Differential interactions of human kallikrein-binding protein and alpha 1-antitrypsin with human tissue kallikrein.
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ABSTRACT:
SUBMITTER: Chen LM
PROVIDER: S-EPMC1131247 | biostudies-other | 1990 Apr
REPOSITORIES: biostudies-other
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Project description:We have identified a tissue-kallikrein-binding protein in human serum and in the serum-free culture media from human lung fibroblasts (WI-38) and rodent neuroblastoma X glioma hybrid cells (NG108-15). Purified and 125I-labelled tissue kallikrein and human serum form an approximately 92,000-Mr SDS-stable complex. The relative quantity of this complex-formation is measured by densitometric scanning of autoradiograms. Complex-formation between tissue kallikrein and the serum binding protein was time-dependent and detectable after 5 min incubation at 37 degrees C, with half-maximal binding at 28 min. Binding of 125I-kallikrein to kallikrein-binding protein is temperature-dependent and can be inhibited by heparin or excess unlabelled tissue kallikrein but not by plasma kallikrein, collagenase, thrombin, urokinase, alpha 1-antitrypsin or kininogens. The kallikrein-binding protein is acid- and heat-labile, as pretreatment of sera at pH 3.0 or at 60 degrees C for 30 min diminishes complex-formation. However, the formed complexes are stable to acid or 1 M-hydroxylamine treatment and can only be partially dissociated with 10 mM-NaOH. When kallikrein was inhibited by the active-site-labelling reagents phenylmethanesulphonyl fluoride or D-Phe-D-Phe-L-Arg-CH2Cl no complex-formation was observed. An endogenous approximately 92,000-Mr kallikrein-kallikrein-binding protein complex was isolated from normal human serum by using a human tissue kallikrein-agarose affinity column. These complexes were recognized by anti-(human tissue kallikrein) antibodies, but not by anti-alpha 1-antitrypsin serum, in Western-blot analyses. The results show that the kallikrein-binding protein is distinct from alpha 1-antitrypsin and is not identifiable with any of the well-characterized plasma proteinase inhibitors such as alpha 2-macroglobulin, inter-alpha-trypsin inhibitor, C1-inactivator or antithrombin III. The functional role of this kallikrein-binding protein and its impact on kallikrein activity or metabolism in vivo remain to be investigated.
| S-EPMC1147284 | biostudies-other
Project description:Alpha 1-antitrypsin (AAT) deficiency, a hereditary disorder characterized by low serum levels of functional AAT, is associated with early development of panacinar emphysema. AAT inhibits serine proteases, including neutrophil elastase, protecting the lung from proteolytic destruction. Cigarette smoke, pollution, and inflammatory cell-mediated oxidation of methionine (M) 351 and 358 inactivates AAT, limiting lung protection. In vitro studies using amino acid substitutions demonstrated that replacing M351 with valine (V) and M358 with leucine (L) on a normal M1 alanine (A) 213 background provided maximum antiprotease protection despite oxidant stress. We hypothesized that a onetime administration of a serotype 8 adeno-associated virus (AAV8) gene transfer vector coding for the oxidation-resistant variant AAT (A213/V351/L358; 8/AVL) would maintain antiprotease activity under oxidant stress compared with normal AAT (A213/M351/M358; 8/AMM). 8/AVL was administered via intravenous (IV) and intrapleural (IPL) routes to C57BL/6 mice. High, dose-dependent AAT levels were found in the serum and lung epithelial lining fluid (ELF) of mice administered 8/AVL or 8/AMM by IV or IPL. 8/AVL serum and ELF retained serine protease-inhibitory activity despite oxidant stress while 8/AMM function was abolished. 8/AVL represents a second-generation gene therapy for AAT deficiency providing effective antiprotease protection even with oxidant stress.
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Project description:The human serine protease high temperature requirement A1 (HTRA1) is highly expressed in the placental tissue, especially in the last trimester of gestation. This suggests that HTRA1 is involved in placental formation and function. With the aim of a better understanding of the role of HTRA1 in the placenta, candidate substrates were screened in a placenta protein extract using a gel-based mass spectrometric approach. Protease inhibitor alpha-1-antitrypsin, actin cytoplasmic 1, tropomyosin beta chain and ten further proteins were identified as candidate substrates of HTRA1. Among the identified candidate substrates, alpha-1-antitrypsin (A1AT) was considered to be of particular interest because of its important role as protease inhibitor. For investigation of alpha-1-antitrypsin as substrate of HTRA1 synthetic peptides covering parts of the sequence of alpha-1-antitrypsin were incubated with HTRA1. By mass spectrometry a specific cleavage site was identified after met-382 (AIPM382↓383SIPP) within the reactive centre loop of alpha-1-antitrypsin, resulting in a C-terminal peptide comprising 36 amino acids. Proteolytic removal of this peptide from alpha-1-antitrypsin results in a loss of its inhibitor function. Beside placental alpha-1-antitrypsin the circulating form in human plasma was also significantly degraded by HTRA1. Taken together, our data suggest a link between the candidate substrates alpha-1-antitrypsin and the function of HTRA1 in the placenta in the syncytiotrophoblast, the cell layer attending to maternal blood in the villous tree of the human placenta. Data deposition: Mass spectrometry (MS) data have been deposited to the ProteomeXchange with identifier PXD000473.
| S-EPMC4203740 | biostudies-literature
Project description:Inflammaging plays an important role in most age-related diseases. However, the mechanism of inflammaging is largely unknown, and therapeutic control of inflammaging is challenging. Human alpha-1 antitrypsin (hAAT) has immune-regulatory, anti-inflammatory, and cytoprotective properties as demonstrated in several disease models including type 1 diabetes, arthritis, lupus, osteoporosis, and stroke. To test the potential anti-inflammaging effect of hAAT, we generated transgenic Drosophila lines expressing hAAT. Surprisingly, the lifespan of hAAT-expressing lines was significantly longer than that of genetically matched controls. To understand the mechanism underlying the anti-aging effect of hAAT, we monitored the expression of aging-associated genes and found that aging-induced expressions of Relish (NF-ĸB orthologue) and Diptericin were significantly lower in hAAT lines than in control lines. RNA-seq analysis revealed that innate immunity genes regulated by NF-kB were significantly and specifically inhibited in hAAT transgenic Drosophila lines. To confirm this anti-inflammaging effect in human cells, we treated X-ray-induced senescence cells with hAAT and showed that hAAT treatment significantly decreased the expression and maturation of IL-6 and IL-8, two major factors of senescence-associated secretory phenotype. Consistent with results from Drosophila,RNA-seq analysis also showed that hAAT treatment significantly inhibited inflammation related genes and pathways. Together, our results demonstrated that hAAT significantly inhibited inflammaging in both Drosophila and human cell models. As hAAT is a FDA-approved drug with a confirmed safety profile, this novel therapeutic potential may make hAAT a promising candidate to combat aging and aging-related diseases.
| S-EPMC5770780 | biostudies-literature
Project description:Expression levels of CX3CR1 (C-X3-C motif chemokine receptor 1) on immune cells have significant importance in maintaining tissue homeostasis under physiological and pathological conditions. The factors implicated in the regulation of CX3CR1 and its specific ligand CX3CL1 (fractalkine) expression remain largely unknown. Recent studies provide evidence that host's misfolded proteins occurring in the forms of polymers or amyloid fibrils can regulate CX3CR1 expression. Herein, a novel example demonstrates that polymers of human ZZ alpha-1 antitrypsin (Z-AAT) protein, resulting from its conformational misfolding due to the Z (Glu342Lys) mutation in SERPINA1 gene, strongly lower CX3CR1 mRNA expression in human peripheral blood mononuclear cells (PBMCs). This parallels with increase of intracellular levels of CX3CR1 and Z-AAT proteins. Presented data indicate the involvement of the CX3CR1 pathway in the Z-AAT-related disorders and further support the role of misfolded proteins in CX3CR1 regulation.
| S-EPMC8205483 | biostudies-literature
Project description:Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17β-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [³H]-17β-estradiol bound ER66 and ER46 with Kd values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17β-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17β-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.
| S-EPMC3639985 | biostudies-literature
Project description:Alpha-1-antitrypsin (AAT), an acute-phase protein encoded by the SERPINA1 gene, is a member of the serine protease inhibitor (SERPIN) superfamily. Its primary function is to protect tissues from enzymes released during inflammation, such as neutrophil elastase and proteinase 3. In addition to its antiprotease activity, AAT interacts with numerous other substances and has various functions, mainly arising from the conformational flexibility of normal variants of AAT. Therefore, AAT has diverse biological functions and plays a role in various pathophysiological processes. This review discusses major molecular forms of AAT, including complex, cleaved, glycosylated, oxidized, and S-nitrosylated forms, in terms of their origin and function.
| S-EPMC7731214 | biostudies-literature
Project description:BackgroundAlpha-1 antitrypsin deficiency (AATD) is characterized by low alpha-1 antitrypsin (AAT) levels, predisposing individuals to lung disease. The standard of care, plasma-derived AAT (pdAAT), is delivered as weekly infusions to maintain serum AAT concentrations ≥11µM (≈50% of those in healthy individuals). INBRX-101, a recombinant human AAT-Fc fusion protein, was designed to have a longer half-life and achieve higher AAT levels than pdAAT.MethodsIn this phase 1 dose-escalation study (N=31), adults with AATD received 1 dose (part 1) or 3 doses (part 2) of 10 (part 1), 40, 80, or 120mg/kg INBRX-101 every 3 weeks (Q3W) via intravenous infusion. The primary endpoint was safety and tolerability. Secondary endpoints were pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of INBRX-101.ResultsINBRX-101 was well tolerated. Most treatment-emergent adverse events were grade ≤2. In part 2 (n=18; each dose, n=6), dose-related increases in serum functional AAT (fAAT) were observed; mean fAAT levels remained above the 21 µM target for up to 4 weeks after the final dose in the 120-mg/kg cohort. Antidrug antibodies had no meaningful impact on PK or PD. INBRX-101 was detected in pulmonary epithelial lining fluid (PELF) from all patients assessed (n=11), and PELF fAAT increased after dosing. PK/PD modeling projected steady-state serum fAAT ≥21µM at 120 mg/kg Q3W (average concentration ≈43µM; trough concentration ≈28µM) and Q4W (≈34µM; ≈21µM).ConclusionThe favorable safety profile and ability to maintain serum fAAT levels >21µM with extended-interval dosing, support a phase 2 trial evaluating Q3W and Q4W dosing of INBRX-101.
| S-EPMC11216232 | biostudies-literature
Project description:Acute ischemic stroke (AIS) remains a major cause of death and disability throughout the world. The most severe form of stroke results from large vessel occlusion of the major branches of the Circle of Willis. The treatment strategies currently available in western countries for large vessel occlusion involve rapid restoration of blood flow through removal of the offending blood clot using mechanical or pharmacological means (e.g. tissue plasma activator; tPA). This review assesses prospects for a novel pharmacological approach to enhance the availability of the natural enzyme tissue kallikrein (KLK1), an important regulator of local blood flow. KLK1 is responsible for the generation of kinins (bradykinin and kallidin), which promote local vasodilation and long-term vascularization. Moreover, KLK1 has been used clinically as a direct treatment for multiple diseases associated with impaired local blood flow including AIS. A form of human KLK1 isolated from human urine is approved in the People's Republic of China for subacute treatment of AIS. Here we review the rationale for using KLK1 as an additional pharmacological treatment for AIS by providing the biochemical mechanism as well as the human clinical data that support this approach.
| S-EPMC6348491 | biostudies-literature
Project description:AimTo identify proteins with which FolBp1 may interact within lipid rafts in tissue derived from embryonic orofacial tissue.MethodsA yeast two-hybrid screen of a cDNA library, derived from orofacial tissue from gestational day 11 to 13 mouse embryos, was conducted.Key findingsUsing the full-length FolBp1 protein as bait, two proteins that bind FolBp1 were identified, Bat2d, and a fibronectin type III-containing domain protein. Results were confirmed by glutathione S-transferase pull-down assays.SignificanceAs a component of membrane lipid raft protein complexes, these binding proteins may represent "helper" or chaperone proteins that associate with FolBp1 in order to facilitate the transport of folate across the plasma membrane. The protein-protein interactions detected, while limited in number, may be critical in mediating the role of FolBp1 in folate transport, particularly in the developing embryo.
| S-EPMC2819649 | biostudies-literature