

Spatial profiling of proteins and protein interactions facilitates understanding of cell functions within tissues and is essential for studies in signaling, immunity, and cancer. We present spatial proximity sequencing (Sprox-seq) for simultaneous profiling of surface proteins, protein complexes, and mRNAs, recording the tissue location of each molecule. Sprox-seq profiled 32 proteins, 528 pairwise interactions, and thousands of mRNAs with spatial resolution across human tonsils and germinal centers. Mapping tissue-wide protein interactions recapitulated RNA-defined tissue architecture but also revealed higher interaction complexity in the light zone. Protein-interaction trajectories uncovered a B cell state transition distinct from that inferred by RNA. Integrated protein-complex and mRNA analysis related spatially enriched complexes with mitotic pathways. Sprox-seq captured cell-cell interactions, such as B cell-follicular dendritic cell interactions mediated by the receptor complex VLA-4-VCAM1. Sprox-seq provides a spatially resolved multi-modal view of cell states and an integrated study of protein and cellular interactions across tissues.

Humans↗
PubMed2006

Detection of individual microbial pathogens by proximity ligation.

BACKGROUND: Nucleic acid amplification allows the detection of single infectious agents. Protein-based assays, although they provide information on ongoing infections, have substantially less detection sensitivity. METHODS: We used proximity ligation reactions to detect proteins on bacteria and virus particles via nucleic acid amplification. Antibodies recognizing viral or bacterial surface proteins were equipped with DNA strands that could be joined by ligation when several antibodies were bound in proximity to surface proteins of individual infectious agents. RESULTS: Detection sensitivities similar to those of nucleic acid-based detection reactions were achieved directly in infected samples for a parvovirus and an intracellular bacterium. CONCLUSIONS: This method enables detection of ligated DNA strands with good sensitivity by real-time PCR and could be of value for early diagnosis of infectious disease and in biodefense.

Animals↗