Rhodamine-based compounds as fluorogenic substrates for serine proteinases.
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ABSTRACT:
SUBMITTER: Leytus SP
PROVIDER: S-EPMC1154094 | biostudies-other | 1983 Feb
REPOSITORIES: biostudies-other
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Project description:A series of dipeptide derivatives of Rhodamine, each containing an arginine residue in the P1 position and one of ten representative benzyloxycarbonyl (Cbz)-blocked amino acids in the P2 position, has been synthesized, purified and characterized as substrates for serine proteinases. These substrates are easily prepared with high yields. Cleavage of a single amide bond converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. Macroscopic kinetic constants for the interaction of these substrates with bovine trypsin, human and dog plasmin, and human thrombin are reported. Certain of these substrates exhibit extremely large specificity constants. For example, the kcat./Km for bovine trypsin with bis-(N-benzyloxycarbonylglycyl-argininamido)-Rhodamine [(Cbz-Gly-Arg-NH)2-Rhodamine] is 1 670 000 M-1 X S-1. Certain of these substrates are also highly selective. For example, the most specific substrate for human plasmin, (Cbz-Phe-Arg-NH2)-Rhodamine, is not hydrolysed by human thrombin, and the most specific substrate for human thrombin, (Cbz-Pro-Arg-NH)2-Rhodamine, is one of the least specific substrates for human plasmin. Comparison of the kinetic constants for hydrolysis of the dipeptide substrates with that of the single amino acid derivative, (Cbz-Arg-NH)2-Rhodamine, indicates that selection of the proper amino acid residue in the P2 position can effect large increases in substrate specificity. This occurs primarily as a result of an increase in kcat. as opposed to a decrease in Km and, in certain cases, is accompanied by a large increase in selectivity. Because of their high degree of sensitivity and selectivity, these Rhodamine-based dipeptide compounds should be extremely useful substrates for studying serine proteinases.
| S-EPMC1152392 | biostudies-other
Project description:Here, we introduced a novel thiourea-based rhodamine compound as a chromo-fluorogenic indicator of nerve agent Soman and its simulant diethyl chlorophosphate (DCP). The synthesized probe N-(rhodamine B)-lactam-2-(4-cyanophenyl) thiourea (RB-CT), which has a rhodamine core linked by a cyanophenyl thiosemicarbazide group, enabled a rapidly and highly sensitive response to DCP with clear fluorescence and color changes. The detection limit was as low as 2 × 10-6 M. The sensing mechanism showed that opening of the spirolactam ring following the phosphorylation of thiosemicarbazides group formed a seven-membered heterocycle adduct, according to MS analysis and TD-DFT calculations. RB-CT exhibited high detecting selectivity for DCP, among other organophosphorus compounds. Moreover, two test kits were employed and successfully used to detect real nerve agent Soman in liquid and gas phase.
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Project description:Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases.
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Project description:Proteasomes are established therapeutic targets for hematological cancers and promising targets for autoimmune diseases. In the past, we have designed and synthesized mechanism-based proteasome inhibitors that are selective for the individual catalytic activities of human constitutive proteasomes and immunoproteasomes: β1c, β1i, β2c, β2i, β5c and β5i. We show here that by taking the oligopeptide recognition element and substituting the electrophile for a fluorogenic leaving group, fluorogenic substrates are obtained that report on the proteasome catalytic activity also targeted by the parent inhibitor. Though not generally applicable (β5c and β2i substrates showing low activity), effective fluorogenic substrates reporting on the individual activity of β1c, β1i, β2c and β5i subunits in Raji (human B cell) lysates and purified 20S proteasome were identified in this manner. Our work thus adds to the expanding proteasome research toolbox through the identification of new and/or more effective subunit-selective fluorogenic substrates.
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Project description:BackgroundDermatomyositis and polymyositis are the best known idiopathic inflammatory myopathies (IIMs). Classic histopathologic findings include the infiltration of inflammatory cells into muscle tissues. Neutrophil serine proteinases (NSPs) are granule-associated enzymes and play roles in inflammatory cell migration by increasing the permeability of vascular endothelial cells. In this study, we aimed to find the roles of NSPs in pathogenesis of IIMs.MethodsRNA and DNA were isolated to measure the relative expression of NSPs and their methylation levels. The expression of NSPs in serum and muscle tissues was tested by enzyme-linked immunosorbent assay, immunohistochemistry, and immunofluorescence, respectively. Serum from patients was used to culture the human dermal microvascular endothelial cells (HDMECs), and then we observed the influence of serum on expression of VE-cadherin, endothelial cell tube formation, and transendothelial migration of peripheral blood mononuclear cells (PBMCs).ResultsWe found that the expression of NSPs was increased in PBMCs, serum, and muscle tissues of IIM patients; these NSPs were hypomethylated in the PBMCs of patients. Serum NSPs were positively correlated with clinical indicators of IIM patients, including lactic dehydrogenase, erythrocyte sedimentation rate, C-reactive protein, immunoglobulin G, immunoglobulin M, and immunoglobulin A. Patients with anti-Jo-1, with anti-Ro-52, or without interstitial lung disease had lower levels of proteinase 3. Serum NSPs degraded the VE-cadherin of HDMECs, and serum NSP application increased the permeability of HDMECs.ConclusionsOur studies indicate, for the first time, that NSPs play an important role in muscle inflammatory cell infiltration by increasing the permeability of vascular endothelial cells in IIM patients.
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Project description:BackgroundWe have recently reported the expression of murine Implantation Serine Proteinase genes in pre-implantation embryos (ISP1) and uterus (ISP1 and ISP2). These proteinases belong to the S1 proteinase family and are similar to mast cell tryptases, which function as multimers.ResultsHere, we report the purification and initial characterization of ISP1 and 2 with respect to their physico-chemical properties and physiological function. In addition to being co-expressed in uterus, we show that ISP1 and ISP2 are also co-expressed in the pre-implantation embryo. Together, they form a heterodimer with an approximate molecular weight of 63 kD. This complex is the active form of the enzyme, which we have further characterized as being trypsin-like, based on substrate and inhibitor specificities. In addition to having a role in embryo hatching and outgrowth, we demonstrate that ISP enzyme is localized to the site of embryo invasion during implantation and that its activity is important for successful implantation in vivo.ConclusionOn the basis of similarities in structural, chemical, and functional properties, we suggest that this ISP enzyme complex represents the classical hatching enzyme, strypsin. Our results demonstrate a critical role for ISP in embryo hatching and implantation.
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Project description:Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson's Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and monitoring the effect of secondary therapeutic agents on lysosomal enzyme activity in drug development for the lysosomal storage disorders and allied diseases.
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Project description:Periodontitis is a chronic destructive infection of the tooth-supportive tissues, which is caused by pathogenic bacteria such as Actinobacillus actinomycetemcomitans. A severe form of periodontitis is found in Papillon-Lefèvre syndrome (PLS), an inheritable disease caused by loss-of-function mutations in the cathepsin C gene. Recently, we demonstrated that these patients lack the activity of the polymorphonuclear leukocyte (PMN)-derived serine proteinases elastase, cathepsin G, and proteinase 3. In the present study we identified possible pathways along which serine proteinases may be involved in the defense against A. actinomycetemcomitans. Serine proteinases are capable to convert the PMN-derived hCAP-18 into LL-37, an antimicrobial peptide with activity against A. actinomycetemcomitans. We found that the PMNs of PLS patients released lower levels of LL-37. Furthermore, because of their deficiency in serine proteases, the PMNs of PLS patients were incapable of neutralizing the leukotoxin produced by this pathogen, which resulted in increased cell damage. Finally, the capacity of PMNs from PLS patients to kill A. actinomycetemcomitans in an anaerobic environment, such as that found in the periodontal pocket, seemed to be reduced. Our report demonstrates a mechanism that suggests a direct link between an inheritable defect in PMN functioning and difficulty in coping with a periodontitis-associated pathogen.
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Project description:Extracellular serine proteinase pathways control immune and homeostatic processes in insects. Our current knowledge of their components is limited-prophenoloxidase-activating proteinases (PAPs) are among the few hemolymph proteinases (HPs) with known functions. To identify components of proteinase systems in the hemolymph of Manduca sexta, we amplified cDNAs from larval fat body or hemocytes using degenerate primers coding for two conserved regions in S1 family serine proteinases. PCR yielded fragments encoding seven known (HP1-HP4, PAP-1, PAP-2 and PAP-3) and 18 unknown (HP5-HP22) serine proteinases. We screened cDNA libraries and isolated clones for 17 of the newly discovered HPs (HP5-HP22 except for HP11) and prepared antibodies to 14 recombinant proteins (HP6, HP8-HP10, HP12, HP14-HP19, HP21 and HP22). Fourteen of the HPs contain regulatory clip domain(s) at their amino-terminus--HP1, HP2, HP6, HP8, HP13, HP17, HP18, HP21, HP22 and PAP-1 have one, whereas HP12, HP15, PAP-2 and PAP-3 have two clip domains. Multiple sequence alignment of catalytic domains in these and other arthropod serine proteinases provided useful clues for future functional analysis. Northern blot and reverse transcription PCR (RT-PCR) analyses showed increases in HP2, HP7, HP9, HP10, HP12-HP22 mRNA levels at 24h after a bacterial challenge, and immunoblot analysis confirmed elevated concentrations of HP12, HP14-HP19, HP21 and HP22 proteins in plasma in response to injected bacteria. Hemocytes express HP13 and HP18; fat body produces HP12, HP20-HP22; both tissues synthesize the other HPs. These results collectively indicate the existence of a complex serine proteinase network in M. sexta hemolymph, predicted to mediate rapid defense responses upon wounding and/or microbial infection.
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Project description:Identification of selective deubiquitinase (DUB) inhibitors is critical for probe development to further understand and explore DUB biological function. Here, we detail the optimization and deployment of an in vitro fluorogenic ubiquitin-rhodamine assay to conduct high-throughput screening of a small molecule library against a panel of DUBs. In screening the compound library against multiple DUBs in parallel, we describe an approach for identifying selective DUB inhibitors and provide a roadmap for enabling selective DUB inhibitor discovery. For complete details on the use and execution of this protocol, please refer to Varca et al. (2021).
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