Disintegrin Domain Research Articles

Corrigendum: A disintegrin and metalloproteinase domain 10 expression inhibition by the small molecules adenosine, cordycepin and N6, N6-dimethyladenosine and immune regulation in malignant cancers

Corrigendum: A disintegrin and metalloproteinase domain 10 expression inhibition by the small molecules adenosine, cordycepin and N6, N6-dimethyladenosine and immune regulation in malignant cancers

Fully human monoclonal antibody targeting the cysteine-rich substrate-interacting region of ADAM17 on cancer cells

ADAM17 sheds EGFR/erbB ligands and triggers oncogenic pathways that lead to the progression of solid tumors. We targeted the ADAM17 disintegrin and cysteine rich domain region (D+C) to generate a panel of single-chain antibody fragments (scFvs) that selectively bind to the D or C domains of ADAM17, but not of ADAM10 or ADAM19. From the panel, we selected one scFv, referred to as C12, based on its high binding affinity towards the target, and re-formatted it to a full IgG for further studies. High-resolution cryo-electron microscopy studies documented that the mAb binds to the ADAM17 C-domain that in ADAM proteases, notably ADAM10 and ADAM17, is known to impart substrate-specificity. The C12 mAb significantly inhibited EGFR phosphorylation in cancer cell lines by hindering the cleavage of EGFR ligands tethered to the cell surface. This inhibition provides a mechanism for potential anti-tumor effects, and indeed C12 diminished the viability of a variety of EGFR-expressing cancer cell lines. Cell-based ELISA studies revealed that C12 preferentially bound to activated ADAM17 present on tumor cells, as compared to the autoinhibited ADAM17 that is the predominant form on HEK293 and other non-tumor cells. C12 also exhibited tumor growth inhibition in an ovarian cancer xenograft mouse model. Consistent with its selective tumor cell binding in vitro, radioimmuno PET (positron emission tomography) imaging with 89Zr-DFO-C12 in mouse xenograft models confirmed tumoral accumulation of the C12 mAb.

Potential probiotic Lactiplantibacillus plantarum strains alleviate TNF-α by regulating ADAM17 protein and ameliorate gut integrity through tight junction protein expression in in vitro model

BackgroundLactiplantibacillus species are extensively studied for their ability to regulate host immune responses and functional therapeutic potentials. Nevertheless, there is a lack of understanding on the mechanisms of interactions with the hosts during immunoregulatory activities.MethodsTwo Lactiplantibacillus plantarum strains MKMB01 and MKMB02 were tested for probiotic potential following Indian Council of Medical Research (ICMR) guidelines. Human colorectal adenocarcinoma cells such as HT-29, caco-2, and human monocytic cell THP-1 were also used to study the potential of MKMB01 and MKMB02 in regulating the host immune response when challenged with enteric pathogen Salmonella enterica typhimurium. Cells were pre-treated with MKMB01 and MKMB02 for 4 h and then stimulated with Salmonella. qRT-PCR and ELISA were used to analyze the genes and protein expression. Confocal microscopy and field emission scanning electron microscopy (FESEM) were used to visualize the effects. An Agilent Seahorse XF analyzer was used to determine real-time mitochondrial functioning.ResultsBoth probiotic strains could defend against Salmonella by maintaining gut integrity via expressing tight junction proteins (TJPs), MUC-2, and toll-like receptors (TLRs) negative regulators such as single Ig IL-1-related receptor (SIGIRR), toll-interacting protein (Tollip), interleukin-1 receptor-associated kinase (IRAK)-M, A20, and anti-inflammatory transforming growth factor-β and interleukin-10. Both strains also downregulated the expression of pro-inflammatory cytokines/chemokines interleukin-1β, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor-alpha (TNF-α), interleukin 6, and nitric oxide (NO). Moreover, TNF-α sheddase protein, a disintegrin and metalloproteinase domain 17 (ADAM17), and its regulator iRhom2 were downregulated by both strains. Moreover, the bacteria also ameliorated Salmonella-induced mitochondrial dysfunction by restoring bioenergetic profiles, such as non-mitochondrial respiration, spare respiratory capacity (SRC), basal respiration, adenosine triphosphate (ATP) production, and maximal respiration.ConclusionsMKMB01 and MKMB02 can reduce pathogen-induced gut-associated disorders and therefore should be further explored for their probiotic potential.

Adam19 Deficiency Impacts Pulmonary Function: Human GWAS Follow-up in a Mouse Knockout Model

PurposeOver 550 loci have been associated with human pulmonary function in genome-wide association studies (GWAS); however, the causal role of most remains uncertain. Single nucleotide polymorphisms in a disintegrin and metalloprotease domain 19 (ADAM19) are consistently related to pulmonary function in GWAS. Thus, we used a mouse model to investigate the causal link between Adam19 and pulmonary function.MethodsWe created an Adam19 knockout (KO) mouse model and validated the gene targeting using RNA-Seq and RT-qPCR. Mouse body composition was assessed using dual-energy X-ray absorptiometry. Mouse lung function was measured using flexiVent.ResultsContrary to prior publications, the KO was not neonatal lethal. KO mice had lower body weight and shorter tibial length than wild-type (WT) mice. Their body composition revealed lower soft weight, fat weight, and bone mineral content. Adam19 KO had decreased baseline respiratory system elastance, minute work of breathing, tissue damping, tissue elastance, and forced expiratory flow at 50% forced vital capacity but higher FEV0.1 and FVC. Adam19 KO had attenuated tissue damping and tissue elastance in response to methacholine following LPS exposure. Adam19 KO also exhibited attenuated neutrophil extravasation into the airway after LPS administration compared to WT. RNA-Seq analysis of KO and WT lungs identified several differentially expressed genes (Cd300lg, Kpna2, and Pttg1) implicated in lung biology and pathogenesis. Gene set enrichment analysis identified negative enrichment for TNF pathways.ConclusionOur murine findings support a causal role of ADAM19, implicated in human GWAS, in regulating pulmonary function.

ADAM10 Alleviates Hypoxia/Reoxygenation-Induced Cardiomyocyte Injury by Activating the Notch Signaling Pathway.

The occurrence of myocardial ischemia/reperfusion injury is commonly observed during cardiac surgery; however, there remains a dearth of effective therapeutic strategies to mitigate this injury. The a disintegrin and metallopeptidase domain 10 (ADAM10) is a transmembrane protein anchored on the cell membrane surface, and its precise mechanism of action in myocardial ischemia/reperfusion injury remains incompletely understood. This study aims to investigate the impact of ADAM10 on cardiomyocyte injury induced by hypoxia/reoxygenation (H/R) and elucidate the underlying mechanisms. The ADAM10 overexpression plasmid was transfected into H9c2 cells, which were subsequently treated with the Notch signaling pathway inhibitor DAPT and cultured under H/R conditions. Cell proliferation activity was assessed using the CCK-8 assay. The levels of LDH, SOD, and MDA were quantified through colorimetric analysis. The levels of ROS and the rate of apoptosis were measured using flow cytometry. The morphological changes in the nucleus of H9c2 cells were observed by employing Hoechst 33258 staining. The mRNA expression levels of ADAM10, Notch1, NICD, and Hes1 in H9c2 cells were determined using qRT-PCR. The expressions of Notch signaling pathway and apoptosis-related proteins were analyzed by Western blot. Overexpression of ADAM10 provided protection to H9c2 cells against injury induced by H/R, leading to an increase in SOD levels and alleviation of oxidative stress caused by the accumulation of ROS and the decrease of SOD activity. Meanwhile, overexpression of ADAM10 inhibited apoptosis in H9c2 cells exposed to H/R by regulating the expression of apoptosis-related proteins, such as Bax, Bcl-2 and Cleaved-caspase-3. Additionally, overexpression of ADAM10 facilitated the activation of the Notch1 signaling pathway in H9c2 cells exposed to H/R by upregulating the protein expression of Notch1, NICD, and Hes1. However, the protective effect of ADAM10 on H/R-induced H9c2 cells was partially reversed by DAPT. Our findings demonstrate that ADAM10 exerts protective effects in H/R-induced H9c2 cells by suppressing oxidative stress and apoptosis via the activation of the Notch signaling pathway.

ADAM9 promotes type I interferon-mediated innate immunity during encephalomyocarditis virus infection.

Viral myocarditis, an inflammatory disease of the heart, causes significant morbidity and mortality. Type I interferon (IFN)-mediated antiviral responses protect against myocarditis, but the mechanisms are poorly understood. We previously identified A Disintegrin And Metalloproteinase domain 9 (ADAM9) as an important factor in viral pathogenesis. ADAM9 is implicated in a range of human diseases, including inflammatory diseases; however, its role in viral infection is unknown. Here, we demonstrate that mice lacking ADAM9 are more susceptible to encephalomyocarditis virus (EMCV)-induced death and fail to mount a characteristic type I IFN response. This defect in type I IFN induction is specific to positive-sense, single-stranded RNA (+ ssRNA) viruses and involves melanoma differentiation-associated protein 5 (MDA5)-a key receptor for +ssRNA viruses. Mechanistically, ADAM9 binds to MDA5 and promotes its oligomerization and thereby downstream mitochondrial antiviral-signaling protein (MAVS) activation in response to EMCV RNA stimulation. Our findings identify a role for ADAM9 in the innate antiviral response, specifically MDA5-mediated IFN production, which protects against virus-induced cardiac damage, and provide a potential therapeutic target for treatment of viral myocarditis.

The Emerging Role of ADAM 12 Regulates Epithelial-mesenchymal Transition by Activating the Wnt/β-catenin Signaling Pathway in Colorectal Cancer.

The objective of this study is to investigate the expression and regulatory mechanisms of A disintegrin and metalloproteinase domain 12 (ADAM12) in colorectal cancer (CRC) tissues and cells. Download and analyze the expression levels of ADAM12 in the TCGA and GSE68468 datasets. Collect paraffin-preserved specimens from the Chongqing University Jiangjin Hospital from April 2017 to December 2019 and detect the expression of ADAM12 through immunohistochemistry. Cell experiments were conducted using colorectal cancer cell lines (SW480, HCT116), and cells with high expression of ADAM12 were selected for silencing experiments, and cell proliferation ability using CCK-8, and migration ability of cells in each group using scratch assay and Transwell invasion assay. The EMT markers (E-cadherin, N-cadherin, Vimentin, Twist) and the Wnt/β-catenin markers (β-catenin, GSK-3β, p-GSK-3β, C-MYC, MMP-7) were detected using western blot. We construct a nude mouse CRC tumor model and validate the effect of ADAM12 on EMT and Wnt/β-catenin through immunohistochemistry and Western blot. Bioinformatics showed that increased expression of ADAM12 was strongly correlated with patient prognosis. Immunohistochemistry showed that elevated ADAM12 was associated with vascular invasion (p < 0.05), neurological invasion (p < 0.01), lymph node metastasis (p < 0.01), and TNM staging (p < 0.001). Experiments on cell function revealed that the ADAM12 overexpression group augmented CRC cells' proliferation and migration. After overexpression of ADAM12, the expression of N-cadherin, Vimentin, and Twist increased, while the expression of E-cadherin decreased (p < 0.01). The expression of Proteins related to Wnt/β-catenin: β-catenin, p-GSK-3 β, C-MYC and MMP-7 increased (p < 0.01), and Wnt/β-catenin inhibitor MSAB can counteract the effect of ADAM12 on EMT in CRC cells. The subcutaneous tumor formation experiment results in nude mice showed that ADAM12 promoted tumor growth and induced EMT compared to the control group. ADAM12 overexpression through the Wnt/β-catenin signal axis controls the EMT of CRC to promote invasion and metastasis.

A Novel Class of Human ADAM8 Inhibitory Antibodies for Treatment of Triple-Negative Breast Cancer.

New targeted treatments are urgently needed to improve triple-negative breast cancer (TNBC) patient survival. Previously, we identified the cell surface protein A Disintegrin And Metalloprotease 8 (ADAM8) as a driver of TNBC tumor growth and spread via its metalloproteinase and disintegrin (MP and DI) domains. In proof-of-concept studies, we demonstrated that a monoclonal antibody (mAb) that simultaneously inhibits both domains represents a promising therapeutic approach. Here, we screened a hybridoma library using a multistep selection strategy, including flow cytometry for Ab binding to native conformation protein and in vitro cell-based functional assays to isolate a novel panel of highly specific human ADAM8 dual MP and DI inhibitory mAbs, called ADPs. The screening of four top candidates for in vivo anti-cancer activity in an orthotopic MDA-MB-231 TNBC model of ADAM8-driven primary growth identified two lead mAbs, ADP2 and ADP13. Flow cytometry, hydrogen/deuterium exchange-mass spectrometry (HDX-MS) and alanine (ALA) scanning mutagenesis revealed that dual MP and DI inhibition was mediated via binding to the DI. Further testing in mice showed ADP2 and ADP13 reduce aggressive TNBC characteristics, including locoregional regrowth and metastasis, and improve survival, demonstrating strong therapeutic potential. The continued development of these mAbs into an ADAM8-targeted therapy could revolutionize TNBC treatment.

The ADAM17 inhibitor ZLDI-8 sensitized hepatocellular carcinoma cells to sorafenib through Notch1-integrin β-talk

ZLDI-8 is an A disintegrin and metalloproteinase domain 17 (ADAM17) inhibitor that suppresses the shedding of Notch1 to the Notch1 intracellular domain (NICD). In previous studies, we found that ZLDI-8 was able to sensitize HCC to sorafenib, but the mechanism of action remains unclear. The sensitizing effects of ZLDI-8 were tested both in vitro and in vivo. EMT-related factors, sorafenib sensitivity-related proteins and ECM-related gene expression were assessed using immunohistochemistry, RTPCR and Western blotting. Knockdown assays were conducted to determine the relationship between the Notch and Integrin pathways. CoIP assays, nuclear and cytoplasmic fractionation and immunofluorescence colocalization were applied to explore the interaction between the Notch and Integrin pathways. Appropriate statistical analysis methods were used to assess the significance of the experimental results and to ensure the scientific validity and reliability of the experimental design. We found that ECM- and EMT-related proteins were downregulated after ZLDI-8 treatment (P<0.05). ZLDI-8 significantly downregulated Integrinβ1 and Integrinβ3 in HCC in vitro and in vivo (P<0.05), possibly through Foxc2-dependent regulation. Mechanistically, interfering with the expression of both Integrin-linked kinase (ILK) and the NICD may downregulate the expression of proteins targeted by sorafenib, thereby sensitizing cells to sorafenib. The retroregulation of Integrinβ by ILK may occur through the interaction between the NICD and ILK and may be the result of the translocation of the complexus. Our study indicates that blocking the Notch pathway may affect Integrinβ through crosstalk between the Notch1 and Integrinβ/ILK signaling pathways, thus providing a potential therapeutic strategy for HCC.

Abstract 1897: Preclinical development of MGC028, an ADAM9-targeted, glycan-linked, exatecan-based antibody-drug conjugate for the treatment of solid cancers

Abstract Introduction: Antibody-drug conjugates (ADCs) seek to increase the therapeutic window of potent cytotoxic agents by linking them with monoclonal antibodies (mAbs) to selectively deliver cytotoxic payloads to tumor cells. The effectiveness and safety of ADCs rely on both the mAb specificity, and the linker-payload employed. Several approved ADCs using microtubule inhibitor payloads are impacted by ocular adverse events that have been observed both preclinically and in patients. A class of linker-payloads incorporating topoisomerase 1 inhibitors (TOP1i) has emerged recently as an effective alternative to tubulin inhibitor-based ADCs. To date, TOP1i ADCs have not been associated with dose-limiting ocular toxicity seen with microtubule inhibitor payloads. Here we report the preclinical development of an ADAM9 (a disintegrin and metalloprotease domain 9)-targeted ADC that incorporates a novel glycan-linked TOP1i. ADAM9, a member of the ADAM family of multifunctional type 1 transmembrane proteins, plays a role in tumorigenesis and cancer progression and is overexpressed in multiple cancers, making it an attractive target for cancer treatment. Methods: MGC028 incorporates the cleavable linker-payload, bicyclononyne carbamoyl sulfamide Val-Ala-PABC exatecan (SYNtecan E™), site-specifically conjugated at asparagine 297 of the heavy chain through enzymatic glycan remodeling and metal-free click chemistry using Synaffix’s GlycoConnect™ technology. In vivo efficacy studies were performed in immunodeficient mice with ADAM9-expressing human tumor cell-line (CDX) or patient-derived (PDX) xenografts. A non-human primate toxicology study was conducted in which MGC028 was administered by 15-minute IV infusion every two weeks at dose levels of 22.5 and 55 mg/kg for a total of two doses. Results: MGC028 exhibited specific, dose-dependent in vivo antitumor activity toward ADAM9-positive CDX models representing gastric, lung, pancreatic and colorectal cancer, and head and neck squamous cell carcinoma. Furthermore, MGC028 demonstrated antitumor activity toward ADAM9-positive PDX models of lung and pancreatic cancer, and cholangiocarcinoma. MGC028 was well tolerated in a repeat-dose non-human primate toxicology study, up to 55 mg/kg, the highest dose level tested. Observations were limited to mild, reversible increases in liver enzymes, without microscopic correlates, and decreased lymphoid cellularity in the thymus. In particular, ocular toxicities were not observed. Conclusions: MGC028 exhibited potent antitumor activity in in vivo models representing various solid cancer indications and was well tolerated in non-human primates at exposure levels exceeding those required for antitumor activity. Our findings support continued investigation of MGC028 as an ADC therapeutic for the treatment of ADAM9-expressing solid cancers. Citation Format: Juniper A. Scribner, Jennifer G. Brown, Thomas Son, Linda Jin, Carroll McKenzie, Viktoriya Nam, Curtis Bush, Dienis Quinonez, Delta Ford, Verlene Gonzalez, James Tamura, Sergey Gorlatov, Hua Li, Shelley Butler, Ezio Bonvini, Deryk Loo. Preclinical development of MGC028, an ADAM9-targeted, glycan-linked, exatecan-based antibody-drug conjugate for the treatment of solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1897.

A disintegrin and metalloproteinase domain 10 expression inhibition by the small molecules adenosine, cordycepin and N6, N6-dimethyladenosine and immune regulation in malignant cancers.

A disintegrin and metalloproteinase domain 10 (ADAM10), a member of the ADAM family, is a cellular surface protein with potential adhesion and protease/convertase functions. The expression regulations in cancers by natural products [adenosine (AD) and its analogs, cordycepin (CD), and N6, N6-dimethyladenosine (m6 2A)], and immune regulation are unclear. As results, AD, CD, and m6 2A inhibited ADAM10 expression in various cancer cell lines, indicating their roles in anti-cancer agents. Further molecular docking with ADAM10 protein found the binding energies of all docking groups were <-7 kcal/mol for all small-molecules (AD, CD and m6 2A), suggesting very good binding activities. In addition, analysis of the immunomodulatory roles in cancer showed that ADAM10 was negatively correlated with immunomodulatory genes such as CCL27, CCL14, CCL25, CXCR5, HLA-B, HLA-DOB1, LAG3, TNFRSF18, and TNFRSF4 in bladder urothelial carcinoma, thymoma, breast invasive carcinoma, TGCT, kidney renal papillary cell carcinoma, SKCM and thyroid carcinoma, indicating the immune-promoting roles for ADAM10. LAG3 mRNA levels were reduced by both AD and CD in vivo. ADAM10 is also negatively associated with tumor immunosuppression and interrelated with the immune infiltration of tumors. Overall, the present study determined ADAM10 expression by AD, CD and m6 2A, and in AD or CD/ADAM10/LAG3 signaling in cancers, and suggested a potential method for immunotherapy of cancers by targeting ADAM10 using the small molecules AD, CD and m6 2A.

ADAM9 drives the immunosuppressive microenvironment by cholesterol biosynthesis-mediated activation of IL6-STAT3 signaling for lung tumor progression.

Chronic inflammation associated with lung cancers contributes to immunosuppressive tumor microenvironments, reducing CD8+ T-cell function and leading to poor patient outcomes. A disintegrin and metalloprotease domain 9 (ADAM9) promotes cancer progression. Here, we aim to elucidate the role of ADAM9 in the immunosuppressive tumor microenvironment. A bioinformatic analysis of TIMER2.0 was used to investigate the correlation of ADAM9 and to infiltrate immune cells in the human lung cancer database and mouse lung tumor samples. Flow cytometry, immunohistochemistry, and RNA sequencing (RNA-seq) were performed to investigate the ADAM9-mediated immunosuppressive microenvironment. The coculture system of lung cancer cells with immune cells, cytokine array assays, and proteomic approach was used to investigate the mechanism. By analyzing the human LUAD database and the mouse lung cancer models, we showed that ADAM9 was associated with the immunosuppressive microenvironment. Additionally, ADAM9 released IL6 protein from cancer cells to inhibit IL12p40 secretion from dendritic cells, therefore leading to dendritic cell dysfunction and further affecting T-cell functions. Proteomic analysis indicated that ADAM9 promoted cholesterol biosynthesis and increased IL6-STAT3 signaling. Mechanistically, ADAM9 reduced the protein stability of LDLR, resulting in reduced cholesterol uptake and induced cholesterol biosynthesis. Moreover, LDLR reduction enhanced IL6-STAT3 activation. We reveal that ADAM9 has a novel biological function that drives the immunosuppressive tumor microenvironment by linking lung cancer's metabolic and signaling axes. Thus, by targeting ADAM9 an innovative and promising therapeutic opportunity was indicated for regulating the immunosuppression of lung cancer.

ADAM8 deficiency in macrophages promotes angiogenesis to antagonize post-myocardial infarction cardiac injury

Abstract Background A disintegrin and metallopeptidase domain 8 (ADAM8) may be closely related to the progress of cardiovascular diseases. ADAM8 was first found in macrophages from mice, and ADAM family is closely related to the secretion function of macrophages. Therefore, ADAM8 may affect the process of myocardial infarction (MI) by regulating macrophage function. Purpose This study aims to explore how ADAM8 regulating the function of macrophages to inhibit the cardiac repair after MI. Methods CRISPR/Cas9 system was used to generate macrophage-specific ADAM8 knockout mice (ADAM8flox/flox,Lyz2-Cre, ADAM8 mKO) and ADAM8flox/flox (control) . Bone marrow transplantation was performed and macrophage-specific ADAM8 overexpressed adeno-associated virus (AAV6-CD68-Adam8) was generated. Proteomics of hearts and RNA-sequencing of bone marrow derived macrophages (BMDM) was performed. Cellular and molecular assays were used to discover the underlying mechanisms of ADAM8 function. Results (1) ADAM8 in serum of AMI patients was significantly higher than control group. ADAM8 was highly expressed in cardiac macrophages after day 3 of AMI. (2) Compared with the control group, the expression of CD31 and VEGFA in the infarcted myocardium was significantly increased in ADAM8 mKO mice, indicating enhanced angiogenesis. The cardiac fibrosis was decreased and heart function was improved in ADAM8 mKO group. ADAM8 overexpression can reverse the increase of angiogenesis caused by ADAM8 mKO and promote cardiac fibrosis in mice. The bone marrow transplantation experiments also showed that donor of ADAM8 mKO mice had increased angiogenesis and reduced cardiac fibrosis in the infarcted area. (3) The proteomics of hearts in AMI day 14, showed the Cardiac muscle contraction pathway and the Citrate cycle (TCA cycle) pathway in ADAM8 mKO mice were both increased significantly, indicating improved cardiac function and maintained the metabolic stability of heart after MI. Myocardial injection in situ of anti-VEGFA antibody can reverse the angiogenesis induced by ADAM8 mKO, and deteriorate heart function. (4) The supernatants and protein lysates of ADAM8 KO BMDM showed the decreased level of VEGFA, and IL-1β/IL-18. (5) RNA-sequencing showed that AMPK signaling pathway and autophagy pathway were both significantly increased in ADAM8 KO BMDM treated with LPS. p-AMPK/AMPK and autophagy-related molecules were obviously elevated in ADAM8 KO BMDM. Application of autophagy inhibitor 3-Methyladenine (3-MA) could reverse ADAM8 KO induced secretion of VEGFA and IL-1β/IL-18. Conclusions Our study has discovered ADAM8 as a new regulator of myocardial infarction. ADAM8 KO may promoting VEGFA secretion and inhibiting inflammatory factors secretion of macrophages via AMPK/autophagy pathway, further promoting angiogenesis and reducing fibrosis of infarcted myocardium, and attenuating heart function and adverse remodeling. ADAM8 may be a new target for treating myocardial infarction.Schematic diagram

Cotadutide improves brown adipose tissue thermogenesis in obese mice

We studied the effect of cotadutide, a dual agonist glucagon-like peptide 1 (GLP1)/Glucagon, on interscapular brown adipose tissue (iBAT) remodeling and thermogenesis of obese mice. Twelve-week-old male C57BL/6 mice were fed a control diet (C group, n = 20) or a high-fat diet (HF group, n = 20) for ten weeks. Then, animals were redivided, adding cotadutide treatment: C, CC, HF, and HFC for four additional weeks. The multilocular brown adipocyte structure showed fat conversion (whitening), hypertrophy, and structural disarray in the HF group, which was reverted in cotadutide-treated animals. Cotadutide enhances the body temperature, thermogenesis, and sympathetic innervation (peroxisome proliferator-activated receptor-α, β3 adrenergic receptor, interleukin 6, and uncoupled protein 1), reduces pro-inflammatory markers (disintegrin and metallopeptidase domain, morphogenetic protein 8a, and neuregulin 4), and improves angiogenesis (vascular endothelial growth factor A, and perlecan). In addition, cotadutide enhances lipolysis (perilipin and cell death-inducing DNA fragmentation factor α), mitochondrial biogenesis (nuclear respiratory factor 1, transcription factor A mitochondrial, mitochondrial dynamin-like GTPase, and peroxisome proliferator-activated receptor gamma coactivator 1α), and mitochondrial fusion/fission (dynamin-related protein 1, mitochondrial fission protein 1, and parkin RBR E3 ubiquitin protein ligase). Cotadutide reduces endoplasmic reticulum stress (activating transcription factor 4, C/EBP homologous protein, and growth arrest and DNA-damage inducible), and extracellular matrix markers (lysyl oxidase, collagen type I α1, collagen type VI α3, matrix metallopeptidases 2 and 9, and hyaluronan synthases 1 and 2). In conclusion, the experimental evidence is compelling in demonstrating cotadutide's thermogenic effect on obese mice's iBAT, contributing to unraveling its action mechanisms and the possible translational benefits.

Mitigative effect of Naringenin in bleomycin induced systemic sclerosis model; role of Notch signaling pathway

Background An immunological, proinflammatory, and vascular disorder called systemic sclerosis (SSc) frequently leading to progressive tissue fibrosis. Reactive oxidizing species are thought to have a substantial influence on disease initiation and progression, based on a variety of studies. Also, Notch signaling is known to modulate fibroblast homeostasis, angiogenesis, and lymphocyte development. Aim This experimental study was designed to shed light on the possible ameliorating mechanism of naringenin as antioxidant in bleomycin-induced SSc model via focusing on Notch signaling cascade. Materials and methods 50 male albino mice were employed for the test, while being allocated randomly to one of five equal groups as follows: Control group (group I), Naringenin-treated group (group II), Bleomycin-treated group (group III), and group IV (Naringenin/Bleomycin co-treated group) and group V (Bleomycin followed by Naringenin treated group). Plasma hydrogen peroxide level, skin tissue hydrogen peroxide and hydroxyproline levels were measured using colorimetric assay. A Disintegrin and metalloproteinase domain containing protein 17 and neuregulin 1 levels in skin tissue were measured by Enzyme-linked immunosorbent assay. Histological evaluation was also performed. Results Plasma hydrogen peroxide, skin tissue hydrogen peroxide, hydroxyproline, a Disintegrin and metalloproteinase domain containing protein 17 and neuregulin 1 levels in the bleomycin-treated group were significantly increased than other studied groups. Naringenin administration in parallel with the induction mitigated the obtained biochemical changes and protected against the chemical induction of SSc. Conclusion Naringenin could protect against bleomycin-induced SSc through its antioxidant role.

Analysis and Identification of Putative Novel Peptides Purified from Iranian Endemic Echis Carinatus Sochureki Snake Venom by MALDI-TOF Mass Spectrometry.

The Iranian Echis Carinatus (IEC) venom is an exclusive natural source of bio-substances for a wide range of purposes in the blood coagulation cascade. The present study for the first time was aimed to assess novel pro-coagulant, anti-coagulant and anti-platelet proteins, named EC1.5 (a), EC5.1 (b) and EC4 (a) from Iranian Echis Carinatus (IEC) venom. These peptides were purified by multi-step chromatography methods. Hematological properties were measured using activated clotting tests, platelet aggregation studies, and hemorrhage assessment. Subsequently, these proteins were identified through both their intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Multiple sequence alignments were performed by ClustalW, Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of proteins.EC1.5 (a), a single-band protein with a molecular mass of 66 and 55 kDa, was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a reduced and non-reduced state, respectively. Based on the Mascot results, we considered that EC1.5 (a) is a metalloproteinase of group ΙΙ which exhibited potent pro-coagulant activity. It is predicted that the EC1.5 (a) with hemorrhagic activity, potentially is a metalloproteinase/disintegrin region that constitutes the disintegrin-like domains. Our findings demonstrate that the disintegrin domain of EC1.5 (a) lacks platelet aggregation inhibitory activity. On the contrary, this factor shows the property of a platelet aggregation inducer. Also, the EC5.1 (b) was observed as a single-band protein with a molecular mass of 7.5 kDa. EC5.1 (b) showed both anti-coagulant and anti-platelet properties. Additionally, the structure of the EC5.1 (b) fraction is expected to be similar to that of phospholipase A2, while EC4 (a) structure is potentially very similar to that of Echistatin with 5 kDa molecular mass. We introduce the predicted structure of P-II snake venom metalloproteinase/ disintegrin domains, phospholipase A2 and Echistatin-like fractions. Further research is therefore needed to determine the complete structure of these novel fractions and elucidate their mechanism of action and future therapeutic applications of cardiovascular and homeostasis disorders.

A disintegrin and metalloproteinase domain 9 facilitates SARS-CoV-2 entry into cells with low ACE2 expression.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the Coronavirus disease-19 (COVID-19) pandemic, utilizes angiotensin-converting enzyme 2 (ACE2) as a receptor for virus infection. However, the expression pattern of ACE2 does not coincide with the tissue tropism of SARS-CoV-2, hinting that other host proteins might be involved in facilitating SARS-CoV-2 entry. To explore potential host factors for SARS-CoV-2 entry, we performed an arrayed shRNA screen in H1650 and HEK293T cells. Here, we identified a disintegrin and a metalloproteinase domain 9 (ADAM9) protein as an important host factor for SARS-CoV-2 entry. Our data showed that silencing ADAM9 reduced virus entry, while its overexpression promoted infection. The knockdown of ADAM9 decreased the infectivity of the variants of concern tested-B.1.1.7 (alpha), B.1.617.2 (delta), and B.1.1.529 (omicron). Furthermore, mechanistic studies indicated that ADAM9 is involved in the binding and endocytosis stages of SARS-CoV-2 entry. Through immunoprecipitation experiments, we demonstrated that ADAM9 binds to the S1 subunit of the SARS-CoV-2 Spike. Additionally, ADAM9 can interact with ACE2, and co-expression of both proteins markedly enhances virus infection. Moreover, the enzymatic activity of ADAM9 facilitates virus entry. Our study reveals an insight into the mechanism of SARS-CoV-2 virus entry and elucidates the role of ADAM9 in virus infection. IMPORTANCE COVID-19, an infectious respiratory disease caused by SARS-CoV-2, has greatly impacted global public health and the economy. Extensive vaccination efforts have been launched worldwide over the last couple of years. However, several variants of concern that reduce the efficacy of vaccines have kept emerging. Thereby, further understanding of the mechanism of SARS-CoV-2 entry is indispensable, which will allow the development of an effective antiviral strategy. Here, we identify a disintegrin and metalloproteinase domain 9 (ADAM9) protein as a co-factor of ACE2 important for SARS-CoV-2 entry, even for the variants of concern, and show that ADAM9 interacts with Spike to aid virus entry. This virus-host interaction could be exploited to develop novel therapeutics against COVID-19.

A disintegrin and metalloproteinase 22 activates integrin β1 through its disintegrin domain to promote the progression of pituitary adenoma.

Approximately 35% of pituitary adenoma (PA) display an aggressive profile, resulting in low surgical total resection rates, high recurrence rates, and worse prognosis. However, the molecular mechanism of PA invasion remains poorly understood. Although "a disintegrin and metalloproteinases" (ADAMs) are associated with the progression of many tumors, there are no reports on ADAM22 in PA. PA transcriptomics databases and clinical specimens were used to analyze the expression of ADAM22. PA cell lines overexpressing wild-type ADAM22, the point mutation ADAM22, the mutated ADAM22 without disintegrin domain, and knocking down ADAM22 were generated. Cell proliferation/invasion assays, flow cytometry, immunohistochemistry, immunofluorescence, co-immunoprecipitation, mass spectrometry, Reverse transcription-quantitative real-time PCR, phos-tag SDS-PAGE, and Western blot were performed for function and mechanism research. Nude mice xenograft models and rat prolactinoma orthotopic models were used to validate in vitro findings. ADAM22 was significantly overexpressed in PA and could promote the proliferation, migration, and invasion of PA cells. ADAM22 interacted with integrin β1 (ITGB1) and activated FAK/PI3K and FAK/ERK signaling pathways through its disintegrin domain to promote PA progression. ADAM22 was phosphorylated by PKA and recruited 14-3-3, thereby delaying its degradation. ITGB1-targeted inhibitor (anti-itgb1) exerted antitumor effects and synergistic effects in combination with somatostatin analogs or dopamine agonists in treating PA. ADAM22 was upregulated in PA and was able to promote PA proliferation, migration, and invasion by activating ITGB1 signaling. PKA may regulate the degradation of ADAM22 through post-transcriptional modification levels. ITGB1 may be a potential therapeutic target for PA.

Analysis and Identification of Putative Novel Peptides Purified from Iranian Endemic Echis Carinatus Sochureki Snake Venom by MALDI-TOF Mass Spectrometry

The Iranian Echis Carinatus (IEC) venom is an exclusive natural source of bio-substances for a wide range of purposes in the blood coagulation cascade. The present study for the first time was aimed to assess novel pro-coagulant, anti-coagulant and anti-platelet proteins, named EC1.5 (a), EC5.1 (b) and EC4 (a) from Iranian Echis Carinatus (IEC) venom.These peptides were purified by multi-step chromatography methods. Hematological properties were measured using activated clotting tests, platelet aggregation studies, and hemorrhage assessment. Subsequently, these proteins were identified through both their intact molecular mass and peptide mass fingerprint (PMF) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Multiple sequence alignments were performed by ClustalW, Bioedit software. Molegro Data Modeller (MDM) 3.0 software was used to predict the putative tertiary structure of proteins.EC1.5 (a), a single-band protein with a molecular mass of 66 and 55 kDa, was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a reduced and non-reduced state, respectively. Based on the Mascot results, we considered that EC1.5 (a) is a metalloproteinase of group ΙΙ which exhibited potent pro-coagulant activity. It is predicted that the EC1.5 (a) with hemorrhagic activity, potentially is a metalloproteinase/disintegrin region that constitutes the disintegrin-like domains. Our findings demonstrate that the disintegrin domain of EC1.5 (a) lacks platelet aggregation inhibitory activity. On the contrary, this factor shows the property of a platelet aggregation inducer. Also, the EC5.1 (b) was observed as a single-band protein with a molecular mass of 7.5 kDa. EC5.1 (b) showed both anti-coagulant and anti-platelet properties. Additionally, the structure of the EC5.1 (b) fraction is expected to be similar to that of phospholipase A2, while EC4 (a) structure is potentially very similar to that of Echistatin with 5 kDa molecular mass. We introduce the predicted structure of P-II snake venom metalloproteinase/ disintegrin domains, phospholipase A2 and Echistatin-like fractions. Further research is therefore needed to determine the complete structure of these novel fractions and elucidate their mechanism of action and future therapeutic applications of cardiovascular and homeostasis disorders.

The G allele of the ADAM33 T1 polymorphism (rs2280091) is a risk factor associated with asthma severity among the Iraqi Arab population

BackgroundThe gene encoding a disintegrin and metalloproteinase domain 33 (ADAM33) is known to be associated with asthma in different ethnic groups. In Iraq, among the Arab ethnic background, this association has not yet been highlighted. MethodsOne hundred and ninety-two asthmatics were examined; 118 males and 74 females (mean age 38.23 ± 9.13 years). The control group was 183; 110 males and the rest were females. The SNP of rs2280091 A/G (T1) was studied here to determine adam33 genotyping status using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP). The level of total IgE was measured using enzyme-linked immunosorbent assay (ELISA). ResultsSignificant differences (p = 0.004) in the frequencies of the ADAM33 mutant allele carriers between patients and controls were found OR = 1.62 (95% CI 1.16–2.27). This association was significant in individuals who carried homozygous (GG) or heterozygous (AG) variant type genotypes in a reference with carriers of wild-type (AA) genotypes. The odds ratios were 1.70 (95% CI 1.11–2.60, p = 0.013), 2.97 (95% CI 1.00–8.75, p = 0.047), 1.79 (95% CI 1.18–2.71 p = 0.005) for those who carried (wt/vt), (vt/vt), and (wt/vt + vt/vt), respectively. Correlation based on gender shows the presence of a significant association (p = 0.01a) for female mutant allele carriers OR = 1.96 (95% CI 1.15–3.36), but not in the case of male. Significant difference (p = 0.02) in the frequencies of mutant G allele carrier compared to wild A allele carrier was also found correlated in patients with severe asthma than moderate or mild asthma, OR = 1.78 (95% CI 1.10–2.89). Serum total IgE level in patients with GG genotype (219.37 ± 108.49) was significantly higher than in patients with AG and AA genotypes, respectively (193.22 ± 85.83), (157.11 ± 92.10), (P = 0.001). ConclusionsCarries of GG and AG alleles T1 ADAM33 polymorphism are at a high risk of developing functional susceptibility of asthma.