Kallikrein-related peptidase 4 activation of protease-activated receptor family members and association with prostate cancer

Two areas of particular importance in prostate cancer progression are primary tumour development and metastasis. These processes involve a number of physiological events, the mediators of which are still being discovered and characterised. Serine proteases have been shown to play a major role in cancer invasion and metastasis. The recently discovered phenomenon of their activation of a receptor family known as the protease activated receptors (PARs) has extended their physiological role to that of signaling molecule. Several serine proteases are expressed by malignant prostate cancer cells, including members of the kallikreinrelated peptidase (KLK) serine protease family, and increasingly these are being shown to be associated with prostate cancer progression. KLK4 is highly expressed in the prostate and expression levels increase during prostate cancer progression. Critically, recent studies have implicated KLK4 in processes associated with cancer. For example, the ectopic over-expression of KLK4 in prostate cancer cell lines results in an increased ability of these cells to form colonies, proliferate and migrate. In addition, it has been demonstrated that KLK4 is a potential mediator of cellular interactions between prostate cancer cells and osteoblasts (bone forming cells). The ability of KLK4 to influence cellular behaviour is believed to be through the selective cleavage of specific substrates. Identification of relevant in vivo substrates of KLK4 is critical to understanding the pathophysiological roles of this enzyme. Significantly, recent reports have demonstrated that several members of the KLK family are able to activate PARs. The PARs are relatively new members of the seven transmembrane domain containing G protein coupled receptor (GPCR) family. PARs are activated through proteolytic cleavage of their N-terminus by serine proteases, the resulting nascent N-terminal binds intramolecularly to initiate receptor activation. PARs are involved in a number of patho-physiological processes, including vascular repair and inflammation, and a growing body of evidence suggests roles in cancer. While expression of PAR family members has been documented in several types of cancers, including prostate, the role of these GPCRs in prostate cancer development and progression is yet to be examined. Interestingly, several studies have suggested potential roles in cellular invasion through the induction of cytoskeletal reorganisation and expression of basement membrane-degrading enzymes. Accordingly, this program of research focussed on the activation of the PARs by the prostate cancer associated enzyme KLK4, cellular processing of activated PARs and the expression pattern of receptor and agonist in prostate cancer. For these studies KLK4 was purified from the conditioned media of stably transfected Sf9 insect cells expressing a construct containing the complete human KLK4 coding sequence in frame with a V5 epitope and poly-histidine encoding sequences. The first aspect of this study was the further characterisation of this recombinant zymogen form of KLK4. The recombinant KLK4 zymogen was demonstrated to be activatable by the metalloendopeptidase thermolysin and amino terminal sequencing indicated that thermolysin activated KLK4 had the predicted N-terminus of mature active KLK4 (31IINED). Critically, removal of the pro-region successfully generated a catalytically active enzyme, with comparable activity to a previously published recombinant KLK4 produced from S2 insect cells. The second aspect of this study was the activation of the PARs by KLK4 and the initiation of signal transduction. This study demonstrated that KLK4 can activate PAR-1 and PAR-2 to mobilise intracellular Ca2+, but failed to activate PAR-4. Further, KLK4 activated PAR-1 and PAR-2 over distinct concentration ranges, with KLK4 activation and mobilisation of Ca2+ demonstrating higher efficacy through PAR-2. Thus, the remainder of this study focussed on PAR-2. KLK4 was demonstrated to directly cleave a synthetic peptide that mimicked the PAR-2 Nterminal activation sequence. Further, KLK4 mediated Ca2+ mobilisation through PAR-2 was accompanied by the initiation of the extra-cellular regulated kinase (ERK) cascade. The specificity of intracellular signaling mediated through PAR-2 by KLK4 activation was demonstrated by siRNA mediated protein depletion, with a reduction in PAR-2 protein levels correlating to a reduction in KLK4 mediated Ca2+mobilisation and ERK phosphorylation. The third aspect of this study examined cellular processing of KLK4 activated PAR- 2 in a prostate cancer cell line. PAR-2 was demonstrated to be expressed by five prostate derived cell lines including the prostate cancer cell line PC-3. It was also demonstrated by flow cytometry and confocal microscopy analyses that activation of PC-3 cell surface PAR-2 by KLK4 leads to internalisation of this receptor in a time dependent manner. Critically, in vivo relevance of the interaction between KLK4 and PAR-2 was established by the observation of the co-expression of receptor and agonist in primary prostate cancer and prostate cancer bone lesion samples by immunohistochemical analysis. Based on the results of this study a number of exciting future studies have been proposed, including, delineating differences in KLK4 cellular signaling via PAR-1 and PAR-2 and the role of PAR-1 and PAR-2 activation by KLK4 in prostate cancer cells and bone cells in prostate cancer progression.

Challenges in mass spectrometry-based quantification of bioactive peptides: A case study exploring the neuropeptide Y family

The study of biologically active peptides is critical to the understanding of physiological pathways, especially those involved in the development of disease. Historically, the measurement of biologically active endogenous peptides has been undertaken by radioimmunoassay, a highly sensitive and robust technique that permits the detection of physiological concentrations in different biofluid and tissue extracts. Over recent years, a range of mass spectrometric approaches have been applied to peptide quantification with limited degrees of success. Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) belong to the NPY family exhibiting regulatory effects on appetite and feeding behavior. The physiological significance of these peptides depends on their molecular forms and in vivo concentrations systemically and at local sites within tissues. In this report, we describe an approach for quantification of individual peptides within mixtures using high-performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of the NPY family peptides. Aspects of quantification including sample preparation, the use of matrix-matched calibration curves, and internal standards will be discussed. This method for the simultaneous determination of NPY, PYY, and PP was accurate and reproducible but lacks the sensitivity required for measurement of their endogenous concentration in plasma. The advantages of mass spectrometric quantification will be discussed alongside the current obstacles and challenges. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 357–366, 2012.

Human Kallikrein 4 signal peptide induces cytotoxic T cell responses in healthy donors and prostate cancer patients.

Immunotherapy is a promising new treatment for patients with advanced prostate and ovarian cancer, but its application is limited by the lack of suitable target antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTL). Human kallikrein 4 (KLK4) is a member of the kallikrein family of serine proteases that is significantly overexpressed in malignant versus healthy prostate and ovarian tissue, making it an attractive target for immunotherapy. We identified a naturally processed, HLA-A*0201-restricted peptide epitope within the signal sequence region of KLK4 that induced CTL responses in vitro in most healthy donors and prostate cancer patients tested. These CTL lysed HLA-A*0201+ KLK4 + cell lines and KLK4 mRNA-transfected monocyte-derived dendritic cells. CTL specific for the HLA-A*0201-restricted KLK4 peptide were more readily expanded to a higher frequency in vitro compared to the known HLA-A*0201-restricted epitopes from prostate cancer antigens; prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA) and prostatic acid phosphatase (PAP). These data demonstrate that KLK4 is an immunogenic molecule capable of inducing CTL responses and identify it as an attractive target for prostate and ovarian cancer immunotherapy.

DrsG from Streptococcus dysgalactiae subsp equisimilis inhibits the antimicrobial peptide LL- 37

SIC and DRS are related proteins present in only four of the more than 200 Streptococcus pyogenes emm-types. These proteins inhibit complement mediated lysis and/or the activity of certain antimicrobial peptides. A gene encoding a homologue of these proteins, herein called DrsG, has been identified in the related bacterium Streptococcus dysgalactiae subsp equisimilis (SDSE). Here we show that geographically dispersed isolates representing 14 of 50 emm-types examined possess variants of drsG. However not all isolates within the drsG-positive emm-types possess the gene. Sequence comparisons also reveal a high degree of conservation in different SDSE emm-types. To examine the biological activity of DrsG, recombinant versions of two major DrsG variants, DrsGS and DrsGL, were expressed and purified. Western blot analysis using antisera raised to these proteins demonstrated both variants to be expressed and secreted into culture supernatant. Unlike SIC, but similar to DRS, DrsG does not inhibit complement mediated lysis. However, like both SIC and DRS, DrsG is a ligand of the cathelcidin LL-37 and is inhibitory to its bactericidal activity in in vitro assays. The greatest similarity between DrsG and DRS/SIC is found in the signal sequence at the amino terminus and proline rich domains in the C-terminal half of the protein. Conservation of prolines in this latter region also suggests these residues are important in the biology of this family of proteins. This is the first report demonstrating the activity of an AMP inhibitory protein in SDSE. These results also suggest that inhibition of AMP activity is the primary function of this family of proteins. The acquisition of complement inhibitory activity of SIC may reflect its continuing evolution.

Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-(X)18-E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K 528R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions.

The Expanded Human Kallikrein (KLK) gene family : genomic organisation, tissue specific expression and potential functions

The tissue kallikreins are serine proteases encoded by highly conserved multigene families. The rodent kallikrein (KLK) families are particularly large, consisting of 13 26 genes clustered in one chromosomal locus. It has been recently recognised that the human KLK gene family is of a similar size (15 genes) with the identification of another 12 related genes (KLK4-KLK15) within and adjacent to the original human KLK locus (KLK1-3) on chromosome 19q13.4. The structural organisation and size of these new genes is similar to that of other KLK genes except for additional exons encoding 5 or 3 untranslated regions. Moreover, many of these genes have multiple mRNA transcripts, a trait not observed with rodent genes. Unlike all other kallikreins, the KLK4-KLK15 encoded proteases are less related (25–44%) and do not contain a conventional kallikrein loop. Clusters of genes exhibit high prostatic (KLK2-4, KLK15) or pancreatic (KLK6-13) expression, suggesting evolutionary conservation of elements conferring tissue specificity. These genes are also expressed, to varying degrees, in a wider range of tissues suggesting a functional involvement of these newer human kallikrein proteases in a diverse range of physiological processes.

Work/Family Balance: HRM Policy and Practice in Australia

An employee's inability to balance work and family responsibilities has resulted in an increase in stress related illnesses. Historically, research into the nexus between work and family has primarily focused on the work/family conflict relationship, predominately investigating the impact of this conflict on parents, usually mothers. To date research has not sufficiently examined the human resource management practices that enable all parents to achieve a balance between their work and family lives. This paper explores the relationship between contemporary family friendly HRM policies and employed parents perceptions of work/family enhancement, work/family satisfaction, propensity to turnover, and work/family conflict. Self-report questionnaire data from 326 men and women is analysed and discussed to enable organisations to consider the use of family friendly policies and thus create a convergence between the well-being of employees and the effectiveness of the organisation.