Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Kallikreins are proteolytic enzymes that constitute a subfamily of serine proteases. Novel kallikrein genes were cloned recently, and it was shown that the human kallikrein family contains 15 genes tandemly aligned on chromosomal locus 19q13.3-q13.4. Based on their altered expression in tumor cells, kallikreins may be involved in the pathogenesis and/or progression of cancer. Evidence is presented that certain kallikreins may be exploited as diagnostic cancer biomarkers. Although the function(s) of novel kallikreins is currently unknown, increasing evidence suggests that kallikreins may participate in regulatory enzymatic cascade(s). Elucidation of the function of novel kallikreins largely depends on the availability of active recombinant proteins. Here, the zymogen for kallikrein 13 was overexpressed in Pichia pastoris and biochemically characterized. It was shown that the kallikrein 13 zymogen displays intrinsic catalytic activity leading to autoactivation. A clipped form of kallikrein 13 was identified, indicating autocatalytic cleavage at the internal bond R114-S115. Mature kallikrein 13 displays trypsin-like activity with restricted specificity on synthetic and protein substrates. Combinatorial P1-Lys libraries of tetrapeptide fluorogenic substrates were synthesized and used for the profiling of the P2 specificity of selected kallikreins. Interestingly, it was shown that human kallikrein 13, similarly to PSA, could specifically cleave human plasminogen to generate angiostatin-like fragments, suggesting that specific kallikreins may have antiangiogenic actions. An understanding of the physiology of human kallikreins is emerging with potential clinical applications.
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PMID:Emerging interest in the kallikrein gene family for understanding and diagnosing cancer. 1272 28

Serine proteases are proteolytic enzymes with an active serine residue in their catalytic site. Kallikreins are a subgroup of the serine protease family and are known to have diverse physiological functions. The human tissue kallikrein gene family has now been fully characterized and includes 15 members, clustered in a 300 kb region on chromosome 19q13.4. In this review, we discuss the common structural features of kallikreins at the DNA, mRNA and protein levels. Kallikreins are secreted as inactive zymogens and are activated by cleavage of an N-terminal peptide. Some kallikreins can undergo autoactivation while others may be activated by other kallikreins or other proteases. Most kallikreins are predicted to have trypsin-like enzymatic activity except for three members which may have chymotrypsin-like activity. Circumstantial evidence suggests that at least some kallikreins may be part of an enzymatic cascade pathway which is activated in aggressive forms of ovarian and probably other cancers. Accumulating evidence suggests potential diagnostic and/or prognostic roles of kallikreins in diverse malignancies. In addition to PSA, many other kallikreins show differential expression in malignancy. For example, hK6, 10 and 11 are promising serological markers for ovarian cancer diagnosis. KLK10 may act as a tumor suppressor. In addition to their diagnostic and prognostic values, kallikreins may also be good therapeutic targets.
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PMID:Tissue kallikreins: new players in normal and abnormal cell growth? 1287 20

Tissue kallikreins are members of the S1 family (clan SA) of trypsin-like serine proteases and are present in at least six mammalian orders. In humans, tissue kallikreins (hK) are encoded by 15 structurally similar, steroid hormone-regulated genes (KLK) that colocalize to chromosome 19q13.4, representing the largest cluster of contiguous protease genes in the entire genome. hKs are widely expressed in diverse tissues and implicated in a range of normal physiologic functions from the regulation of blood pressure and electrolyte balance to tissue remodeling, prohormone processing, neural plasticity, and skin desquamation. Several lines of evidence suggest that hKs may be involved in cascade reactions and that cross-talk may exist with proteases of other catalytic classes. The proteolytic activity of hKs is regulated in several ways including zymogen activation, endogenous inhibitors, such as serpins, and via internal (auto)cleavage leading to inactivation. Dysregulated hK expression is associated with multiple diseases, primarily cancer. As a consequence, many kallikreins, in addition to hK3/PSA, have been identified as promising diagnostic and/or prognostic biomarkers for several cancer types, including ovarian, breast, and prostate. Recent data also suggest that hKs may be causally involved in carcinogenesis, particularly in tumor metastasis and invasion, and, thus, may represent attractive drug targets to consider for therapeutic intervention.
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PMID:Human tissue kallikreins: physiologic roles and applications in cancer. 1519 20

Protein cleavage-isotope dilution mass spectrometry (PC-IDMS) can be used to quantify proteins, with an isotope-labeled analogue of the peptide fragment used as an internal standard. Here, we investigate use of a standard LC-MS/MS platform for quantifying a model biomarker directly from serum by this technique. We synthesized a peptide (IVGGWECEK) identical to the N-terminal tryptic fragment of PSA but with each glycine containing two 13C atoms and one 15N atom. PSA-free human serum was denatured with urea followed by the introduction of PSA standard and the stable isotope labeled internal standard peptide. The sample was then proteolyzed with trypsin and subjected to quantification using LC-MS/ MS on a triple quadrupole mass spectrometer. A linear least squares calibration curve made from five different concentrations of PSA added to serum and digested (each made in triplicate and randomly injected three times) had a mean slope of 0.973 (SE = 0.023), intercept of -0.003 (SE = 0.022), and R2 of 0.971. Recovery of calibrators ranged from 70 to 85% with a mean run-to-run CV of 13% and a mean within-run CV of 5.7%. PC-IDMS is a promising technique for quantifying proteins covering a broad range of applications from standardizing immunoassays to monitoring post-translational modifications to quantifying newly discovered biomarkers prior to the development and implementation of an immunoassay, just to name a few. Issues surrounding the application of PC-IDMS for the absolute quantification of proteins include selection of a proteolytic fragment for quantification that can be cleaved and isolated reproducibly over a broad dynamic range, stable isotope labeled synthetic peptide standards that give consistent results, and LC-MS/MS methods that provide adequate sensitivity and reproducibility without creating impractical analysis times. The results presented here show that absolute quantification can be performed on the model biomarker PSA introduced into denatured serum when analyzed by LC-MS/MS. However, concerns still exist regarding sensitivity compared to existing immunoassays as well as the reproducibility of PC-IDMS performed in different matrixes.
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PMID:Absolute quantification of the model biomarker prostate-specific antigen in serum by LC-Ms/MS using protein cleavage and isotope dilution mass spectrometry. 1525 48

Photonic induced immobilization is a novel technology that results in spatially oriented and spatially localized covalent coupling of biomolecules onto thiol-reactive surfaces. Immobilization using this technology has been achieved for a wide selection of proteins, such as hydrolytic enzymes (lipases/esterases, lysozyme), proteases (human plasminogen), alkaline phosphatase, immunoglobulins' Fab fragment (e.g., antibody against PSA [prostate specific antigen]), Major Histocompability Complex class I protein, pepsin, and trypsin. The reaction mechanism behind the reported new technology involves "photonic activation of disulfide bridges," i.e., light-induced breakage of disulfide bridges in proteins upon UV illumination of nearby aromatic amino acids, resulting in the formation of free, reactive thiol groups that will form covalent bonds with thiol-reactive surfaces (see Fig. 1). Interestingly, the spatial proximity of aromatic residues and disulfide bridges in proteins has been preserved throughout molecular evolution. The new photonic-induced method for immobilization of proteins preserves the native structural and functional properties of the immobilized protein, avoiding the use of one or more chemical/thermal steps. This technology allows for the creation of spatially oriented as well as spatially defined multiprotein/DNA high-density sensor arrays with spot size of 1 microm or less, and has clear potential for biomedical, bioelectronic, nanotechnology, and therapeutic applications.
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PMID:Photonic activation of disulfide bridges achieves oriented protein immobilization on biosensor surfaces. 1643 46

Human tissue kallikreins (hKs) form a family of 15 closely related (chymo)trypsin-like serine proteinases. These tissue kallikreins are expressed in a wide range of tissues including the central nervous system, the salivary gland, and endocrine-regulated tissues, such as prostate, breast, or testis, and may have diverse physiological functions. For several tissue kallikreins, a clear correlation has been established between expression and different types of cancer. For example, the prostate-specific antigen (PSA or hK3) serves as tumor marker and is used to monitor therapy response. Using a novel strategy, we have cloned, expressed in Escherichia coli or in insect cells, refolded, activated, and purified the seven human tissue kallikreins hK3/PSA, hK4, hK5, hK6, hK7, hK10, and hK11. Moreover, we have determined their extended substrate specificity for the nonprime side using a positional scanning combinatorial library of tetrapeptide substrates. hK3/PSA and hK7 exhibited a chymotrypsin-like specificity preferring large hydrophobic or polar residues at the P1 position. In contrast, hK4, hK5, and less stringent hK6 displayed a trypsin-like specificity with strong preference for P1-Arg, whereas hK10 and hK11 showed an ambivalent specificity, accepting both basic and large aliphatic P1 residues. The extended substrate specificity profiles are in good agreement with known substrate cleavage sites but also in accord with experimentally solved (hK4, hK6, and hK7) or modeled structures. The specificity profiles may lead to a better understanding of human tissue kallikrein functions and assist in identifying their physiological protein substrates as well as in designing more selective inhibitors.
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PMID:Specificity profiling of seven human tissue kallikreins reveals individual subsite preferences. 1674 Jun 31

Although flow cytometry is useful for studying neural lineage relationships, the method of dissociation can potentially bias cell analysis. We compared dissociation methods on viability and antigen recognition of mouse central nervous system (CNS) tissue and human CNS tumor tissue. Although nonenzymatic dissociation yielded poor viability, papain, purified trypsin replacement (TrypLE), and two purified collagenase/neutral protease cocktails (Liberase-1 or Accutase) each efficiently dissociated fetal tissue and postnatal tissue. Mouse cells dissociated with Liberase-1 were titrated with antibodies identifying distinct CNS precursor subtypes, including CD133, CD15, CD24, A2B5, and PSA-NCAM. Of the enzymes tested, papain most aggressively reduced antigenicity for mouse and human CD24. On human CNS tumor cells, CD133 expression remained highest after Liberase-1 and was lowest after papain or Accutase treatment; Liberase-1 digestion allowed magnetic sorting for CD133 without the need for an antigen re-expression recovery period. We conclude that Liberase-1 and TrypLE provide the best balance of dissociation efficiency, viability, and antigen retention. One implication of this comparison was confirmed by dissociating E13.5 mouse cortical cells and performing prospective isolation and clonal analysis on the basis of CD133/CD24 or CD15/CD24 expression. Highest fetal expression of CD133 or CD15 occurred in a CD24(hi) population that was enriched in neuronal progenitors. Multipotent cells expressed CD133 and CD15 at lower levels than did these neuronal progenitors. We conclude that CD133 and CD15 can be used similarly as selectable markers, but CD24 coexpression helps to distinguish fetal mouse multipotent stem cells from neuronal progenitors and postmitotic neurons. This particular discrimination is not possible after papain treatment. Disclosure of potential conflicts of interest is found at the end of this article.
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PMID:Optimized flow cytometric analysis of central nervous system tissue reveals novel functional relationships among cells expressing CD133, CD15, and CD24. 1733 13

Two forms of the same commercial product (SORBIAL, Allonnes, France), one with live bacteria (PSA) and the other with heat-inactivated bacteria (PSI), containing a mixture of 2 strains of lactobacilli and their growth medium were tested as a diet complement for juvenile sea bass (Dicentrarchus labrax) during a 103-day experiment. In addition to zootechnical parameters (survival, growth, conformation), some effects on digestive metabolism were studied, including enzymatic, ultrastructural and microbial aspects. Microbial preparations improved survival rate. The ventral, dorsal and operculum malformations which usually occur in juveniles did not appear in those receiving PSA and PSI. Furthermore, they stimulated, but not constantly, trypsin and acid phosphatase activities. Intestinal ultrastructure showed an increase in the number of endocytosis vesicles at the apical pole of enterocytes in fishes receiving enrichments. Bacterial flora was not modified in terms of quantity, especially the lactic acid bacteria counts, which were not changed in fishes receiving live lactobacilli (PSA). The mode of action of these multiple beneficial effects appears complex and could be caused by different molecules inside the bacterial cell or excreted into their medium.
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PMID:Preliminary study of the effects of commercial lactobacilli preparations on digestive metabolism of juvenile sea bass (Dicentrarchus labrax). 1795 16

Mass spectrometry-based strategies for the quantification of low-abundance putative protein biomarkers in human blood currently require extensive sample fractionation steps which hamper their implementation in a routine and robust way across clinical laboratories. We demonstrate that a technique using MS(3) reconstructed chromatograms on a signature of secondary ions issued from a trapped primary product ion, termed multiple reaction monitoring cubed (MRM(3)), enables targeting protein biomarkers in the low nanogram/milliliter range in nondepleted human serum. The simple two-step workflow is based on a trypsin proteolysis of whole serum (100 microL) followed by enrichment of targeted proteotypic peptides on a solid phase extraction column using mixed-cation exchange resin. MRM(3)'s fidelity of peak detection extends the dynamic range and limit of quantitation (LOQ) of protein biomarkers to the low nanogram/milliliter range, corresponding to a concentration that is 10(6)-fold lower than the concentration of the most abundant proteins in serum. The power of the MRM(3) method is illustrated by the assay of prostate specific antigen in nondepleted human sera of patients. The results correlate well with the established method for determining PSA levels in serum, i.e., enzyme-linked immunosorbent assay (ELISA) tests.
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PMID:Multiple reaction monitoring cubed for protein quantification at the low nanogram/milliliter level in nondepleted human serum. 1983 94

Poly(L-lysine), (PLL), and poly(DL-amino serinate), (PSA), are respectively enzymatically and hydrolytically degradable polycations. This work was aimed at investigating their degradability when they are complexed with polyanions, namely poly(acrylic acid) and poly(L-lysine citramide), taken as simple models of DNA in polyplexes. Comparison was made with degradation characteristics of the same polycations in solution in the absence of polyanion on the basis of size exclusion chromatography and capillary zone electrophoresis. Complexed PLL remained enzymatically degradable by trypsin, an endopeptidase, but was no longer degradable by aminopeptidase, an exopeptidase. Trypsin yielded a mixture of trilysin and tetralysin. Complexed PSA remained hydrolytically degradable in aqueous media. The hydrolysis of PSA led to DL-serine. However, traces of anionic species were also detected that were identified as residues of constituting repeating units issued from the N-benzyloxycarbonyl polyaminoserinate precursor (PSAZ).
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PMID:Degradability of poly(L-lysine) and poly(DL-aminoserinate) complexed with a polyanion under conditions modelling physico-chemical characteristics of body fluids. 2067 6


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