Cell-cell Adhesion Proteins Research Articles

Promising Support Coming from Nature: Antioxidant and Anti-Inflammatory Potential of Castanea sativa Wood Distillate on Skin Cells.

Tissue homeostasis, function recovery, and protection mechanisms are boosted by the balanced and timely control of inflammation and oxidative stress. Nowadays, many natural products and bio-derivates exhibit antioxidant and anti-inflammatory activity, supporting medical care and tissue wellness against inflammation, oxidative stress, and inflammaging. Castanea sativa wood distillate (WD) is a bio-derivative used as a corroborant and biofertilizer in agriculture. Based on the safety profile of low concentrations of WD on human cells, the present study aims to assess the anti-inflammatory and antioxidant activity of WD on different cell types in the integumentary system. Human keratinocytes, mucosal epithelium, dermal fibroblasts, and endothelial cells were exposed to WD, and the concentrations devoid of pro-apoptotic potential were profiled. Then, the effect of nontoxic doses of WD revealed an anti-inflammatory effect, observed through the immunodetection of prostanoid cascade markers in experimentally induced inflammation. A reduction in endothelial hyperpermeability was evidenced by the immunofluorescence analysis of cell-cell adhesion proteins, VE-cadherin and ZO-1. In addition, WD buffered the exogenously produced oxidative stress. On the whole, WD showed both anti-inflammatory and antioxidant activities on the various cell types, preserving endothelial barrier integrity. Overall, this study supports the involvement of this bio-derivative in novel exploitable fields, such as therapeutic dermatological applications for human and animal medical care.

Outside-in engineering of cadherin endocytosis using a conformation strengthening antibody.

P-cadherin, a crucial cell-cell adhesion protein which is overexpressed in numerous malignant cancers, is a popular target for drug delivery antibodies. However, molecular guidelines for engineering antibodies that can be internalized upon binding to P-cadherin are unknown. Here, we use a combination of biophysical, biochemical, and cell biological methods to demonstrate that trapping the cadherin extracellular region in an X-dimer adhesive conformation, triggers cadherin endocytosis via a novel outside-in signaling mechanism. We show that the monoclonal antibody CQY684 traps P-cadherin in an X-dimer conformation and strengthens this adhesive structure. Formation of stable X-dimers results in the dissociation of p120-catenin, a suppressor of cadherin endocytosis, from the X-dimer cytoplasmic region. This increases the turnover of P-cadherin and targets the cadherin-antibody complex to the lysosome. Our results establish a previously unknown outside-in signaling mechanism that provides fundamental insights into how cells regulate adhesion and that can be exploited by anti-cadherin antibodies for intracellular drug delivery.

Molecular signatures of normal pressure hydrocephalus: a large-scale proteomic analysis of cerebrospinal fluid

Given the persistent challenge of differentiating idiopathic Normal Pressure Hydrocephalus (iNPH) from similar clinical entities, we conducted an in-depth proteomic study of cerebrospinal fluid (CSF) in 28 shunt-responsive iNPH patients, 38 Mild Cognitive Impairment (MCI) due to Alzheimer’s disease, and 49 healthy controls. Utilizing the Olink Explore 3072 panel, we identified distinct proteomic profiles in iNPH that highlight significant downregulation of synaptic markers and cell-cell adhesion proteins. Alongside vimentin and inflammatory markers upregulation, these results suggest ependymal layer and transependymal flow dysfunction. Moreover, downregulation of multiple proteins associated with congenital hydrocephalus (e.g., L1CAM, PCDH9, ISLR2, ADAMTSL2, and B4GAT1) points to a possible shared molecular foundation between congenital hydrocephalus and iNPH. Through orthogonal partial least squares discriminant analysis (OPLS-DA), a panel comprising 13 proteins has been identified as potential diagnostic biomarkers of iNPH, pending external validation. These findings offer novel insights into the pathophysiology of iNPH, with implications for improved diagnosis.

Hepatic GDP-fucose transporter SLC35C1 attenuates cholestatic liver injury and inflammation by inducing CEACAM1 N153 fucosylation.

Inflammatory response is crucial for bile acid (BA)-induced cholestatic liver injury, but molecular mechanisms remain to be elucidated. Solute Carrier Family 35 Member C1 (SLC35C1) can transport GDP-fucose into the Golgi to facilitate protein glycosylation. Its mutation leads to the deficiency of leukocyte adhesion and enhances inflammation in humans. However, little is known about its role in liver diseases. Hepatic SLC35C1 mRNA transcripts and protein expression were significantly increased in patients with obstructive cholestasis (OC) and mouse models of cholestasis. Immunofluorescence revealed that the upregulated SLC35C1 expression mainly occurred in hepatocytes. Liver-specific ablation of Slc35c1 (Slc35c1 cKO) significantly aggravated liver injury in mouse models of cholestasis induced by bile duct ligation and 1% cholic acid-feeding, evidenced by increased liver necrosis, inflammation, fibrosis, and bile ductular proliferation. The Slc35c1 cKO increased hepatic chemokine Ccl2 and Cxcl2 expression and T-cell, neutrophil and F4/80 macrophage infiltration, but did not affect the levels of serum and liver BA in mouse models of cholestasis. LC-MS/MS analysis revealed that hepatic Slc35c1 deficiency substantially reduced the fucosylation of cell-cell adhesion protein CEACAM1 at N153. Mechanistically, cholestatic levels of conjugated BAs stimulated SLC35C1 expression by activating the STAT3 signaling to facilitate CEACAM1 fucosylation at N153, and deficiency in the fucosylation of CEACAM1 at N135 enhanced the BA-stimulated CCL2 and CXCL2 mRNA expression in primary mouse hepatocytes and PLC/PRF/5-ASBT cells. Elevated hepatic SLC35C1 expression attenuates cholestatic liver injury by enhancing CEACAM1 fucosylation to suppress CCL2 and CXCL2 expression and liver inflammation.

Cadherin Expression Profiles Define Glioblastoma Differentiation and Patient Prognosis.

Cadherins are cell-cell adhesion proteins which have been strongly implicated in cancer invasion, dissemination and metastasis capacity; thus, they are key players in the epithelial-to-mesenchymal transition (EMT) program. However, their role in glioblastoma (GBM), a primary central nervous system aggressive tumor, remains to be clarified. N-, E- and P-cadherin expression was analyzed on a large series of GBMs, characterized with clinical, imaging and neuropathological parameters, as well as with patients' survival data. In addition, cadherins' expression was studied in match-recurrent cases. Using TCGA data, cadherin expression profiles were also evaluated according to GBM transcription subtypes. N-cadherin expression was observed in 81.5% of GBM, followed by E-cadherin in 31% and P-cadherin in 20.8%. Upon tumor recurrence, P-cadherin was the only significantly upregulated cadherin compared with the primary tumor, being positive in 65.8% of the cases. Actually, P-cadherin gain was observed in 51.4% of matched primary-recurrent cases. Cadherins' co-expression was also explored. Interestingly, E- and N-cadherin co-expression identified a GBM subgroup with frequent epithelial differentiation and a significant survival benefit. On the other hand, subgroups with P-cadherin expression carried the worse prognosis. P- and N-cadherin co-expression correlated with the presence of a mesenchymal phenotype. Expressions of isolated P-cadherin or E- and P-cadherin co-expression were associated with imaging characteristics of aggressiveness, to highly heterogeneous tumors, an d to worse patient survival. Classical cadherins co-expression subgroups present consistent clinical, imaging, neuropathological and survival differences, which probably reflect different states of an EMT-like program in GBM.

A novel noncanonical function for IRF6 in the recycling of E-cadherin.

Interferon Regulatory Factor 6 (IRF6) is a transcription factor essential for keratinocyte cell-cell adhesions. Previously, we found that recycling of E-cadherin was defective in the absence of IRF6, yet total E-cadherin levels were not altered, suggesting a previously unknown, nontranscriptional function for IRF6. IRF6 protein contains a DNA binding domain (DBD) and a protein binding domain (PBD). The transcriptional function of IRF6 depends on its DBD and PBD, however, whether the PBD is necessary for the interaction with cytoplasmic proteins has yet to be demonstrated. Here, we show that an intact PBD is required for recruitment of cell-cell adhesion proteins at the plasma membrane, including the recycling of E-cadherin. Colocalizations and coimmunoprecipitations reveal that IRF6 forms a complex in recycling endosomes with Rab11, Myosin Vb, and E-cadherin, and that the PBD is required for this interaction. These data indicate that IRF6 is a novel effector of the endosomal recycling of E-cadherin and demonstrate a non-transcriptional function for IRF6 in regulating cell-cell adhesions.

Actin Cytoskeleton Remodeling Accompanied by Redistribution of Adhesion Proteins Drives Migration of Cells in Different EMT States.

Epithelial-mesenchymal transition (EMT) is a process during which epithelial cells lose epithelial characteristics and gain mesenchymal features. Here, we used several cell models to study migratory activity and redistribution of cell-cell adhesion proteins in cells in different EMT states: EGF-induced EMT of epithelial IAR-20 cells; IAR-6-1 cells with a hybrid epithelial-mesenchymal phenotype; and their more mesenchymal derivatives, IAR-6-1-DNE cells lacking adherens junctions. In migrating cells, the cell-cell adhesion protein α-catenin accumulated at the leading edges along with ArpC2/p34 and α-actinin. Suppression of α-catenin shifted cell morphology from fibroblast-like to discoid and attenuated cell migration. Expression of exogenous α-catenin in MDA-MB-468 cells devoid of α-catenin drastically increased their migratory capabilities. The Y654 phosphorylated form of β-catenin was detected at integrin adhesion complexes (IACs). Co-immunoprecipitation studies indicated that α-catenin and pY654-β-catenin were associated with IAC proteins: vinculin, zyxin, and α-actinin. Taken together, these data suggest that in cells undergoing EMT, catenins not participating in assembly of adherens junctions may affect cell migration.

The type of fatty acid dictates their impact on gut permeability and nutrient absorption

We showed that short- and long-term (3 to 12 weeks) high-fat diet (HFD) feeding reduced protein expression of nutrient transporters in the proximal jejunum of mice. The most abundant fatty acids (FA) in HFD were oleic (OA), palmitic (PA) and stearic (EA) acid. Because PA and EA are generally regarded as pro-inflammatory, we hypothesize that dietary PA and EA alter gut permeability and absorption via inflammatory activation. To test this hypothesis, we orally administered HFD-matched doses of OA, PA, and EA to male C57BL/6 mice, fed on purified diet control (LFD), for 3 weeks. Food intake and body weight were monitored, and composition were also evaluated. We next qPCR-assessed the mRNA expression of cytokines ( TNF-α, IL-6, IL-1β, IFN-γ, IL-10) and inflammatory signaling transducers ( MD-2 and Myd88) as well as the protein content of nutrient transporters (Glut2, Pept1, Fat/cd36 and Npc1l1) and cell-cell adhesion proteins (Occludin) in the gut. Small intestinal permeability was assessed ex vivo and gut morphology was histologically determined. In addition, Caco-2 enterocytes were used to assess the impact of specific cytokines on nutrient transport and gut barrier integrity. All dietary FA reduced food intake without affecting lean or fat mass. In the jejunum, OA increased TNF-α gene expression whereas PA upregulated IFN-γ and MD-2 gene expression. Furthermore, OA reduced the nutrient transporters Glut2, Pept1, and Fat/cd36 without affecting Npc1l1, whereas PA and EA reduced Occludin protein content. PA, but not OA and EA, increased jejunum paracellular permeability ex vivo and increased villus length. In Caco-2 cells, while Tnf-α did not affect Glut2, Pept1 and Fat/cd36 or Npc1l1 content, Ifn-γ downregulated Occludin protein content. We conclude that OA and, more potently, PA exert inflammatory effects which contribute to damage gut barrier integrity and downregulate nutrient absorption. Our data point to IFN-γ as a main player coupling dietary stress from PA to altered gut homeostasis. Funding: FAPESP (grants number: 2014/12871-9, 2019/05086-7, 2020/12201-4, and 2022/14545). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Strengthening E-cadherin adhesion via antibody-mediated binding stabilization

E-cadherins (Ecads) are a crucial cell-cell adhesion protein with tumor suppression properties. Ecad adhesion can be enhanced by the monoclonal antibody 66E8, which has potential applications in inhibiting cancer metastasis. However, the biophysical mechanisms underlying 66E8-mediated adhesion strengthening are unknown. Here, we use molecular dynamics simulations, site-directed mutagenesis, and single-molecule atomic force microscopy experiments to demonstrate that 66E8 strengthens Ecad binding by stabilizing the primary Ecad adhesive conformation: the strand-swap dimer. By forming electrostatic interactions with Ecad, 66E8 stabilizes the swapped β-strand and its hydrophobic pocket and impedes Ecad conformational changes, which are necessary for rupture of the strand-swap dimer. Our findings identify fundamental mechanistic principles for strengthening of Ecad binding using monoclonal antibodies.

Light-activated BioID - an optically activated proximity labeling system to study protein-protein interactions.

Proximity labeling with genetically encoded enzymes is widely used to study protein-protein interactions in cells. However, the accuracy of proximity labeling is limited by a lack of control over the enzymatic labeling process. Here, we present a light-activated proximity labeling technology for mapping protein-protein interactions at the cell membrane with high accuracy and precision. Our technology, called light-activated BioID (LAB), fuses the two halves of the split-TurboID proximity labeling enzyme to the photodimeric proteins CRY2 and CIB1. We demonstrate, in multiple cell lines, that upon illumination with blue light, CRY2 and CIB1 dimerize, reconstitute split-TurboID and initiate biotinylation. Turning off the light leads to the dissociation of CRY2 and CIB1 and halts biotinylation. We benchmark LAB against the widely used TurboID proximity labeling method by measuring the proteome of E-cadherin, an essential cell-cell adhesion protein. We show that LAB can map E-cadherin-binding partners with higher accuracy and significantly fewer false positives than TurboID.

Identification of Rare Genetic Variants in Familial Spontaneous Coronary Artery Dissection and Evidence for Shared Biological Pathways.

Rare familial spontaneous coronary artery dissection (SCAD) kindreds implicate genetic disease predisposition and provide a unique opportunity for candidate gene discovery. Whole-genome sequencing was performed in fifteen probands with non-syndromic SCAD who had a relative with SCAD, eight of whom had a second relative with extra-coronary arteriopathy. Co-segregating variants and associated genes were prioritized by quantitative variant, gene, and disease-level metrics. Curated public databases were queried for functional relationships among encoded proteins. Fifty-four heterozygous coding variants in thirteen families co-segregated with disease and fulfilled primary filters of rarity, gene variation constraint, and predicted-deleterious protein effect. Secondary filters yielded 11 prioritized candidate genes in 12 families, with high arterial tissue expression (n = 7), high-confidence protein-level interactions with genes associated with SCAD previously (n = 10), and/or previous associations with connective tissue disorders and aortopathies (n = 3) or other vascular phenotypes in mice or humans (n = 11). High-confidence associations were identified among 10 familial SCAD candidate-gene-encoded proteins. A collagen-encoding gene was identified in five families, two with distinct variants in COL4A2. Familial SCAD is genetically heterogeneous, yet perturbations of extracellular matrix, cytoskeletal, and cell-cell adhesion proteins implicate common disease-susceptibility pathways. Incomplete penetrance and variable expression suggest genetic or environmental modifiers.

LCK and resistance to cytotoxic chemotherapy via activating an AXL-PEAK1 signaling axis in cholangiocarcinoma.

27 Background: Cholangiocarcinoma (CCA) is a devastating cancer with few effective treatment options. We have previously discovered LCK can activate AXL, a TAM receptor tyrosine kinase (RTK), by phosphorylation of AXL-Y866 ( J Hepatol 2022). AXL is known to confer therapeutic resistance to solid cancers. However, the role of AXL in CCA, and the impact of AXL-Y866 activation by LCK still remains to be elucidated. Here, we investigated the significance of AXL expression, signaling, and activation by LCK on Y866. Methods: We first evaluated the expression levels of AXL in CCA and its association with patient outcome using The Cancer Genome Atlas (TCGA) dataset. Next, to evaluate whether AXL inactivation sensitizes CCA cells to gemcitabine and cisplatin (GC), AXL downregulation was achieved via siRNA approach and the selective AXL inhibitor bemcentinib (Bem) using human CCA cell lines, HuCCT1 and RBE, and murine SB-1. We examined 50% inhibitory concentration (IC50) value on GC with or without AXL knockdown (KD) using cell viability assay. Then we assessed the efficacy of the combination of GC and Bem utilizing Calcusyn software and apoptosis by Annexin V assay. I n vivo efficacy was assessed using both a patient derived CCA xenograft and SB-1 a syngeneic model of CCA treated with vehicle, GC, Bem, or the combination. Next, we defined the interactome of wildtype AXL- Y866 compared to AXL-Y866F using biotin proximity labelling methods (BioID), validation of downstream target proteins using Western blot (WB), and a functional study with AXL-/- cells reconstituted with either AXL WT, or AXL Y866F. Results: In TCGA cohorts, AXL transcripts are more abundant than in normal adjacent liver, and higher levels are correlated with worse clinical outcome. In in vitro study, IC50 values of GC decreased after AXL KD. Synergistic effects were observed in the combination. The combination caused increased apoptosis compared to other treatments. In the in vivo studies, the combination suppressed tumor growth and the Ki67 levels, and the phosphorylation levels of PEAK1 decreased in the combination group compared to other groups. In BioID experiments, among the 222 proteins which were detected in both AXL WT and AXL Y866F interactomes, PEAK1 kinase, which localizes to actin cytoskeleton and focal adhesions, was most altered when Y866 was inactivated by the Y866F mutation. In gene ontology analysis, cell-cell adhesion and focal adhesion related proteins were enriched. In WB, we validated that the phosphorylation of PEAK1 decreased after AXL or LCK knockout (KO) and after Bem treatment in above in vivo study. In cell viability assay, reconstitution of AXL KO cells with WT AXL increased the IC50 to GC while reconstitution with AXL-Y866F did not. Conclusions: AXL inhibition sensitizes CCA cells to cytotoxic chemotherapy in preclinical model. LCK-AXL Y866-PEAK1 signaling axis is a potential target for the treatment of CCA.

Optically activated proximity labeling for spatially and temporally resolved proteomics.

Membrane protein interactions are essential for the proliferation, differentiation and survival of cells. However, due to the lack of appropriate technologies, the interactomes of membrane proteins, at distinct spatial locations within the cell and at different stages of the cell cycle, are poorly understood. Here, we develop a light-activated proximity labeling method for high-precision mapping of membrane protein interactions with high spatial and temporal resolution. Our method combines a split version of the proximity biotinylation enzyme, TurboID, with the optically activated protein dimerization pair CRY2 and CIB1. As proof of principle, we engineer cells expressing CIB1 and an inactive TurboID fragment on the cell membrane along with the complementary TurboID fused to CRY2 in the cytoplasm. Using immunofluorescence imaging and mass spectrometry, we demonstrate that the split-TurboID fragments assemble, become functional and biotinylate proteins, only upon exposure to light. Next we tag E-cadherin, an essential cell-cell adhesion protein, with our optically activated proximity labeling system and validate it by comparing the measured interactome upon light exposure with known E-cadherin binding partners. Our results demonstrate that light-triggered proximity labeling can map the E-cadherin interactome with high precision. We anticipate that our optically activated technique will serve as a revolutionary tool to map junctional protein interactions within cells, with high fidelity, in a previously unprecedented, spatially and temporally resolved fashion.

Characterization of TGF-β signaling in a human organotypic skin model reveals that loss of TGF-βRII induces invasive tissue growth.

Transforming growth factor-β (TGF-β) signaling regulates various aspects of cell growth and differentiation and is often dysregulated in human cancers. We combined genetic engineering of a human organotypic three-dimensional (3D) skin model with global quantitative proteomics and phosphoproteomics to dissect the importance of essential components of the TGF-β signaling pathway, including the ligands TGF-β1, TGF-β2, and TGF-β3, the receptor TGF-βRII, and the intracellular effector SMAD4. Consistent with the antiproliferative effects of TGF-β signaling, the loss of TGF-β1 or SMAD4 promoted cell cycling and delayed epidermal differentiation. The loss of TGF-βRII, which abrogates both SMAD4-dependent and SMAD4-independent downstream signaling, more strongly affected cell proliferation and differentiation than did loss of SMAD4, and it induced invasive growth. TGF-βRII knockout reduced cell-matrix interactions, and the production of matrix proteins increased the production of cancer-associated cell-cell adhesion proteins and proinflammatory mediators and increased mitogen-activated protein kinase (MAPK) signaling. Inhibiting the activation of the ERK and p38 MAPK pathways blocked the development of the invasive phenotype upon the loss of TGF-βRII. This study provides a framework for exploring TGF-β signaling pathways in human epithelial tissue homeostasis and transformation using genetic engineering, 3D tissue models, and high-throughput quantitative proteomics and phosphoproteomics.

425 GRPR is an effective target for melanoma

While tremendous progress has been made in the understanding of the mechanisms leading to cancers, the understanding of the mechanisms governing the formation of metastases remains poorly understood. The cell-cell adhesion protein E-cadherin (E-cad) has been associated with the metastatic development of melanoma, but the mechanisms have not yet been described nor has the actual impact of E-cad been evaluated. Here, using a novel mouse model expressing the oncogenic form NRASQ61K in melanocyte, we show that the conditional deletion of Cdh1 (which encode E-cad) does not influence melanoma initiation but dramatically promotes metastases.

Role of TM3 in claudin-15 strand flexibility: A molecular dynamics study.

Claudins are cell-cell adhesion proteins within tight junctions that connect epithelial cells together. Claudins polymerize into a network of strand-like structures within the membrane of adjoining cells and create ion channels that control paracellular permeability to water and small molecules. Tight junction morphology and barrier function is tissue specific and regulated by claudin subtypes. Here, we present a molecular dynamics study of claudin-15 strands within lipid membranes and the role of a single-point mutation (A134P) on the third transmembrane helix (TM3) of claudin-15 in determining the morphology of the strand. Our results indicate that the A134P mutation significantly affects the lateral flexibility of the strands, increasing the persistence length of claudin-15 strands by a factor of three. Analyses of claudin-claudin contact in our μsecond-long trajectories show that the mutation does not alter the intermolecular contacts (interfaces) between claudins. However, the dynamics and frequency of interfacial contacts are significantly affected. The A134P mutation introduces a kink in TM3 of claudin-15 similar to the one observed in claudin-3 crystal structure. The kink on TM3 skews the rotational flexibility of the claudins in the strands and limits their fluctuation in one direction. This asymmetric movement in the context of the double rows reduces the lateral flexibility of the strand and leads to higher persistence lengths of the mutant.

Molecular mechanism for strengthening E-cadherin adhesion using a monoclonal antibody

E-cadherin (Ecad) is an essential cell-cell adhesion protein with tumor suppression properties. The adhesive state of Ecad can be modified by the monoclonal antibody 19A11, which has potential applications in reducing cancer metastasis. Using X-ray crystallography, we determine the structure of 19A11 Fab bound to Ecad and show that the antibody binds to the first extracellular domain of Ecad near its primary adhesive motif: the strand-swap dimer interface. Molecular dynamics simulations and single-molecule atomic force microscopy demonstrate that 19A11 interacts with Ecad in two distinct modes: one that strengthens the strand-swap dimer and one that does not alter adhesion. We show that adhesion is strengthened by the formation of a salt bridge between 19A11 and Ecad, which in turn stabilizes the swapped β-strand and its complementary binding pocket. Our results identify mechanistic principles for engineering antibodies to enhance Ecad adhesion.

The guidance and adhesion protein FLRT2 dimerizes in cis via dual small-X3-small transmembrane motifs

Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate cell adhesion and guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact at the surface of the same cell, in cis, remains unknown. Here, molecular dynamics simulations reveal two dimerization motifs in the FLRT2 transmembrane helix. Single particle tracking experiments show that these Small-X3-Small motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and co-diffusion patterns of FLRT2 receptors on cells. These results may point to a competitive switching mechanism between in cis and in trans interactions, which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules.

Cadherins can dimerize via asymmetric interactions.

Cadherins are essential cell-cell adhesion proteins that interact in two distinct conformations: X-dimers and strand-swap dimers. Both X-dimers and strand-swap dimers are thought to exclusively rely on symmetric sets of interactions between key amino acids on both cadherin binding partners. Here, we use single-molecule atomic force microscopy and computer simulations to show that symmetry in cadherin binding is dispensable and that cadherins can also interact in a novel conformation that asymmetrically incorporates key elements of both strand-swap dimers and X-dimers. Our results clarify the biophysical rules for cadherin binding and demonstrate that cadherins interact in a more diverse range of conformations than previously understood.

Multi-omics analysis of the cervical epithelial integrity of women using depot medroxyprogesterone acetate.

Depot medroxyprogesterone acetate (DMPA) is an injectable hormonal contraceptive used by millions of women worldwide. However, experimental studies have associated DMPA use with genital epithelial barrier disruption and mucosal influx of human immunodeficiency virus (HIV) target cells. We explored the underlying molecular mechanisms of these findings. Ectocervical biopsies and cervicovaginal lavage (CVL) specimens were collected from HIV-seronegative Kenyan sex workers using DMPA (n = 32) or regularly cycling controls (n = 64). Tissue samples were assessed by RNA-sequencing and quantitative imaging analysis, whereas protein levels were measured in CVL samples. The results suggested a DMPA-associated upregulation of genes involved in immune regulation, including genes associated with cytokine-mediated signaling and neutrophil-mediated immunity. A transcription factor analysis further revealed DMPA-associated upregulation of RELA and NFKB1 which are involved in several immune activation pathways. Several genes significantly downregulated in the DMPA versus the control group were involved in epithelial structure and function, including genes encoding keratins, small proline-rich proteins, and cell-cell adhesion proteins. Pathway analyses indicated DMPA use was associated with immune activation and suppression of epithelium development, including keratinization and cornification processes. The cervicovaginal microbiome composition (Lactobacillus dominant and non-Lactobacillus dominant) had no overall interactional impact on the DMPA associated tissue gene expression. Imaging analysis verified that DMPA use was associated with an impaired epithelial layer as illustrated by staining for the selected epithelial junction proteins E-cadherin, desmoglein-1 and claudin-1. Additional staining for CD4+ cells revealed a more superficial location of these cells in the ectocervical epithelium of DMPA users versus controls. Altered protein levels of SERPINB1 and ITIH2 were further observed in the DMPA group. Identification of specific impaired epithelial barrier structures at the gene expression level, which were verified at the functional level by tissue imaging analysis, illustrates mechanisms by which DMPA adversely may affect the integrity of the genital mucosa.