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International Journal of Molecular... Jul 2023Metal-organic frameworks (MOFs) are a class of porous two- or three-dimensional infinite structure materials consisting of metal ions or clusters and organic linkers,...
Metal-organic frameworks (MOFs) are a class of porous two- or three-dimensional infinite structure materials consisting of metal ions or clusters and organic linkers, which are connected via coordination bonds [...].
Topics: Animals; Metal-Organic Frameworks; Cyprinidae; Immunosuppressive Agents; Insulysin; Metals
PubMed: 37446366
DOI: 10.3390/ijms241311188 -
Frontiers in Immunology 2023Unlike glycosylation of proteins expressed in mammalian systems, bacterial glycosylation is often neglected in the development of recombinant vaccines.
INTRODUCTION
Unlike glycosylation of proteins expressed in mammalian systems, bacterial glycosylation is often neglected in the development of recombinant vaccines.
METHODS
Here, we compared the effects of glycosylation of YghJ, an protein important for mucus attachment of bacteria causing in urinary tract infections (UTIs). A novel method based on statistical evaluation of phage display for the identification and comparison of epitopes and mimotopes of anti-YghJ antibodies in the sera was used. This is the first time that the effect of glycosylation of a recombinant bacterial antigen has been studied at the peptide epitope level.
RESULTS
The study identifies differences in the immune response for (non)-glycosylated antigens in rabbits and pigs and compares them to a large group of patients with UTI, which have been diagnosed as positive for various bacterial pathogens. We identified glycosylation-specific peptide epitopes, a large immunological similarity between different UTI pathogens, and a broad peptide epitope pattern in patients and animals, which could result in a variable response in patients upon vaccination.
DISCUSSION
This epitope analysis indicates that the vaccination of rabbits and pigs raises antibodies that translate well into the human immune system. This study underlines the importance of glycosylation in bacterial vaccines and provides detailed immune diagnostic methods to understand individual immune responses to vaccines.
Topics: Humans; Rabbits; Swine; Animals; Epitopes; Antigens, Bacterial; Glycosylation; Escherichia coli; Urinary Tract Infections; Peptides; Mammals; Escherichia coli Proteins; Metalloproteases
PubMed: 37954588
DOI: 10.3389/fimmu.2023.1258136 -
Matrix Biology : Journal of the... Nov 2023Extracellular proteolysis and turnover are core processes of tissue homeostasis. The predominant matrix-degrading enzymes are members of the Matrix Metalloproteinase...
Extracellular proteolysis and turnover are core processes of tissue homeostasis. The predominant matrix-degrading enzymes are members of the Matrix Metalloproteinase (MMP) family. MMPs extensively degrade core matrix components in addition to processing a range of other factors in the extracellular, plasma membrane, and intracellular compartments. The proteolytic activity of MMPs is modulated by the Tissue Inhibitors of Metalloproteinases (TIMPs), a family of four multi-functional matrisome proteins with extensively characterized MMP inhibitory functions. Thus, a well-regulated balance between MMP activity and TIMP levels has been described as critical for healthy tissue homeostasis, and this balance can be chronically disturbed in pathological processes. The relationship between MMPs and TIMPs is complex and lacks the constraints of a typical enzyme-inhibitor relationship due to secondary interactions between various MMPs (specifically gelatinases) and TIMP family members. We illustrate a new complexity in this system by describing how MMP9 can cleave members of the TIMP family when in molar excess. Proteolytic processing of TIMPs can generate functionally altered peptides with potentially novel attributes. We demonstrate here that all TIMPs are cleaved at their C-terminal tails by a molar excess of MMP9. This processing removes the N-glycosylation site for TIMP3 and prevents the TIMP2 interaction with latent proMMP2, a prerequisite for cell surface MMP14-mediated activation of proMMP2. TIMP2/4 are further cleaved producing ∼14 kDa N-terminal proteins linked to a smaller C-terminal domain through residual disulfide bridges. These cleaved TIMP2/4 complexes show perturbed MMP inhibitory activity, illustrating that MMP9 may bear a particularly prominent influence upon the TIMP:MMP balance in tissues.
Topics: Matrix Metalloproteinase 9; Proteolysis; Tissue Inhibitor of Metalloproteinases; Gelatinases; Proteins
PubMed: 37804930
DOI: 10.1016/j.matbio.2023.09.002 -
Life Sciences Jan 2024Hepatocellular carcinoma (HCC) is a challenging and very fatal liver cancer. The signal transducer and activator of transcription 3 (STAT3) pathway is a crucial... (Review)
Review
Hepatocellular carcinoma (HCC) is a challenging and very fatal liver cancer. The signal transducer and activator of transcription 3 (STAT3) pathway is a crucial regulator of tumor development and are ubiquitously active in HCC. Therefore, targeting STAT3 has emerged as a promising approach for preventing and treating HCC. Various natural bioactive compounds (NBCs) have been proven to target STAT3 and have the potential to prevent and treat HCC as STAT3 inhibitors. Numerous kinds of STAT3 inhibitors have been identified, including small molecule inhibitors, peptide inhibitors, and oligonucleotide inhibitors. Due to the undesirable side effects of the conventional therapeutic drugs against HCC, the focus is shifted to NBCs derived from plants and other natural sources. NBCs can be broadly classified into the categories of terpenes, alkaloids, carotenoids, and phenols. Most of the compounds belong to the family of terpenes, which prevent tumorigenesis by inhibiting STAT3 nuclear translocation. Further, through STAT3 inhibition, terpenes downregulate matrix metalloprotease 2 (MMP2), matrix metalloprotease 9 (MMP9) and vascular endothelial growth factor (VEGF), modulating metastasis. Terpenes also suppress the anti-apoptotic proteins and cell cycle markers. This review provides comprehensive information related to STAT3 abrogation by NBCs in HCC with in vitro and in vivo evidences.
Topics: Humans; Carcinoma, Hepatocellular; Liver Neoplasms; STAT3 Transcription Factor; Vascular Endothelial Growth Factor A; Cell Line, Tumor; Cell Proliferation; Terpenes; Metalloproteases
PubMed: 38103726
DOI: 10.1016/j.lfs.2023.122351 -
Medical Oncology (Northwood, London,... Nov 2023Lung cancer continues to be a major health problem worldwide owing to its incidence, and causes physical, psychological, social, and economic problems. Activated...
Lung cancer continues to be a major health problem worldwide owing to its incidence, and causes physical, psychological, social, and economic problems. Activated cytotoxic T cells (ACTC) are positively correlated with the tumor microenvironment (TME), improving the prognosis of cancer patients. Recently, ACTC-derived exosomes (ACTC-dExo) were implicated in this effect by inhibiting mesenchymal stem cells, which may promote metastasis in the TME. Exosomes are thought to be advantageous for the specific delivery of drugs to cancer cells because they have the characteristics of natural liposomes, are nanosized, and remain largely stable in the blood due to the protein and lipid content they carry on their membranes. In this study, we aimed to determine the cytotoxic and metastatic inhibitory effects of ACTC-dExo in A549 cells in vitro. Cytotoxic CD8 T cells were isolated from whole blood obtained from healthy individuals and cultured for 5-7 days after stimulation. The ACTC-dExo serum-free culture medium was collected by ultracentrifugation. Characterization and quantification of the isolated exosomes were performed using flow cytometry, electron microscopy, zeta-sizer measurements, and bicinchoninic acid (BCA) assays. We co-cultured ACTC and ACTC-dExo with A549 cells for 48 h. The viability of A549 cells was evaluated using a WST-1 assay. The metastasis-related genes MMP2, MMP9, TWIST, SNAI1, and CDH1 were detected by qRT-PCR, and MMP2 and MMP9 proteins were evaluated by confocal microscopy. In addition, changes in cell migration were investigated using a scratch assay. ACTC-dExo were found to have anti-proliferative and anti-metastatic effects and reduced cancer cell proliferation and metastatic properties.
Topics: Humans; CD8-Positive T-Lymphocytes; Lung Neoplasms; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Exosomes; Antineoplastic Agents; Tumor Microenvironment
PubMed: 37966661
DOI: 10.1007/s12032-023-02198-0 -
International Journal of Laboratory... Oct 2023The objective of the present study was to evaluate and compare the validity and utility of two fully automated ADAMTS13 (a disintegrin and metalloproteinase with a... (Comparative Study)
Comparative Study
INTRODUCTION
The objective of the present study was to evaluate and compare the validity and utility of two fully automated ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity assays for clinical diagnostic decision-making and to compare their performance.
METHODS
Two automated ADAMTS13 activity assays (Werfen HemosIL® AcuStar ADAMTS13 Activity, Technoclone Technofluor ADAMTS13 Activity) were compared with a manual FRET assay (BioMedica ACTIFLUOR ADAMTS13 Activity). The following samples were used: 13 acute phase TTP (thrombotic thrombocytopenic purpura) samples from 11 different patients, one sample from a patient with congenital ADAMTS13 deficiency, 16 samples from control patients, three follow-up samples from TTP patients in long-term remission and one sample from a patient with stem cell transplantation related thrombotic microangiopathy (TMA). The WHO 1st International Standard for ADAMTS13 and several dilutions of normal plasma with ADAMTS13-depleted normal plasma were also tested. Statistical analysis included descriptive statistics, sensitivity and specificity, Passing & Bablok regression and Bland-Altman plot.
RESULTS
The quantitative comparison between the HemosIL® (x) and Technofluor (y) methods showed a strong correlation (Pearson r = 0.98, n = 49). When considering an ADAMTS13 activity of <10% as a hallmark for the diagnosis of TTP, two fully automated assays were both able to identify all TTP- and non-TTP-samples correctly, resulting in sensitivities and specificities of 100%.
CONCLUSION
Both fully automated ADAMTS13 activity assays showed a good diagnostic performance and quantitative correlation among themselves, discriminating reliably between TTP- and non-TTP-patients.
Topics: Humans; ADAM Proteins; ADAMTS13 Protein; Fluorescence Resonance Energy Transfer; Hematopoietic Stem Cell Transplantation; Purpura, Thrombotic Thrombocytopenic; Thrombotic Microangiopathies
PubMed: 37194625
DOI: 10.1111/ijlh.14090 -
The Journal of Biological Chemistry Dec 2023Increasing evidence suggests that aberrant regulation of sortilin ectodomain shedding can contribute to amyloid-β pathology and frontotemporal dementia, although the...
Increasing evidence suggests that aberrant regulation of sortilin ectodomain shedding can contribute to amyloid-β pathology and frontotemporal dementia, although the mechanism by which this occurs has not been elucidated. Here, we probed for novel binding partners of sortilin using multiple and complementary approaches and identified two proteins of the neuron-specific gene (NSG) family, NSG1 and NSG2, that physically interact and colocalize with sortilin. We show both NSG1 and NSG2 induce subcellular redistribution of sortilin to NSG1- and NSG2-enriched compartments. However, using cell surface biotinylation, we found only NSG1 reduced sortilin cell surface expression, which caused significant reductions in uptake of progranulin, a molecular determinant for frontotemporal dementia. In contrast, we demonstrate NSG2 has no effect on sortilin cell surface abundance or progranulin uptake, suggesting specificity for NSG1 in the regulation of sortilin cell surface expression. Using metalloproteinase inhibitors and A disintegrin and metalloproteinase 10 KO cells, we further show that NSG1-dependent reduction of cell surface sortilin occurred via proteolytic processing by A disintegrin and metalloproteinase 10 with a concomitant increase in shedding of sortilin ectodomain to the extracellular space. This represents a novel regulatory mechanism for sortilin ectodomain shedding that is regulated in a neuron-specific manner. Furthermore, this finding has implications for the development of strategies for brain-specific regulation of sortilin and possibly sortilin-driven pathologies.
Topics: Adaptor Proteins, Vesicular Transport; Biotinylation; Brain; Carrier Proteins; Disintegrins; Frontotemporal Dementia; Metalloproteases; Nerve Tissue Proteins; Neurons; Progranulins; Protein Binding; Proteolysis; Cell Membrane; Amyloid beta-Peptides
PubMed: 37949230
DOI: 10.1016/j.jbc.2023.105446 -
Tissue Engineering and Regenerative... Aug 2023Extracellular matrix (ECM) components confer biomechanical properties, maintain cell phenotype and mediate tissue homeostasis. ECM remodeling is complex and plays a key... (Review)
Review
Extracellular matrix (ECM) components confer biomechanical properties, maintain cell phenotype and mediate tissue homeostasis. ECM remodeling is complex and plays a key role in both physiological and pathological processes. Matrix metalloproteinases (MMPs) are a group of enzymes responsible for ECM degradation and have been accepted as a key regulator in ECM remodeling. In this mini-review, we summarize MMPs categories, functions and the targeted substrates. We then discuss current understanding of the role of MMPs-mediated events, including inflammation reaction, angiogenesis, cellular activities, etc., in ECM remodeling in the context of regenerative medicine.
Topics: Humans; Regenerative Medicine; Matrix Metalloproteinases; Extracellular Matrix; Inflammation
PubMed: 37160567
DOI: 10.1007/s13770-023-00536-x -
Anatomia, Histologia, Embryologia Sep 2023The functional roles of the a disintegrin and metalloprotease with a thrombospondin type motifs (ADAMTS) gene family in reproductive physiology, reproductive organs...
Expression of placental growth factor and a disintegrin and metalloprotease with a thrombospondin type motifs 1-4-8 during the three trimesters of rat pregnancy at the maternal-fetal interface.
The functional roles of the a disintegrin and metalloprotease with a thrombospondin type motifs (ADAMTS) gene family in reproductive physiology, reproductive organs developments and adult reproductive health are still under investigation. The expression of the anti-angiogenic proteases ADAMTS-1, ADAMTS-4 and ADAMTS-8 in placental angiogenesis at various stages of pregnancy also remains unclear. The purpose of this study was therefore to determine the localization and expression of the ADAMTS-1, ADAMTS-4 and ADAMTS-8 proteins during the three stages of pregnancy in rats. Maternal-fetal tissue samples were collected on Days 5, 12 and 19 of each trimester, corresponding to the first, second and third trimesters. The expression of placental growth factor (PlGF) and ADAMTS-1, ADAMTS-4 and ADAMTS-8 at the maternal-fetal interface was examined using immunohistochemistry and western blot at three distinct phases of pregnancy. ADAMTS-1, ADAMTS-4 and ADAMTS-8 were detected in all three trimesters of pregnancy. The relative amount of PIGF increased in the first trimester and decreased significantly in the third trimester (p < 0.05). The expression of ADAMTS-1 and ADAMTS-4 was significantly higher in the second (p < 0.05) and third trimesters (p < 0.01) compared to the first trimester. However, no statistically significant change was observed in ADAMTS-8 expression between trimesters. The ADAMTS exhibiting the highest expression during the first trimester was ADAMTS8. These findings indicate that the expression of ADAMTS-1, ADAMTS-4 and ADAMTS-8 in the three different stages of rat pregnancy may be involved in the modulation of decidualization, morphogenesis and angiogenesis. Periodic changes in ADAMTS expression are thought to be regulated by gonadal steroids.
Topics: Animals; Female; Pregnancy; Rats; Disintegrins; Placenta; Placenta Growth Factor; ADAMTS Proteins
PubMed: 37424113
DOI: 10.1111/ahe.12948 -
Biochemistry Nov 2023Intracellular leucine aminopeptidases (PepA) are metalloproteases from the family M17. These enzymes catalyze peptide bond cleavage, removing N-terminal residues from...
Intracellular leucine aminopeptidases (PepA) are metalloproteases from the family M17. These enzymes catalyze peptide bond cleavage, removing N-terminal residues from peptide and protein substrates, with consequences for protein homeostasis and quality control. While general mechanistic studies using model substrates have been conducted on PepA enzymes from various organisms, specific information about their substrate preferences and promiscuity, choice of metal, activation mechanisms, and the steps that limit steady-state turnover remain unexplored. Here, we dissected the catalytic and chemical mechanisms of PepA: a leucine aminopeptidase from . Cleavage assays using peptides and small-molecule substrate mimics allowed us to propose a mechanism for catalysis. Steady-state and pre-steady-state kinetics, pH rate profiles, solvent kinetic isotope effects, and biophysical techniques were used to evaluate metal binding and activation. This revealed that metal binding to a tight affinity site is insufficient for enzyme activity; binding to a weaker affinity site is essential for catalysis. Progress curves for peptide hydrolysis and crystal structures of free and inhibitor-bound PepA revealed that PepA cleaves peptide substrates in a processive manner. We propose three distinct modes for activity regulation: tight packing of PepA in a hexameric assembly controls substrate length and reaction processivity; the product leucine acts as an inhibitor, and the high concentration of metal ions required for activation limits catalytic turnover. Our work uncovers catalysis by a metalloaminopeptidase, revealing the intricacies of metal activation and substrate selection. This will pave the way for a deeper understanding of metalloenzymes and processive peptidases/proteases.
Topics: Leucine; Leucyl Aminopeptidase; Peptides; Hydrolysis; Metals; Catalysis; Kinetics; Substrate Specificity
PubMed: 37924287
DOI: 10.1021/acs.biochem.3c00420