Molecular characterization of zyme/protease M/neurosin (PRSS9), a hormonally regulated kallikrein-like serine protease

The cDNA for the zyme/protease M/neurosin gene (HGMW-approved symbol PRSS9) has recently been identified. Zyme appears to play a role in Alzheimer disease as well as in breast cancer. In this paper, we describe the complete genomic organization of the zyme gene. Zyme spans 10.5 kb of genomic sequence on chromosome 19q13.3-q13.4. The gene consists of seven exons, the first two of which are untranslated. All splice junctions follow the GT/AG rule, and the intron phases are identical to those of many other genes belonging to the same family, i.e., the kallikreins, NES1, and neuropsin. Fine-mapping of the genomic locus indicates that zyme lies upstream of the NES1 gene and downstream from the PSA and KLK2 genes. Tissue expression studies indicate that zyme is expressed mainly in brain tissue, including spinal cord and cerebellum, in mammary gland, and in kidney and uterus. Zyme is regulated by steroid hormones in the breast carcinoma cell line BT-474. Estrogens and progestins, and to a lesser extent androgens, up-regulate the zyme gene in a dose-dependent manner.     

The new kallikrein-like gene, KLK-L2. Molecular characterization, mapping, tissue expression, and hormonal regulation

Since in rodents the kallikreins are represented by a large multi-gene family, the restriction of this family in humans to three genes is somewhat surprising. In an effort to identify new human kallikrein genes, we examined a genomic area of about 300 kilobases on chromosome 19q13.3-q13.4, a region that contains most of the currently known kallikreins. By using the positional candidate approach, we were able to identify a new gene named KLK-L2 (for kallikrein- like gene 2). Screening of human EST libraries allowed us to delineate the full genomic and cDNA structure of the new gene. KLK-L2 consists of 5 coding exons and 4 introns and has significant similarities to other members of the kallikrein multi-gene family. Homology studies suggest that the protein is likely secreted. KLK-L2 is expressed mainly in breast, brain, and testis and to a lesser extent in many other tissues. KLK-L2 is up-regulated by estrogens and progestins in the breast cancer cell line BT-474.     

Human tissue kallikreins: a new enzymatic cascade pathway?

Serine proteases are proteolytic enzymes with an active serine residue in their catalytic site. Kallikreins are a subgroup of the serine protease family which is known to have diverse physiological functions. The human kallikrein gene family has now been fully characterized and includes 15 members tandemly located on chromosome 19q13.4. Here we discuss the common structural features of kallikreins at the DNA, mRNA and protein levels and summarize their tissue expression and hormonal regulation patterns. Kallikreins are expressed in many tissues including the salivary gland, endocrine tissues such as testis, prostate, breast and endometrium, and in the central nervous system. Most genes appear to be under steroid hormone regulation. The occurrence of several splice variants is common among kallikreins, and some of the splice variants seem to be tissue-specific and might be related to certain pathological conditions. Kallikreins are secreted in an inactive 'zymogen' form which is activated by cleavage of an N-terminal peptide. Some kalikreins 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 three which are probably chymotrypsin-like. New, but mainly circumstantial evidence, suggests that at least some kallikreins may be part of a novel enzymatic cascade pathway which is turned-on in aggressive forms of ovarian and probably other cancers.     

Human tissue kallikreins: physiologic roles and applications in cancer

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.     

KLK12 is a novel serine protease and a new member of the human kallikrein gene family-differential expression in breast cancer

Kallikreins are a subgroup of serine proteases that are involved in the posttranslational processing of polypeptide precursors. Growing evidence suggests that many kallikreins are implicated in carcinogenesis. In rodents, kallikreins are encoded by a large multigene family, but in humans, only three genes have been identified. By using the positional candidate approach, we were able to identify a new kallikrein-like gene, tentatively named KLK12 (for kallikrein gene 12). This new gene maps to chromosome 19q13.3-q13.4, is formed of five coding exons, and shows structural similarity to serine proteases and other known kallikreins. KLK12 is expressed in a variety of tissues including salivary gland, stomach, uterus, lung, thymus, prostate, colon, brain, breast, thyroid, and trachea. We identified three splicing forms of KLK12 that are expressed in many tissues. Our preliminary results indicate that the expression of KLK12 is down-regulated at the mRNA level in breast cancer tissues and is up-regulated by steroid hormones in breast and prostate cancer cell lines. This gene may be involved in the pathogenesis and/or progression of certain cancer types and may find applicability as a novel cancer biomarker.     

Identification and characterization of KLK-L4, a new kallikrein-like gene that appears to be down-regulated in breast cancer tissues

Kallikreins are a subgroup of serine proteases and these proteolytic enzymes have diverse physiological functions in many tissues. Growing evidence suggests that many kallikreins are implicated in carcinogenesis. In rodents, kallikreins constitute a large multigene family, but in humans, only three genes were identified. By using the positional candidate gene approach, we were able to identify a new kallikrein-like gene, tentatively named KLK-L4 (for kallikrein-like gene 4). This new gene maps to chromosome 19q13. 3-q13.4, is formed of five coding exons and four introns, and shows structural similarity to other kallikreins and kallikrein-like genes. KLK-L4 is expressed in a variety of tissues including prostate, salivary gland, breast, and testis. Our preliminary results show that KLK-L4 is down-regulated, at the mRNA level, in breast cancer tissues and breast cancer cell lines. Its expression is regulated by steroid hormones in the breast cancer cell line BT-474. This gene may be involved in the pathogenesis and/or progression of breast cancer and may find applicability as a novel cancer biomarker.     

Evaluating the evidence for targeting FOXO3a in breast cancer: a systematic review

Background

Tumour cells show greater dependency on glycolysis so providing a sufficient and rapid energy supply for fast growth. In many breast cancers, estrogen, progesterone and epidermal growth factor receptor-positive cells proliferate in response to growth factors and growth factor antagonists are a mainstay of treatment. However, triple negative breast cancer (TNBC) cells lack receptor expression, are frequently more aggressive and are resistant to growth factor inhibition. Downstream of growth factor receptors, signal transduction proceeds via phosphatidylinositol 3-kinase (PI3k), Akt and FOXO3a inhibition, the latter being partly responsible for coordinated increases in glycolysis and apoptosis resistance. FOXO3a may be an attractive therapeutic target for TNBC. Therefore we have undertaken a systematic review of FOXO3a as a target for breast cancer therapeutics.

Methods

Articles from NCBI were retrieved systematically when reporting primary data about FOXO3a expression in breast cancer cells after cytotoxic drug treatment.

Results

Increased FOXO3a expression is common following cytotoxic drug treatment and is associated with apoptosis and cell cycle arrest. There is some evidence that metabolic enzyme expression is also altered and that this effect is also elicited in TNBC cells. FOXO3a expression serves as a positive prognostic marker, especially in estrogen (ER) receptor positive cells.

Discussion

FOXO3a is upregulated by a number of receptor-dependent and -independent anti-cancer drugs and associates with apoptosis. The identification of microRNA that regulate FOXO3a directly suggest that it offers a tangible therapeutic target that merits wider evaluation.     

Quantitative tandem mass-spectrometry of skin tissue reveals putative psoriatic arthritis biomarkers

Background

Psoriatic arthritis (PsA) is a distinct inflammatory arthritis occurring in 30% of psoriasis patients. There is a high prevalence of undiagnosed PsA in psoriasis patients; therefore, identifying soluble biomarkers for PsA could help in screening psoriasis patients for appropriate referral to a rheumatologist. Potential PsA biomarkers likely originate in sites of inflammation, such as the skin, and subsequently enter systemic circulation. Our goal was to identify candidate PsA biomarkers by comparing the proteome of skin biopsies obtained from patients with PsA to that from patients with psoriasis without PsA.

Methods

Skin biopsies were obtained from involved and uninvolved skin of 10 PsA and 10 age/gender-matched psoriasis patients without PsA (PsC). Using strong cation exchange chromatography, followed by label-free quantitative tandem mass spectrometry, we characterized the proteomes of pooled skin samples. Extracted ion current intensities were used to calculate protein abundance ratios, and these were utilized to identify differentially regulated proteins.

Results

Forty-seven proteins were elevated in PsA-derived skin compared to PsC-derived skin. Selected reaction monitoring assays were developed to quantify these potential PsA markers in individual skin samples, and 8 markers were confirmed in an independent sample set. ITGB5 and POSTN were measured in serum samples from 33 PsA and 15 PsC patients, using enzyme-linked immunosorbent assays. ITGB5 was significantly elevated in PsA serum (P < 0.01), and POSTN showed a trend. ITGB5 and POSTN correlated significantly in both patient groups (r = 0.472, P < 0.001).

Conclusion

Proteomic analysis of PsA and PsC skin identified eight new candidate biomarkers. These markers need to be validated with a larger and independent cohort, in order to delineate their clinical utility in PsA patients. These proteins may also uncover unknown aspects of PsA pathobiology.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-12-1) contains supplementary material, which is available to authorized users.     

Defining the extended substrate specificity of kallikrein 1-related peptidases

Human kallikrein 1-related peptidases (KLKs) form a subfamily of 15 extracellular (chymo)tryptic-like serine proteases. KLKs 4, 5, 13 and 14 display altered expression/activity in diverse pathological conditions, including cancer. However, their distinct (patho)physiological roles remain largely uncharacterized. As a step toward distinguishing their proteolytic functions, we attempt to define their primary and extended substrate specificities and identify candidate biological targets. Heterologously expressed KLKs 4, 5, 13 and 14 were screened against fluorogenic 7-amino-4-carbamoylmethylcoumarin positional scanning-synthetic combinatorial libraries with amino acid diversity at the P1-P4 positions. Our results indicate that these KLKs share a P1 preference for Arg. However, each KLK exhibited distinct P2-P4 specificities, attributable to structural variations in their surface loops. The preferred P4-P1 substrate recognition motifs based on optimal subsite occupancy were as follows: VI-QSAV-QL-R for KLK4; YFWGPV-RK-NSFAM-R for KLK5; VY-R-LFM-R for KLK13; and YW-KRSAM-HNSPA-R for KLK14. Protein database queries using these motifs yielded many extracellular targets, some of which represent plausible KLK substrates. For instance, cathelicidin, urokinase-type plasminogen activator, laminin and transmembrane protease serine 3 were retrieved as novel putative substrates for KLK4, 5, 13 and 14, respectively. Our findings may facilitate studies on the role of KLKs in (patho)physiology and can be used in the development of selective KLK inhibitors.