The intron-containing gene for yeast profilin (PFY) encodes a vital function

The gene coding for profilin (PFY), an actin-binding protein, occurs as a single copy in the haploid genome of Saccharomyces cerevisiae and is required for spore germination and cell viability. Displacement of one gene copy in a diploid cell by a nonfunctional allele is recessively lethal: tetrad analysis yields only two viable spores per ascus. The PFY gene maps on chromosome XV and is linked to the ADE2 marker. The primary transcript of about 1,000 bases contains an intron of 209 bases and is spliced into a messenger of about 750 bases. The intron was identified by comparison with a cDNA clone, which also revealed the 3' end of the transcript. The 5' end of the mRNA was mapped by primer elongation. The gene is transcribed constitutively and has a coding capacity for a protein of 126 amino acids. The deduced molecular weight o

RAB31 (Ras-related protein in brain 31)

Rab31 is a member of the large Rab protein family (66 human members) of the Ras superfamily of small GTPases. Rab31 is expressed fairly ubiquitously in normal human tissue and regulates membrane traffic between the Golgi/TGN and the plasma membrane and/or endosomes. Dysregulated expression of Rab31 has not only been observed in several types of cancer, including breast, ovarian, cervical and liver cancer as well as glioblastoma, but also in skin diseases such as psoriasis and atopic dermatitis

Overexpression of the urokinase receptor splice variant uPAR-del4/5 in breast cancer cells affects cell adhesion and invasion in a dose-dependent manner and modulates transcription of tumor-associated genes

mRNA levels of the urokinase receptor splice variant uPAR-del4/5 are associated with prognosis in breast cancer. Its overexpression in cancer cells affects tumor biologically relevant processes. In the present study, individual breast cancer cell clones displaying low vs. high uPAR-del4/5 expression were analyzed demonstrating that uPAR-del4/5 leads to reduced cell adhesion and invasion in a dose-dependent manner. Additionally, matrix metalloproteinase-9 (MMP-9) was found to be strongly upregulated in uPAR-del4/5 overexpressing compared to vector control cells. uPAR-del4/5 may thus play an important role in the regulation of the extracellular proteolytic network and, by this, influence the metastatic potential of breast cancer cells

Cyclo19,31[D-Cys19]-uPA19-31 is a potent competitive antagonist of the interaction of urokinase-type plasminogen activator with its receptor (CD87)

Urokinase-type plasminogen activator (uPA) represents a central molecule in pericellular proteolysis and is implicated in a variety of physiological and pathophysiological processes such as tissue remodelling, wound healing, tumor invasion, and metastasis. uPA binds with high affinity to a specific cell surface receptor, uPAR (CD87), via a well defined sequence within the N-terminal region of uPA (uPA(19-31)). This interaction directs the proteolytic activity of uPA to the cell surface which represents an important step in tumor cell proliferation, invasion, and metastasis. Due to its fundamental role in these processes, the uPA/uPAR-system has emerged as a novel target for tumor therapy. Previously, we have identified a synthetic, cyclic, uPA-derived peptide, cyclo(19,31)uPA(19-31), as a lead structure for the development of low molecular weight uPA-analogues, capable of blocking uPA/uPAR-interaction {[}Burgle et al., Biol. Chem. 378 (1997), 231-237]. We now searched for peptide variants of cyclo(19,31)uPA(19-31) with elevated affinities for uPAR binding. Among other tasks, we performed a systematic D-amino acid scan of quPA(19-31), in which each of the 13 L-amino acids was individually substituted by the corresponding D-amino acid. This led to the identification of cyclo(19,31) {[}D-Cys(19)]-uPA(19-31) as a potent inhibitor of uPA/uPAR-interaction, displaying only a 20 to 40-fold lower binding capacity as compared to the naturally occurring uPAR-ligands uPA and its amino-terminal fragment. Cyclo(19,31)[D-Cys(19)]-uPA(19-31) not only blocks binding of uPA to uPAR but is also capable of efficiently displacing uPAR-bound uPA from the cell surface and to inhibit uPA-mediated, tumor cell-associated plasminogen activation and fibrin degradation. Thus, cyclo(19,31)[D-Cys(19)]-uPA(19-31) represents a promising therapeutic agent to significantly affect the tumor-associated uPA/uPAR-system

Biological Chemistry / Structural basis for the Zn2+ inhibition of the zymogen-like kallikrein-related peptidase 10

Although kallikrein-related peptidase 10 (KLK10) is expressed in a variety of human tissues and body fluids, knowledge of its physiological functions is fragmentary. Similarly, the pathophysiology of KLK10 in cancer is not well understood. In some cancer types, a role as tumor suppressor has been suggested, while in others elevated expression is associated with poor patient prognosis. Active human KLK10 exhibits a unique, three residue longer N-terminus with respect to other serine proteases and an extended 99-loop nearly as long as in tissue kallikrein KLK1. Crystal structures of recombinant ligand-free KLK10 and a Zn2+ bound form explain to some extent the mixed trypsin- and chymotrypsin-like substrate specificity. Zn2+-inhibition of KLK10 appears to be based on a unique mechanism, which involves direct binding and blocking of the catalytic triad. Since the disordered N-terminus and several loops adopt a zymogen-like conformation, the active protease conformation is very likely induced by interaction with the substrate, in particular at the S1 subsite and at the unusual Ser193 as part of the oxyanion hole. The KLK10 structures indicate that the N-terminus, the nearby 75-, 148-, and the 99-loops are connected in an allosteric network, which is present in other trypsin-like serine proteases with several variations.(VLID)223339

Rab31 expression levels modulate tumor-relevant characteristics of breast cancer cells

BACKGROUND: Rab proteins constitute a large family of monomeric GTP-binding proteins that regulate intracellular vesicle transport. Several Rab proteins, including rab31, have been shown to affect cancer progression and are related with prognosis in various types of cancer including breast cancer. Recently, the gene encoding rab31 was found to be overexpressed in estrogen receptor-positive breast cancer tissue. In a previous study we found a significant association of high rab31 mRNA expression with poor prognosis in node-negative breast cancer patients. In the present study, we aimed to investigate the impact of rab31 (over)-expression on important aspects of tumor progression in vitro and in vivo. METHODS: Breast cancer cells displaying low (MDA-MB-231) or no (CAMA-1) endogenous rab31 expression were stably transfected with a rab31 expression plasmid. Batch-transfected cells as well as selected cell clones, expressing different levels of rab31 protein, were analyzed with regard to proliferation, cell adhesion, the invasive capacity of tumor cells, and in vivo in a xenograft tumor model. Polyclonal antibodies directed to recombinantly expressed rab31 were generated and protein expression analyzed by immunohistochemistry, Western blot analysis, and a newly developed sensitive ELISA. RESULTS: Elevated rab31 protein levels were associated with enhanced proliferation of breast cancer cells. Interestingly, weak to moderate overexpression of rab31 in cell lines with no detectable endogenous rab31 expression was already sufficient to elicit distinct effects on cell proliferation. By contrast, increased expression of rab31 in breast cancer cells led to reduced adhesion towards several extracellular matrix proteins and decreased invasive capacity through Matrigel(TM). Again, the rab31-mediated effects on cell adhesion and invasion were dose-dependent. Finally, in a xenograft mouse model, we observed a significantly impaired metastatic dissemination of rab31 overexpressing MDA-MB-231 breast cancer cells to the lung. CONCLUSIONS: Overexpression of rab31 in breast cancer cells leads to a switch from an invasive to a proliferative phenotype as indicated by an increased cell proliferation, reduced adhesion and invasion in vitro, and a reduced capacity to form lung metastases in vivo

Kallikrein-Related Peptidase 6 Is Associated with the Tumour Microenvironment of Pancreatic Ductal Adenocarcinoma

As cancer-associated factors, kallikrein-related peptidases (KLKs) are components of the tumour microenvironment, which represents a rich substrate repertoire, and considered attractive targets for the development of novel treatments. Standard-of-care therapy of pancreatic cancer shows unsatisfactory results, indicating the need for alternative therapeutic approaches. We aimed to investigate the expression of KLKs in pancreatic cancer and to inhibit the function of KLK6 in pancreatic cancer cells. KLK6, KLK7, KLK8, KLK10 and KLK11 were coexpressed and upregulated in tissues from pancreatic cancer patients compared to normal pancreas. Their high expression levels correlated with each other and were linked to shorter survival compared to low KLK levels. We then validated KLK6 mRNA and protein expression in patient-derived tissues and pancreatic cancer cells. Coexpression of KLK6 with KRT19, αSMA or CD68 was independent of tumour stage, while KLK6 was coexpressed with KRT19 and CD68 in the invasive tumour area. High KLK6 levels in tumour and CD68+ cells were linked to shorter survival. KLK6 inhibition reduced KLK6 mRNA expression, cell metabolic activity and KLK6 secretion and increased the secretion of other serine and aspartic lysosomal proteases. The association of high KLK levels and poor prognosis suggests that inhibiting KLKs may be a therapeutic strategy for precision medicine

A single glycan at the 99-loop of human kallikrein-related Peptidase 2 regulates activation and enzymatic activity

Human kallikrein-related peptidase 2 (KLK2) is a key serine protease in semen liquefaction and prostate cancer together with KLK3/prostate-specific antigen. In order to decipher the function of its potential N-glycosylation site, we produced pro-KLK2 in Leishmania tarentolae cells and compared it with its non-glycosylated counterpart from Escherichia coli expression. Mass spectrometry revealed that Asn-95 carries a core glycan, consisting of two GlcNAc and three hexoses. Autocatalytic activation was retarded in glyco-pro-KLK2, whereas the activated glyco-form exhibited an increased proteolytic resistance. The specificity patterns obtained by the PICS (proteomic identification of protease cleavage sites) method are similar for both KLK2 variants, with a major preference for P1-Arg. However, glycosylation changes the enzymatic activity of KLK2 in a drastically substrate-dependent manner. Although glyco-KLK2 has a considerably lower catalytic efficiency than glycan-free KLK2 toward peptidic substrates with P2-Phe, the situation was reverted toward protein substrates, such as glyco-pro-KLK2 itself. These findings can be rationalized by the glycan-carrying 99-loop that prefers to cover the active site like a lid. By contrast, the non-glycosylated 99-loop seems to favor a wide open conformation, which mostly increases the apparent affinity for the substrates (i.e. by a reduction of Km). Also, the cleavage pattern and kinetics in autolytic inactivation of both KLK2 variants can be explained by a shift of the target sites due to the presence of the glycan. These striking effects of glycosylation pave the way to a deeper understanding of kallikrein-related peptidase biology and pathology.(VLID)239213