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Frontiers in Immunology 2019Envenomation by viperid snakes is characterized by systemic thrombotic syndrome and prominent local inflammation. To date, the mechanisms underlying inflammation and... (Review)
Review
Envenomation by viperid snakes is characterized by systemic thrombotic syndrome and prominent local inflammation. To date, the mechanisms underlying inflammation and blood coagulation induced by venoms have been viewed as distinct processes. However, studies on the mechanisms involved in these processes have revealed several factors and signaling molecules that simultaneously act in both the innate immune and hemostatic systems, suggesting an overlap between both systems during viper envenomation. Moreover, distinct classes of venom toxins involved in these effects have also been identified. However, the interplay between inflammation and hemostatic alterations, referred as to thromboinflammation, has never been addressed in the investigation of viper envenomation. Considering that platelets are important targets of viper snake venoms and are critical for the process of thromboinflammation, in this review, we summarize the inflammatory effects and mechanisms induced by viper snake venoms, particularly from the genus, which strongly activate platelet functions and highlight selected venom components (metalloproteases and C-type lectins) that both stimulate platelet functions and exhibit pro-inflammatory activities, thus providing insights into the possible role(s) of thromboinflammation in viper envenomation.
Topics: Animals; Blood Coagulation; Hemostasis; Humans; Inflammation; Lectins, C-Type; Metalloproteases; Platelet Activation; Snake Bites; Snake Venoms; Thrombosis; Viperidae
PubMed: 31572356
DOI: 10.3389/fimmu.2019.02082 -
Molecular Biology of the Cell Jul 2019Hemodynamic forces activate the Von Willebrand factor (VWF) and facilitate its cleavage by a disintegrin and metalloprotease with thrombospondin motifs-13 (ADAMTS13),...
Hemodynamic forces activate the Von Willebrand factor (VWF) and facilitate its cleavage by a disintegrin and metalloprotease with thrombospondin motifs-13 (ADAMTS13), reducing the adhesive activity of VWF. Biochemical assays have mapped the binding sites on both molecules. However, these assays require incubation of two molecules for a period beyond the time allowed in flowing blood. We used a single-molecule technique to examine these rapid, transient, and mechanically modulated molecular interactions in short times under forces to mimic what happens in circulation. Wild-type ADAMTS13 and two truncation variants that either lacked the C-terminal thrombospondin motif-7 to the CUB domain (MP-TSP6) or contained only the two CUB domains (CUB) were characterized for interactions with coiled VWF, flow-elongated VWF, and a VWF A1A2A3 tridomain. These interactions exhibited distinctive patterns of calcium dependency, binding affinity, and force-regulated lifetime. The results suggest that 1) ADAMTS13 binds coiled VWF primarily through CUB in a calcium-dependent manner via a site(s) outside A1A2A3, 2) ADAMTS13 binds flow-extended VWF predominantly through MP-TSP6 via a site(s) different from the one(s) at A1A2A3; and 3) ADAMTS13 binds A1A2A3 through MP-TSP6 in a Ca-dependent manner to autoinhibit another Ca-independent binding site on CUB. These data reveal that multiple sites on both molecules are involved in mechanically modulated VWF-ADAMTS13 interaction.
Topics: ADAMTS13 Protein; Binding Sites; Biomechanical Phenomena; Cations; HEK293 Cells; Humans; Kinetics; Protein Binding; Protein Domains; von Willebrand Factor
PubMed: 31067148
DOI: 10.1091/mbc.E19-01-0021 -
Cell Death & Disease May 2022Bone marrow (BM) fibrosis was thought to be induced exclusively by mesenchymal stromal cells (MSCs). However, we and others found that neoplastic fibrocytes induce BM...
Bone marrow (BM) fibrosis was thought to be induced exclusively by mesenchymal stromal cells (MSCs). However, we and others found that neoplastic fibrocytes induce BM fibrosis in myelofibrosis (MF). Because glioma-associated oncogene-1 (GLI1), an effector of the Hedgehog pathway, plays a role in the induction of BM fibrosis, we wondered whether GLI1 affects fibrocyte-induced BM fibrosis in MF. Multiplexed fluorescence immunohistochemistry analysis of MF patients' BM detected high levels of GLI1 in MF fibrocytes compared to MSCs or normal fibrocytes. Immunostaining, RNA in situ hybridization, gene expression analysis, and western immunoblotting detected high levels of GLI1 and GLI1-induced matrix metalloproteases (MMP) 2 and 9 in MF patients BM-derived cultured fibrocytes. Similarly, MF patients' BM-derived GLI1 fibrocytes were found in BMs and spleens of MF xenograft mice. GLI1 silencing reduced the levels of MMP2/9, phosphorylated SMAD2/3, and procollagen-I, and knockdown or inhibition of GLI1 decreased fibrocyte formation and induced apoptosis of both fibrocytes and fibrocyte progenitors. Because Janus kinase (JAK)2-induced STAT3 is constitutively activated in MF and because STAT3 induces GLI1 expression, we sought to determine whether STAT3 activates GLI1 in MF fibrocytes. Imaging analysis detected phosphotyrosine STAT3 in MF patients' BM fibrocytes, and transfection of fibrocytes with STAT3-siRNA or treatment with a JAK1/2 inhibitor ruxolitinib reduced GLI1 and MMP2/9 levels. Chromatin immunoprecipitation and a luciferase assay revealed that STAT3 induced the expression of the GLI1 gene in both MF BM fibrocytes and fibrocyte progenitors. Together, our data suggest that STAT3-activated GLI1 contributes to the induction of BM fibrosis in MF.
Topics: Animals; Fibrosis; Hedgehog Proteins; Humans; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Primary Myelofibrosis; STAT3 Transcription Factor; Zinc Finger Protein GLI1
PubMed: 35595725
DOI: 10.1038/s41419-022-04932-4 -
International Journal of Molecular... Mar 2021Experimental evidence for enzymatic mechanisms is often scarce, and in many cases inadvertently biased by the employed methods. Thus, apparently contradictory model... (Review)
Review
Experimental evidence for enzymatic mechanisms is often scarce, and in many cases inadvertently biased by the employed methods. Thus, apparently contradictory model mechanisms can result in decade long discussions about the correct interpretation of data and the true theory behind it. However, often such opposing views turn out to be special cases of a more comprehensive and superior concept. Molecular dynamics (MD) and the more advanced molecular mechanical and quantum mechanical approach (QM/MM) provide a relatively consistent framework to treat enzymatic mechanisms, in particular, the activity of proteolytic enzymes. In line with this, computational chemistry based on experimental structures came up with studies on all major protease classes in recent years; examples of aspartic, metallo-, cysteine, serine, and threonine protease mechanisms are well founded on corresponding standards. In addition, experimental evidence from enzyme kinetics, structural research, and various other methods supports the described calculated mechanisms. One step beyond is the application of this information to the design of new and powerful inhibitors of disease-related enzymes, such as the HIV protease. In this overview, a few examples demonstrate the high potential of the QM/MM approach for sophisticated pharmaceutical compound design and supporting functions in the analysis of biomolecular structures.
Topics: Algorithms; Cysteine Proteases; Metalloproteases; Molecular Dynamics Simulation; Molecular Structure; Peptide Hydrolases; Protease Inhibitors; Protein Conformation; Quantum Theory; Serine Proteases; Thermodynamics
PubMed: 33810118
DOI: 10.3390/ijms22063232 -
Pharmacological Research Feb 2022The proteases of the mitochondrial inner membrane are challenging yet highly desirable drug targets for complex, multifactorial diseases prevalent mainly in the elderly.... (Review)
Review
The proteases of the mitochondrial inner membrane are challenging yet highly desirable drug targets for complex, multifactorial diseases prevalent mainly in the elderly. Among them, OMA1 with its substrates OPA1 and DELE1 safeguards mitochondrial homeostasis at the intersection of energy metabolism and apoptosis, which may have relevance for neurodegeneration, malignancy and heart failure, among other diseases. Little is known about OMA1. Its structure has not been solved and we are just beginning to understand the enzyme's context-dependent regulation. OMA1 appears dormant under physiological conditions as judged by OPA1's processing pattern. The protease is rapidly activated, however, when cells experience stress or undergo apoptosis. Intriguingly, genetic OMA1 ablation can delay or even prevent apoptosis in animal models for diseases that can be broadly categorized as ischemia-reperfusion related disorders. Three groups have reported their efforts implementing OMA1 drug screens. This article reviews some of the technical challenges encountered in these assays and highlights what can be learned for future screening campaigns, and about the OMA1 protease more broadly. OMA1 does not exists in a vacuum and potent OMA1 inhibitors are needed to tease apart OMA1's intricate interactions with the other mitochondrial proteases and enzymes. Furthermore, OMA1 inhibitors hold the promise of becoming a new class of cytoprotective medicines for disorders influenced by dysfunctional mitochondria, such as heart failure or Alzheimer's Disease.
Topics: Animals; Drug Design; High-Throughput Screening Assays; Humans; Metalloendopeptidases
PubMed: 34999225
DOI: 10.1016/j.phrs.2022.106063 -
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 -
BMC Genomics Jul 2023A Disintegrin and Metalloproteinase (ADAM) and A Disintegrin and Metalloproteinase with Thrombospondin Motif (ADAMTS) have been reported potentially involved in bone...
PURPOSE
A Disintegrin and Metalloproteinase (ADAM) and A Disintegrin and Metalloproteinase with Thrombospondin Motif (ADAMTS) have been reported potentially involved in bone metabolism and related to bone mineral density. This Mendelian Randomization (MR) analysis was performed to determine whether there are causal associations of serum ADAM/ADAMTS with BMD in rid of confounders.
METHODS
The genome-wide summary statistics of four site-specific BMD measurements were obtained from studies in individuals of European ancestry, including forearm (n = 8,143), femoral neck (n = 32,735), lumbar spine (n = 28,498) and heel (n = 426,824). The genetic instrumental variables for circulating levels of ADAM12, ADAM19, ADAM23, ADAMTS5 and ADAMTS6 were retrieved from the latest genome-wide association study of European ancestry (n = 5336 ~ 5367). The estimated causal effect was given by the Wald ratio for each variant, the inverse-variance weighted model was used as the primary approach to combine estimates from multiple instruments, and sensitivity analyses were conducted to assess the robustness of MR results. The Bonferroni-corrected significance was set at P < 0.0025 to account for multiple testing, and a lenient threshold P < 0.05 was considered to suggest a causal relationship.
RESULTS
The causal effects of genetically predicted serum ADAM/ADAMTS levels on BMD measurements at forearm, femoral neck and lumbar spine were not statistically supported by MR analyses. Although causal effect of ADAMTS5 on heel BMD given by the primary MR analysis (β = -0.006, -0.010 to 0.002, P = 0.004) failed to reach Bonferroni-corrected significance, additional MR approaches and sensitivity analyses indicated a robust causal relationship.
CONCLUSION
Our study provided suggestive evidence for the causal effect of higher serum levels of ADAMTS5 on decreased heel BMD, while there was no supportive evidence for the associations of ADAM12, ADAM19, ADAM23, and ADAMTS6 with BMD at forearm, femoral neck and lumbar spine in Europeans.
Topics: Humans; Bone Density; Mendelian Randomization Analysis; Genome-Wide Association Study; Disintegrins; Polymorphism, Single Nucleotide; Metalloproteases
PubMed: 37468870
DOI: 10.1186/s12864-023-09449-4 -
Cell Death & Disease Nov 2022Genetic studies have proved the involvement of Tuberous sclerosis complex subunit 2 (Tsc2) in aortic aneurysm. However, the exact role of macrophage Tsc2 in the vascular...
RATIONALE
Genetic studies have proved the involvement of Tuberous sclerosis complex subunit 2 (Tsc2) in aortic aneurysm. However, the exact role of macrophage Tsc2 in the vascular system remains unclear. Here, we examined the potential function of macrophage Tsc2 in the development of aortic remodeling and aortic aneurysms.
METHODS AND RESULTS
Conditional gene knockout strategy combined with histology and whole-transcriptomic analysis showed that Tsc2 deficiency in macrophages aggravated the progression of aortic aneurysms along with an upregulation of proinflammatory cytokines and matrix metallopeptidase-9 in the angiotensin II-induced mouse model. G protein-coupled receptor 68 (Gpr68), a proton-sensing receptor for detecting the extracellular acidic pH, was identified as the most up-regulated gene in Tsc2 deficient macrophages compared with control macrophages. Additionally, Tsc2 deficient macrophages displayed higher glycolysis and glycolytic inhibitor 2-deoxy-D-glucose treatment partially attenuated the level of Gpr68. We further demonstrated an Tsc2-Gpr68-CREB network in macrophages that regulates the inflammatory response, proteolytic degradation and vascular homeostasis. Gpr68 inhibition largely abrogated the progression of aortic aneurysms caused by Tsc2 deficiency in macrophages.
CONCLUSIONS
The findings reveal that Tsc2 deficiency in macrophages contributes to aortic aneurysm formation, at least in part, by upregulating Gpr68 expression, which subsequently drives proinflammatory processes and matrix metallopeptidase activation. The data also provide a novel therapeutic strategy to limit the progression of the aneurysm resulting from Tsc2 mutations.
Topics: Mice; Animals; Angiotensin II; Tuberous Sclerosis; Aortic Aneurysm; Metalloproteases; Receptors, G-Protein-Coupled
PubMed: 36400753
DOI: 10.1038/s41419-022-05423-2 -
Biochimica Et Biophysica Acta.... Oct 2019Several membrane-bound proteins with a single transmembrane domain are subjected to limited proteolysis at the cell surface. This cleavage leads to the release of their... (Review)
Review
Several membrane-bound proteins with a single transmembrane domain are subjected to limited proteolysis at the cell surface. This cleavage leads to the release of their biologically active ectodomains, which can trigger different signalling pathways. In many cases, this ectodomain shedding is mediated by members of the family of a disintegrins and metalloproteinases (ADAMs). ADAM17 in particular is responsible for the cleavage of several proinflammatory mediators, growth factors, receptors and adhesion molecules. Due to its direct involvement in the release of these signalling molecules, ADAM17 can be positively and negatively involved in various physiological processes as well as in inflammatory, fibrotic and malignant pathologies. This central role of ADAM17 in a variety of processes requires strict multi-level regulation, including phosphorylation, various conformational changes and endogenous inhibitors. Recent research has shown that an early, crucial control mechanism is interaction with certain adapter proteins identified as iRhom1 and iRhom2, which are pseudoproteases of the rhomboid superfamily. Thus, iRhoms have also a decisive influence on physiological and pathophysiological signalling processes regulated by ADAM17. Their characteristic gene expression profiles, the specific consequences of gene knockouts and finally the occurrence of disease-associated mutations suggest that iRhom1 and iRhom2 undergo different gene regulation in order to fulfil their function in different cell types and are therefore only partially redundant. Therefore, there is not only interest in ADAM17, but also in iRhoms as therapeutic targets. However, to exploit the therapeutic potential, the regulation of ADAM17 activity and in particular its interaction with iRhoms must be well understood.
Topics: ADAM17 Protein; Animals; Carrier Proteins; Gene Knockout Techniques; Genetic Predisposition to Disease; Humans; Inflammation; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Metalloproteases; Mice; Mutation; Phosphorylation; Protein Conformation; Signal Transduction; Transcriptome
PubMed: 31330158
DOI: 10.1016/j.bbamcr.2019.06.017 -
Viruses Oct 2022Bovine viral diarrhea virus (BVDV) is one of the most hazardous viruses, which causes huge economic losses in the cattle industry around the world. In recent years,... (Review)
Review
Bovine viral diarrhea virus (BVDV) is one of the most hazardous viruses, which causes huge economic losses in the cattle industry around the world. In recent years, there has been a continuous increase in the diversity of pestivirus worldwide. As a member of the genus in the family, BVDV has a wide range of host animals including cattle, goat, sheep, pig, camel and other cloven-hoofed animals, and it has multi-tissue tropism as well. The recognition of their permissive cells by viruses via interaction with the cellular receptors is a prerequisite for successful infection. So far, little is known about the cellular receptors essential for BVDV entry and their detailed functions during BVDV infection. Thus, discovery of the cellular receptors involved in the entry of BVDV and other pestiviruses is significant for development of the novel intervention. The viral envelope glycoprotein E and E2 are crucial determinants of the cellular tropism of BVDV. The cellular proteins bound with E and E2 potentially participate in BVDV entry, and their abundance might determine the cellular tropism of BVDV. Here, we summarize current knowledge regarding the cellular molecules have been described for BVDV entry, such as, complement regulatory protein 46 (CD46), heparan sulfate (HS), the low-density lipoprotein (LDL) receptor, and a disintegrin and metalloproteinase 17 (ADAM17). Furthermore, we focus on their implications of the recently identified cellular receptors for pestiviruses in BVDV life cycle. This knowledge provides a theoretical basis for BVDV prevention and treatment by targeting the cellular receptors essential for BVDV infection.
Topics: Cattle; Animals; Swine; Sheep; Viral Envelope Proteins; Disintegrins; Diarrhea Viruses, Bovine Viral; Diarrhea Virus 1, Bovine Viral; Pestivirus; Receptors, Cell Surface; Heparitin Sulfate; Lipoproteins, LDL; Metalloproteases; Tropism
PubMed: 36298858
DOI: 10.3390/v14102302