Proximity Ligation Research Articles

Proxi-RIMS-seq2 applied to native microbiomes uncovers hundreds of known and novel m5C methyltransferase specificities.

Methylation patterns in bacteria can be used to study restriction-modificationor other defense systems with novel properties. While m4C and m6A methylation are well characterized mainly through PacBio sequencing, the landscape of m5C methylation is under-characterized. To bridge this gap, we performed RIMS-seq2 (rapid identification of methyltransferase specificity sequencing) on microbiomes composed of resolved assemblies of distinct genomes through proximity ligation. This high-throughput approach enables the identification of m5C methylated motifs and links them to cognate methyltransferases directly on native microbiomes without the need to isolate bacterial strains. Methylation patterns can also be identified on bacteriophage DNA and compared withhost DNA, strengthening evidence for phage-host interactions. Applied to three different microbiomes, the method unveiled over 1900 motifs that were deposited in REBASE. The motifs include a novel eight-base recognition site (CATm5CGATG) that was experimentally validated by characterizing its cognate methyltransferase. Our findings suggest that microbiomes harbor arrays of untapped m5C methyltransferase specificities, providing insights intobacterial biology and biotechnological applications.

A Novel Hidden Protein p-414aa Encoded by circSETD2(14,15) Inhibits Vascular Remodeling.

Phenotypic switching of vascular smooth muscle cells (VSMCs), leading to neointimal hyperplasia, is a fundamental cause of vascular remodeling diseases such as atherosclerosis and hypertension. Novel hidden proteins encoded by circular RNAs play crucial roles in disease progression, yet their involvement in vascular remodeling diseases has not been comprehensively studied. This study identifies a novel protein derived from a circular RNA in VSMCs and demonstrates its potential role in regulating vascular remodeling. Cell proliferation assays were performed to investigate the effects of circSETD2(14,15) on VSMC proliferation. Techniques such as vector construction, immunoprecipitation-mass spectrometry, and dual-luciferase reporter gene were used to confirm that circSETD2(14,15) encoded a novel protein, p-414aa. The interaction between p-414aa and HuR (human antigen R) was validated with techniques such as coimmunoprecipitation, mass spectrometry, and proximity ligation assay. Through experiments including RNA sequencing and RNA immunoprecipitation, the interaction between HuR and C-FOS (C-Fos proto-oncogene) mRNA was revealed. The role of p-414aa in neointimal hyperplasia was assessed with a carotid artery ligation model in male mice. Overexpression of circSETD2(14,15) inhibits VSMC phenotypic switching. The novel protein p-414aa, encoded by circSETD2(14,15), interacts with HuR to reduce C-FOS mRNA stability, thereby suppressing VSMC proliferation and ultimately inhibiting neointimal hyperplasia in male mice. We uncover a novel hidden protein derived from circSETD2(14,15), called p-414aa, that inhibits vascular remodeling. CircSETD2(14,15) and p-414aa may serve as potential therapeutic targets for vascular remodeling diseases.

Chromosome-level genome assembly of the spangled emperor, Lethrinus nebulosus (Forsskål 1775)

Spangled emperor, Lethrinus nebulosus (Forsskål 1775), is a tropical marine fish of economic and cultural importance throughout the Indo-West Pacific. It is one of the most targeted recreational fishes in the Gascoyne Coast Bioregion of Western Australia where it serves as an indicator species for recreational fishing. Here, we present a highly accurate, near-gapless, chromosome-level, haplotype-phased reference genome assembly of L. nebulosus (Lethrinus nebulosus (Spangled Emperor) genome, fLetNeb1.1; PRJNA1074345), the first for the species and the first high-quality genome representative of the family Lethrinidae. The 1.09 Gb genome was assembled from PacBio HiFi and Dovetail Omni-C proximity ligation sequencing data. The contig N50 is 21–24 Mbp and BUSCO completeness greater than 99%. A preliminary gene annotation identified 24,583 genes with the predicted transcriptome achieving a BUSCO completeness score of 99.1% This resource will facilitate genomic studies to inform the sustainable management of L. nebulosus and other Lethrinids.

Domain-specific folding of the tandem β-propeller protein Coronin 7 (Coro7) by CCT/TRiC.

The Chaperonin containing tailless complex polypeptide 1 (CCT) or TCP-1 ring complex (TRiC) plays a central role in maintaining cellular homeostasis by supporting protein folding and damping protein aggregation. Besides the abundant cytoskeletal proteins, actin and tubulin, CCT/TRiC is emerging as an obligate chaperone for WD40 proteins, which are comprised of one or multiple β-propeller domains. To date, only WD40 proteins consisting of a single β-propeller domain have been described as CCT/TRiC substrates. Using a combination of biotin proximity ligation, mass spec analysis and co-immunoprecipitation, we here identify the tandem β-propeller protein, Coronin 7 (Coro7), as a novel CCT/TRiC interactor. Transient knockdown of CCT/TRiC further severely diminished expression of Coro7, suggesting that Coro7 is a bona fide CCT/TRiC substrate. Interestingly, co-immunoprecipitation of truncated Coro7 proteins demonstrated that CCT/TRiC only interacts with the first β-propeller domain of Coro7. In line with this, fusion of a miniTurboID tag to the N- or C-terminus of Coro7 showed significant enrichment of all CCT/TRiC subunits for the first, but not the second β-propeller domain. Similarly, co-immunoprecipitation with individual Coro7 β-propeller domains generated by introduction of a protease cleavage site in full length Coro7, confirmed that CCT/TRiC only binds to the first β-propeller domain. Altogether, our study shows that CCT/TRiC can also function as a chaperone for multi-β-propeller domain proteins, likely by initiating the folding of the first β-propeller domain, which can then help template autonomous folding of consecutive β-propeller domains.

Detection of rare plasmid hosts using a targeted Hi-C approach.

Despite the significant role plasmids play in microbial evolution, there is limited knowledge of their ecology, evolution, and transfer in microbial communities. This is partly due to the limitations of current methods in associating a plasmid with its host in microbiomes. To address this knowledge gap, we developed and implemented a novel approach to identify rare plasmid hosts by combining Hi-C, a proximity ligation method, with enrichment for plasmid-specific DNA. We hereafter refer to this approach as Hi-C+. We applied Hi-C and Hi-C+ to soil microbial communities in which we mimicked increasingly rare transfer of an antimicrobial resistance plasmid from a donor to a recipient. This was achieved by inoculating agricultural soil with mixtures of known plasmid-containing and plasmid-free cells at different proportions. We demonstrated that Hi-C can link a plasmid to its host in soil when the relative abundance of that plasmid-host pair is as low as 0.001%. Hi-C+ further improved the detection limit of Hi-C 100-fold and allowed the identification of plasmid hosts at the genus level. As a culture-independent approach, Hi-C+ will significantly improve our understanding of the range and frequency of spread of antibiotic resistance and other genes that are introduced into soil and other microbiomes.

Maf-family bZIP transcription factor NRL interacts with RNA-binding proteins and R-loops in retinal photoreceptors.

RNA-binding proteins (RBPs) perform diverse functions including the regulation of chromatin dynamics and the coupling of transcription with RNA processing. However, our understanding of their actions in mammalian neurons remains limited. Using affinity purification, yeast-two-hybrid and proximity ligation assays, we identified interactions of multiple RBPs with neural retina leucine (NRL) zipper, a Maf-family transcription factor critical for retinal rod photoreceptor development and function. In addition to splicing, many NRL-interacting RBPs are associated with R-loops, which form during transcription and increase during photoreceptor maturation. Focusing on DHX9 RNA helicase, we demonstrate that its expression is modulated by NRL and that the NRL-DHX9 interaction is positively influenced by R-loops. ssDRIP-Seq analysis reveals both stranded and unstranded R-loops at distinct genomic elements, characterized by active and inactive epigenetic signatures and enriched at neuronal genes. NRL binds to both types of R-loops, suggesting an epigenetically independent function. Our findings suggest additional functions of NRL during transcription and highlight complex interactions among transcription factors, RBPs, and R-loops in regulating photoreceptor gene expression in the mammalian retina.

Targeting the Galectin-7/TRPM2/Zn2+/DRP-1 Signaling Pathway: A Potential Therapeutic Intervention in the Pathogenesis of SJS/TEN.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) represent a spectrum of severe drug-induced cutaneous reactions. These conditions are characterized by widespread and confluent keratinocyte apoptosis, which differentiates them from erythema multiforme (EM). Mounting evidence has implicated the mitochondrial-dependent apoptosis pathway in the pathogenesis of SJS/TEN, but the potential roles and specific mechanisms of these pathways in SJS/TEN remain largely unexplored. Proteomic analyses were conducted to investigate differential protein expression in blister fluid (BF)-derived exosomes from suction surgery in healthy individuals (Con Exo) or patients with EM (EM Exo) or SJS/TEN (TEN Exo). Further analysis involved glutathione S-transferase (GST) pull-down assay, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and validation of MS results through proximity ligation assay (PLA) and coimmunoprecipitation (co-IP). Phenotypic and mechanistic analyses were performed using immunohistochemistry (IHC) staining, enzyme-linked immunosorbent assay (ELISA), western blotting, reverse transcription-polymerase chain reaction (RT-PCR), co-IP, CCK-8 assay, adenosine triphosphate (ATP) level measurements, and flow cytometry. Galectin-7 was markedly upregulated in BF-derived exosomes from SJS/TEN patients and showed a correlation with disease severity. Further analysis confirmed the interaction between galectin-7 and transient receptor potential (melastatin) 2 (TRPM2). BF-derived exosomes from SJS/TEN patients induced an imbalance in mitochondrial dynamics via galectin-7/TRPM2 upregulation. Activation of TRPM2 led to an elevation in mitochondrial Zn2+, which facilitated the recruitment of the fission factor dynamin-related protein-1 (DRP-1) to mitochondria to trigger mitochondrial fission in the keratinocyte. In addition, the recruitment of DRP-1-dependent mitochondrial fission via the voltage-dependent anion channel 1 (VDAC1)/hexokinase 2 (HK2)-mediated opening of the mitochondrial permeability transition pore (mPTP)-triggered cytochrome c release. These effects ultimately induce activation of the intrinsic mitochondrial apoptotic pathway and contribute to the pathogenesis of SJS/TEN. Targeting the galectin-7/TRPM2/Zn2+/DRP-1 signaling pathway in keratinocytes presents a prospective therapeutic strategy for mitigating SJS/TEN in the future.

Puromycin Proximity Ligation Assay (Puro-PLA) to Assess Local Translation in Axons From Human Neurons.

Local mRNA translation in axons is crucial for the maintenance of neuronal function and homeostasis, particularly in processes such as axon guidance and synaptic plasticity, due to the long distance from axon terminals to the soma. Recent studies have shown that RNA granules can hitchhike on the surface of motile lysosomal vesicles, facilitating their transport within the axon. Accordingly, disruption of lysosomal vesicle trafficking in the axon, achieved by knocking out the lysosome-kinesin adaptor BLOC-one-related complex (BORC), decreases the levels of a subset of mRNAs in the axon. This depletion impairs the local translation of mitochondrial and ribosomal proteins, leading to mitochondrial dysfunction and axonal degeneration. Various techniques have been developed to visualize translation in cells, including translating RNA imaging by coat protein knock-off (TRICK), SunTag, and metabolic labeling using the fluorescent non-canonical amino acid tagging (FUNCAT) systems. Here, we describe a sensitive technique to detect newly synthesized proteins at subcellular resolution, the puromycin proximity ligation assay (Puro-PLA). Puromycin, a tRNA analog, incorporates into nascent polypeptide chains and can be detected with an anti-puromycin antibody. Coupling this method with the proximity ligation assay (PLA) allows for precise visualization of newly synthesized target proteins. In this article, we describe a step-by-step protocol for performing Puro-PLA in human induced pluripotent stem cell (iPSC)-derived neuronal cultures (i3Neurons), offering a powerful tool to study local protein synthesis in the axon. This tool can also be applied to rodent neurons in primary culture, enabling the investigation of axonal protein synthesis across species and disease models. Key features • Establishment of quantitative local translation assay in axons of human iPSC-derived neurons. • Microscopy-based direct visualization of local translation events in neurons. • Puro-PLA is a sensitive method for detecting new protein synthesis occurring within minutes in neurons, enabling precise temporal analysis of translation dynamics.

Targeting uPARAP Modifies Lymphatic Vessel Architecture and Attenuates Lymphedema.

Lymphedema is an incurable disease associated with lymphatic dysfunction that causes tissue swelling and fibrosis. We investigated whether lymphedema could be attenuated by interfering with uPARAP (urokinase plasminogen activator receptor-associated protein; Mrc2 gene), an endocytic receptor involved in fibrosis and lymphangiogenesis. We generated mice with lymphatic endothelial cell (LEC)-specific uPARAP deficiency and compared them with constitutive knockout mice by applying a preclinical model of secondary lymphedema (SL). Computerized methods were applied for 2-dimensional and 3-dimensional image quantifications. Cellular effects of uPARAP deletion on lymphatic permeability were assessed by small interfering RNA-mediated silencing in human dermal LECs and a pharmacologic treatment targeting ROCK (rho-associated coiled coil containing kinase), an established regulator of cell junctions. The uPARAP and vascular endothelial cadherin partnership was investigated through proximity ligation assay, coimmunoprecipitation, and immunostaining. An in silico model was generated to analyze the fluid-absorbing function of the lymphatic vasculature. To interfere with uPARAP, its downregulation was achieved in vivo through a gapmer approach. uPARAP deficiency mitigated several key pathologic features of SL, including hindlimb swelling, epidermal thickening, and the accumulation and size of adipocytes. In both global and LEC-conditional uPARAP-deficient mice, induction of SL led to a distinctive labyrinthine vasculature, defined herein by twisted and hyperbranched vessels with overlapping cells. This topology, mainly composed of pre-collecting vessels, correlated with reduced SL, but not with change in fibrosis, highlighting the importance of uPARAP in regulating LEC functions in a lymphedematous context. In vitro, uPARAP knockdown in LECs impaired vascular endothelial growth factor C-mediated endosomal trafficking of vascular endothelial cadherin and induced overlapping cell junctions. The pharmacologic inhibition of ROCK recapitulated cell superimposition in vitro and the labyrinthine vasculature in vivo with attenuated SL. Computational modeling of labyrinthine lymphatic vasculature supported the observation on their improved fluid-absorbing function in comparison with a normal hierarchic network. These data provide proof of concept of inducing a labyrinthine topology to treat SL. For therapeutic purposes, we validated the use of an anti-uPARAP gapmer to induce a labyrinthine vasculature and attenuate SL formation. Our findings provide evidence that downregulating uPARAP expression can induce a beneficial remodeling of lymphatic vasculature that attenuates lymphedema through a cell junction-based mechanism, offering a novel therapeutic pathway for lymphedema.

Aggregated proinsulin in pancreatic β-cells is degraded by the autophagy pathway.

Aggregated proinsulin in pancreatic β-cells is degraded by the autophagy pathway.

ALKBH5 promotes cardiac fibroblasts pyroptosis after myocardial infarction through Notch1/NLRP3 pathway.

ALKBH5 promotes cardiac fibroblasts pyroptosis after myocardial infarction through Notch1/NLRP3 pathway.

Nanobody-assisted nanoluciferase fragment complementation for in situ measurement and visualization of endogenous protein-protein interaction.

Nanobody-assisted nanoluciferase fragment complementation for in situ measurement and visualization of endogenous protein-protein interaction.

GCIP and SIRT6 cooperatively suppress ITGAV gene expression by modulating c-myc transcription ability.

GCIP and SIRT6 cooperatively suppress ITGAV gene expression by modulating c-myc transcription ability.

Translesion-synthesis-mediated bypass of DNA lesions occurs predominantly behind replication forks restarted by PrimPol.

Translesion-synthesis-mediated bypass of DNA lesions occurs predominantly behind replication forks restarted by PrimPol.

Shotgun and Hi-C Sequencing Datasets for Binning Wheat Rhizosphere Microbiome

Binning is a crucial process in metagenomics studies, where sequenced reads are combined to form longer contigs and assigned to individual genomes. Conventional methods, such as shotgun binning, rely on similarity measurements and abundance profiles across multiple samples. However, cost constraints for sequencing and limited sample collection capacity hinder their effectiveness. High-throughput chromosome conformation capture (Hi-C), a DNA proximity ligation technique, has been adapted to accurately bin metagenome-assembled genomes (MAGs) from a single sample, addressing challenges like chimeric MAGs. In this study, we generated over 190 Gb of metagenomic data from wheat rhizospheres grown in two highly calcareous soils of South Australian region and compared conventional and Hi-C binning methods. Two shotgun metagenomes and Hi-C libraries were generated, assembling 1089 shotgun MAGs across 39 bacterial and one archaeal taxon, including 94 Hi-C based bins. Binning performed using only short read sequences was prone to high contamination, while the addition of Hi-C binning improved MAG quality and identified mobile element-host-infection interaction. This dataset provides important tools for studying microbial communities in wheat rhizosphere soils.

Spatiotemporal translation of sperm acrosome associated proteins during early capacitation modulates sperm fertilizing ability.

Spatiotemporal translation of sperm acrosome associated proteins during early capacitation modulates sperm fertilizing ability.

Mannose-6-phosphate attenuates acute lung injury by competitive release of acid sphingomyelinase from the mannose-6-phosphate receptor in endothelial caveolae.

Platelet-activating factor (PAF)-induced pulmonary endothelial barrier failure is mediated by acid sphingomyelinase (ASM) translocation to caveolae. ASM, however, lacks a transmembrane domain for anchoring inside caveolae. We hypothesized that ASM may anchor to cation-independent mannose-6-phosphate receptor (CI-M6PR) in caveolae from where it can be competitively released by M6P. We probed for ASM-CI-M6PR interaction by co-immunoprecipitation (Co-IP) and proximity ligation assay (PLA) in isolated lungs and human pulmonary microvascular endothelial cells (hPMVECs). ASM release by M6P was determined in hPMVECs, isolated lungs and in vivo. Effects of M6P i) on PAF-induced lung edema formation and endothelial Ca2+ concentration ([Ca2+]i), and ii) lung injury in acid instilled, overventilated mouse lungs were tested. ASM levels were measured in serum and bronchoalveolar lavage fluid (BALF) of ARDS patients. The TriNetX database was probed for the association of ASM-inhibiting tricyclic antidepressants (TCA) with outcome. Co-IP and PLA revealed ASM interaction with CI-M6PR in endothelial caveolae, which was further increased by PAF. M6P, but not glucose-6-phosphate (Glu6P) caused ASM release, thereby decreasing ASM content and activity in caveolae in vitro, in situ and in vivo. Analogously, M6P, yet not Glu6P attenuated PAF-induced endothelial [Ca2+]i signaling and lung edema in situ, and acute lung injury in vivo. ASM levels were increased in serum, yet not BALF in ARDS patients. Use of TCA was associated with better outcome in patients with severe respiratory infections. CI-M6PR anchors ASM in caveolae. M6P may hence present a promising strategy against ASM-related lung injury and edema.

Parasitic plasmids are anchored to inactive regions of eukaryotic chromosomes through a nucleosome signal

Natural plasmids are common in prokaryotes, but few have been documented in eukaryotes. The natural 2µ plasmid present in the yeast Saccharomyces cerevisiae is one of these best-characterized exceptions. This highly stable genetic element has coexisted with its host for millions of years, faithfully segregating at each cell division through a mechanism that remains unclear. Using proximity ligation methods (such as Hi-C, Micro-C) to map the contacts between 2µ plasmid and yeast chromosomes under dozens of different biological conditions, we found that the plasmid is tethered preferentially to regions with low transcriptional activity, often corresponding to long, inactive genes. These contacts do not depend on common chromosome-structuring factors, such as members of the structural maintenance of chromosome complexes (SMC) but depend on a nucleosome-encoded signal associated with RNA Pol II depletion. They appear stable throughout the cell cycle and can be established within minutes. This chromosome hitchhiking strategy may extend beyond the 2µ plasmid/S. cerevisiae pair, as suggested by the binding pattern of the natural eukaryotic plasmid Ddp5 along silent chromosome regions of the amoeba Dictyostelium discoideum.

Regulation of CXCR4 function by S1P1 through heteromerization

BackgroundThe trafficking of immune cells between lymphoid organs and circulation depends on gradients of CXCL12 and sphingosine-1-phosphate (S1P), mediated through their cognate receptors C-X-C chemokine receptor type 4 (CXCR4) and S1P receptor type 1 (S1P1). S1P1 facilitates the egress of hematopoietic stem cells and lymphocytes by counteracting CXCR4-mediated retention signals. However, the molecular mechanisms underlying this interplay remain poorly understood. In this study, we uncover CXCR4-S1P1 heteromerization and explore their functional interactions.MethodsBimolecular fluorescence complementation (BiFC) assay, proximity ligation assay (PLA), and quantitative bioluminescence resonance energy transfer (BRET) assay were employed to detect CXCR4-S1P1 heteromerization. Functional properties of the heteromers were assessed using cAMP assay, G protein activation, β-arrestin recruitment, ligand binding, calcium mobilization, and transwell migration assays. S1P1-overexpressing Jurkat T cells were generated via lentiviral transduction, while S1P1-deficient KARPAS299 cells and β-arrestin1/2-deficient HEK293A cells were constructed using the CRISPR/Cas9 system.ResultsCXCR4-S1P1 heteromerization was observed in HEK293A cells overexpressing both receptors. The S1P/S1P1 axis interfered with CXCR4-mediated signaling, while CXCR4 did not affect S1P1-mediated signaling, indicating a unidirectional modulation of CXCR4 by S1P1. CXCL12 binding to CXCR4 remained unchanged in the presence of S1P1, and interference of CXCL12-induced Gαi activation by S1P1 was observed in β-arrestin1/2-deficient cells. BRET analysis revealed that S1P1 interfered with CXCR4-Gαi pre-association and CXCR4 oligomerization, both of which are critical for CXCR4 function. Domain-swapping experiments identified transmembrane domain 3 of S1P1 as essential for this modulation. In Jurkat T cells overexpressing S1P1, CXCR4-mediated signaling and cell migration were diminished, whereas these functions were enhanced in S1P1-deficient KARPAS299 cells. Co-activation of S1P1 attenuated CXCL12-induced migration, while pretreatment with S1P or FTY720-phosphate increased CXCR4-mediated migration by downregulating surface S1P1 in KARPAS299 cells. In primary T cells, PLA confirmed CXCR4-S1P1 heteromerization, and S1P interfered with CXCL12-induced migration.ConclusionsThis study identifies CXCR4-S1P1 heteromers and demonstrates a unidirectional modulation of CXCR4 by S1P1. S1P1 affects CXCR4 function by disrupting its G protein pre-association and oligomerization. These findings underscore the regulatory role of the S1P/S1P1 axis in CXCR4 signaling within the heteromeric context and provide novel insights into the intricate mechanisms governing immune cell trafficking.

Chromosome level genome assembly and annotation of the Swanson rainbow trout homozygous line

The genome of the Swanson doubled haploid (DH) YY male line of rainbow trout was de novo assembled using the Canu pipeline, high-coverage PacBio long-read sequence data, Bionano optical maps, and Hi-C proximity ligation sequence data, resulting in 29 major scaffolds aligning with the karyotype of the Swanson line (2 N = 58). This assembly, totaling 2.3 Gb with an N50 of 52.4 Mb, represents approximately 95% of the genome in 29 chromosome sequences with only 109 gaps between scaffolds. Notably, corrections to previous errors in the Swanson line genome assembly were made, including the identification of a double large inversion on the Omy05 chromosome (~57 Mb), the absence of the Omy20 inversion between the Arlee and Swanson assemblies, and the discovery of a ~6.7 Mb inversion on Omy26. This comprehensive assembly contributes to refining the rainbow trout reference genome and serves as a valuable resource for future genetic studies within this species.