Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0376358 (prostate cancer)
 59,338 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Kallikrein-related peptidase 4 (KLK4) is one of the 15 members of the human KLK family and a trypsin-like, prostate cancer-associated serine protease. Signaling initiated by trypsin-like serine proteases are transduced across the plasma membrane primarily by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. Here we show, using Ca(2+) flux assays, that KLK4 signals via both PAR-1 and PAR-2 but not via PAR-4. Dose-response analysis over the enzyme concentration range 0.1-1000 nM indicated that KLK4-induced Ca(2+) mobilization via PAR-1 is more potent than via PAR-2, whereas KLK4 displayed greater efficacy via the latter PAR. We confirmed the specificity of KLK4 signaling via PAR-2 using in vitro protease cleavage assays and anti-phospho-ERK1/2/total ERK1/2 Western blot analysis of PAR-2-overexpressing and small interfering RNA-mediated receptor knockdown cell lines. Consistently, confocal microscopy analyses indicated that KLK4 initiates loss of PAR-2 from the cell surface and receptor internalization. Immunohistochemical analysis indicated the co-expression of agonist and PAR-2 in primary prostate cancer and bone metastases, suggesting that KLK4 signaling via this receptor will have pathological relevance. These data provide insight into KLK4-mediated cell signaling and suggest that signals induced by this enzyme via PARs may be important in prostate cancer.
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PMID:Kallikrein-related peptidase 4 (KLK4) initiates intracellular signaling via protease-activated receptors (PARs). KLK4 and PAR-2 are co-expressed during prostate cancer progression. 1830 30

The prostate is a site of high expression of serine proteinases including members of the kallikrein-related peptidase (KLK) family, as well as other secreted and membrane-anchored serine proteinases. It has been known for some time that members of this enzyme family elicit cellular responses by acting directly on cells. More recently, it has been recognised that for serine proteinases with specificity for cleavage after arginine and lysine residues (trypsin-like or tryptic enzymes) these cellular responses are often mediated by cleavage of members of the proteinase-activated receptor (PAR) family--a four member sub-family of G protein-coupled receptors. Here, we review the expression of PARs in prostate, the ability of prostatic trypsin-like KLKs and other prostate-expressed tryptic enzymes to cleave PARs, as well as the prostate cancer-associated consequences of PAR activation. In addition, we explore the dysregulation of trypsin-like serine proteinase activity through the loss of normal inhibitory mechanisms and potential interactions between these dysregulated enzymes leading to aberrant PAR activation, intracellular signalling and cancer-promoting cellular changes.
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PMID:Prostatic trypsin-like kallikrein-related peptidases (KLKs) and other prostate-expressed tryptic proteinases as regulators of signalling via proteinase-activated receptors (PARs). 1862 86

Proteomics discovery leads to a list of potential protein biomarkers that have to be subsequently verified and validated with a statistically viable number of patients. Although the most sensitive, the development of an ELISA test is time-consuming when antibodies are not available and need to be conceived. Mass spectrometry analysis driven in quantitative multiple reaction monitoring mode is now appearing as a promising alternative to quantify proteins in biological fluids. However, all the studies published to date describe limits of quantitation in the low microg/ml range when no immunoenrichment of the target protein is applied, whereas the concentration of known clinical biomarkers is usually in the ng/ml range. Using prostate-specific antigen as a model biomarker, we now provide proof of principle that mass spectrometry enables protein quantitation in a concentration range of clinical interest without immunoenrichment. We have developed and optimized a robust sample processing method combining albumin depletion, trypsin digestion, and solid phase extraction of the proteotypic peptides starting from only 100 microl of serum. For analysis, mass spectrometry was coupled to a conventional liquid chromatography system using a 2-mm-internal diameter reverse phase column. This mass spectrometry-based strategy was applied to the quantitation of prostate-specific antigen in sera of patients with either benign prostate hyperplasia or prostate cancer. The quantitation was performed against an external calibration curve by interpolation, and results showed good correlation with existing ELISA tests applied to the same samples. This strategy might now be implemented in any clinical laboratory or certified company for further evaluation of any putative biomarker in the low ng/ml range of serum or plasma.
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PMID:Clinical quantitation of prostate-specific antigen biomarker in the low nanogram/milliliter range by conventional bore liquid chromatography-tandem mass spectrometry (multiple reaction monitoring) coupling and correlation with ELISA tests. 1906 76

Prostate tumor cells over-express a prostate-specific membrane antigen (PSMA) that can be used as a marker to select these cells from highly heterogeneous clinical samples, even when found in low abundance. Antibodies and aptamers have been developed that specifically bind to PSMA. In this study, anti-PSMA aptamers were immobilized onto the surface of a capture bed poised within a PMMA, microchip, which was fabricated into a high-throughput micro-sampling unit (HTMSU) used for the selective isolation of rare circulating prostate tumor cells resident in a peripheral blood matrix. The HTMSU capture bed consisted of 51 ultra-high-aspect ratio parallel curvilinear channels with a width similar to the prostate cancer cell dimensions. The surface density of the PSMA-specific aptamers on an ultraviolet-modified PMMA microfluidic capture bed surface was determined to be 8.4 x 10(12) molecules/cm(2). Using a linear velocity for optimal cell capture in the aptamer-tethered HTMSU (2.5 mm/s), a recovery of 90% of LNCaP cells (prostate cancer cell line; used as a model in this example) was found. Due to the low abundance of these cells, the input volume required was 1 mL and this could be processed in approximately 29 min using an optimized linear flow rate of 2.5 mm/s. Captured cells were subsequently released intact from the affinity surface using 0.25% w/w trypsin followed by counting individual cells using a contact conductivity sensor integrated into the HTMSU that provided high detection and sampling efficiency (approximately 100%) and did not require staining of the cells for enumeration.
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PMID:Highly efficient capture and enumeration of low abundance prostate cancer cells using prostate-specific membrane antigen aptamers immobilized to a polymeric microfluidic device. 1972 12

Ecotin is a bidentate, fold-specific inhibitor of mammalian serine-proteases produced by Escherichia coli. This molecule may be engineered to increase and/or change its affinity and specificity providing significant biotechnological potential. Since ecotin binds tightly to serine proteases of the trypsin fold, it may help to identify the role of these enzymes in different biological processes. In this work, we tested ecotin variants as an affinity purification reagent for identifying enzymes in samples of tumor progression and mammary gland involution. Initially, we used a commercial source of urokinase-type plasminogen activator (u-PA) that remained fully active after elution from an affinity column of the ecotin variant (M84R, M85R). We then successfully identified u-PA from more complex mixtures including lysates from a prostate cancer cell line and involuting mouse mammary glands. Interestingly, a membrane-type serine protease 1 was isolated from the Triton X-100-solubilized PC-3 cell lysates, and surprisingly, haptoglobin, a serine-protease homolog protein, was also identified in mammary gland lysates and in blood. Haptoglobin does not prevent ecotin inhibition of u-PA, but it may act as a carrier within blood when ecotin is used in vivo. Finally, this affinity purification matrix was also able to identify a thrombin-like enzyme from snake venom using an ecotin variant directed against thrombin. Overall, the ecotin variants acted as robust tools for the isolation and characterization of proteins with a trypsin fold. Thus, they may assist in the understanding of the role of these serine proteases and homologous proteins in different biological processes.
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PMID:Engineering ecotin for identifying proteins with a trypsin fold. 1972 73

Reversible protein phosphorylation forms the basis of cell signaling networks. Aberrations in protein phosphorylation have been linked to human diseases including cancer. Phosphoproteomics has recently emerged as an approach that focuses on analysis of protein phosphorylation on a global scale. We have recently developed a new methodology, termed in-gel IEF LC-MS/MS, and we have adapted this methodology for phosphoproteome analysis. Here, we report on the application of in-gel IEF LC-MS/MS to the mapping of the phosphoproteome in the LNCaP human prostate cancer cell line. The analytical methodology used in the study included separation of the LNCaP proteins by in-gel isoelectric focusing (IEF), digestion of the proteins with trypsin, enrichment of the digests for phosphopeptides with Immobilized Metal Ion Affinity Chromatography (IMAC), analysis of the enriched digests by LC-MS/MS, and identification of the phosphorylated peptides/proteins through searches of a protein sequence database. With this analytical platform, we have characterized over 600 different phosphorylation sites in 296 phosphoproteins. This panel of the LNCaP phosphoproteins is 3-fold larger than the panel obtained in our previous work, which attests to the power of the chosen analytical methodology. The characterized phosphoproteins are functionally diverse and include a number of proteins relevant to cancer.
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PMID:Characterization of the phosphoproteome in LNCaP prostate cancer cells by in-gel isoelectric focusing and tandem mass spectrometry. 2004 36

KLK4 is a member of the human kallikrein-related peptidase family of (chymo)trypsin-like serine proteases. The aim of the present study was to generate polyclonal antibodies (pAb) directed against KLK4 for the analysis of KLK4 by immunohistochemistry in human tissues. Recombinantly expressed human mature KLK4 was used for immunization of chickens. pAb 617A is an affinity-purified monospecific pAb fraction reacting with a linear epitope within a flexible surface-exposed loop of KLK4. pAb 617C is the KLK-directed pAb fraction completely depleted from pAb 617A. In healthy adult tissues, KLK4 was immunodetected by both antibody fractions in kidney, liver, and prostate, but not in other organs such as colon and lung. To evaluate protein expression of KLK4 in prostate cancer, samples of tumor tissue plus corresponding tumor-free areas of 44 prostate cancer patients, represented on a tissue microarray, were investigated. Distinct KLK4 immunostaining was observed with both antibodies in cancerous glandular epithelial cells, but not in surrounding stromal cells. KLK4 expression was lower in stage pT3+4 than in pT1+2 tumors, which was highly significant when employing pAb 617A. Thus, our results indicate that KLK4, which is expressed in the healthy prostate, is upregulated in early-stage but not late-stage prostate cancer.
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PMID:Polyclonal antibodies against kallikrein-related peptidase 4 (KLK4): immunohistochemical assessment of KLK4 expression in healthy tissues and prostate cancer. 2018 Jun 34

Kallikrein-related peptidase 2 (KLK2) degrades insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) in vitro. IGFBP-3 forms complexes with IGFs, preventing them from binding to their receptors and stimulating cell proliferation and survival. IGF-independent actions have also been described for IGFBP-3. The degradation of IGFBP-3 by KLK2 or other proteases in the prostate may promote the growth of prostate cancer. We studied IGFBP-3 degradation by immunoblotting and two specific immunoassays, one recognizing only native non-fragmented IGFBP-3 and the other one recognizing both intact and proteolytically cleaved IGFBP-3. Peptides were used to inhibit the enzyme activity of KLK2 and cleavage sites in IGFBP-3 were identified by mass spectrometry. KLK2 proteolyzed IGFBP-3 into several small fragments, mostly after Arg residues, in keeping with the trypsin-like activity of KLK2. The fragmentation could be inhibited by KLK2-inhibiting peptides in a dose-dependent fashion. As degradation of IGFBP-3 could lead to a more aggressive cancer phenotype, inhibition of KLK2 activity might be useful for treatment of prostate cancer and other diseases associated with increased KLK2 activity.
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PMID:Identification of IGFBP-3 fragments generated by KLK2 and prevention of fragmentation by KLK2-inhibiting peptides. 2018 Jun 40

CPA4 (carboxypeptidase A4) is a member of the metallocarboxypeptidase family. CPA4 was originally found in a screen of mRNAs up-regulated by sodium butyrate-induced differentiation of cancer cells. Further studies suggested a relation between CPA4 and prostate cancer aggressiveness. In the present study, we determined that CPA4 is secreted from cells as a soluble proenzyme (pro-CPA4) that can be activated by endoproteases, such as trypsin. Three complementary approaches were used to study the substrate specificity of CPA4; kinetic analysis was performed using a new series of chromogenic substrates and some biologically relevant peptides, the cleavage of synthetic peptides was tested individually, and the cleavage of a mixture of >100 mouse brain peptides was examined using a quantitative peptidomics mass spectrometry-based approach. CPA4 was able to cleave hydrophobic C-terminal residues with a preference for Phe, Leu, Ile, Met, Tyr, and Val. However, not all peptides with C-terminal hydrophobic residues were cleaved, indicating the importance of additional residues within the peptide. Aliphatic, aromatic, and basic residues in the P1 position have a positive influence on the cleavage specificity. In contrast, acidic residues, Pro, and Gly have a negative influence in the P1 position. Some of the peptides identified as CPA4 substrates (such as neurotensin, granins, and opioid peptides) have been previously shown to function in cell proliferation and differentiation, potentially explaining the link between CPA4 and cancer aggressiveness. Taken together, these studies suggest that CPA4 functions in neuropeptide processing and regulation in the extracellular environment.
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PMID:Characterization of the substrate specificity of human carboxypeptidase A4 and implications for a role in extracellular peptide processing. 2038 63

We present here the results from MS peptide profiling experiments of prostate carcinoma patients and controls with a specific focus on protease activity-related protein fragments. After purification with surface-active magnetic beads, MALDI-TOF profiling experiments were performed on tryptic digests of serum samples of prostate cancer patients with metastases (n=27) and controls (n=30). This resulted in the reproducible detection of eight differentially expressed peptides, which were then identified by nanoLC-MALDI-TOF/TOF and confirmed by MALDI-FTMS exact mass measurements. All differentially expressed peptides are derived from two homologous parts of human serum albumin; two of the eight peptides were tryptic and six were nontryptic. The presence of the nontryptic fragments indicates that a proteolysis process occurs which is not mediated by trypsin. Since the nontryptic fragments were found at significantly higher levels in control samples compared with metastases samples, it is proposed that a specific proteolytic inhibition process is in effect in the serum of prostate cancer patients. Experiments using synthetic peptides showed that this proteolytic activity occurs ex vivo and is sequence specific. Importantly, the observed prostate carcinoma-related inhibition of the proteolysis was reproduced ex vivo using synthetic peptides.
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PMID:Differential expression of protease activity in serum samples of prostate carcinoma patients with metastases. 2039 78


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