Yves Courty

Purification and characterization of active recombinant rat kallikrein rK9

The rat tissue kallikrein rK9 is most abundant in the submandibular gland and the prostate. It has been successfully expressed in the Pichia pastoris yeast expression system. A full-length cDNA coding for the mature rK9 was fused in frame with yeast alpha-factor cDNA. The fusion protein was secreted into the medium with high yield without being processed by the yeast KEX2 signal peptidase. Mature rK9 was efficiently released from the fusion protein by trypsin and was purified to homogeneity by one-step affinity chromatography using soya bean trypsin inhibitor (SBTI) as affinity ligand. The identity of the recombinant enzyme was checked by N-terminal amino acid sequencing, Western blot analysis and kinetic studies. The dual trypsin- and chymotrypsin-like enzymatic specificity of rK9 was assessed by determining specificity constants (k(cat)/K(m)) for the hydrolysis of fluorogenic substrates, the peptide sequences of which were derived from proparathyroid hormone (pro-PTH) and from semenogelin-I. Our results confirmed the presence of an extended binding site in the rK9 active site. We also identified a far more sensitive substrate of this enzyme than those previously described, Abz-VKKRSARQ-EDDnp, which was hydrolysed with a catalytic efficiency k(cat)/K(m) of 420000 M(-1)s(-1). Finally, we showed that four of the five major proteins contained in secretions of rat seminal vesicles were rapidly degraded by recombinant rK9.

Interleukin-22 receptor is overexpressed in nonsmall cell lung cancer and portends a poor prognosis

The recent decade has witnessed a change of view about cancer mechanisms. Considerable evidence now suggests that the tumour microenvironment status is well correlated with disease outcome. The presence of particular immune cell types or molecules determines whether a pro- or antitumour immune response predominates within the microenvironment [1]. Accordingly, improved understanding of the factors that modulate the tumour microenvironment will be critical for the development of effective future strategies for cancer management. High expression of IL-22R1 in nonsmall cell lung cancer is an independent indicator of poor overall survival http://ow.ly/WZxKh

Altered expression of the CCN genes in the lungs of mice in response to cigarette smoke exposure and viral and bacterial infections.

The CCN proteins are key signaling and regulatory molecules involved in many biological functions and contribute to malignant and non-malignant lung diseases. Despite the high morbidity and mortality of the lung respiratory infectious diseases, there is very little data related to the expression of the CCNs during infection. We investigated in mice the pulmonary mRNA expression levels of five CCNs (1 to 5) in response to influenza A virus (IAV) and bacterial agents (Nontypeable Haemophilus influenzae (NTHi), lipopolysaccharide (LPS) and lipoteichoic acid (LTA)). IAV, NTHi, LPS or LTA were instilled intranasally into mice. Mice were also exposed for 4days or 8weeks to cigarette smoke alone or prior infection to IAV in order to determine if CS modifies the CCN response to a viral infection. All challenges induced a robust inflammation. The mRNA expression of CCN1, CCN2 and CCN3 was decreased after short exposure to CS whereas prolonged exposure altered the expression of CCN1, CCN3 and CCN4. Influenza A virus infection increased CCN1, 2, 4 and 5 mRNA levels but expression of CCN3 was significantly decreased. Acute CS exposure prior infection had little effect on the expression of CCN genes but prolonged exposure abolished the IAV-dependent induction. Treatment with LPS or LTA and infection with NTHi revealed that both Gram-positive and Gram-negative bacteria rapidly modulate the expression of the CCN genes. Our findings reveal that several triggers of lung inflammation influence differently the CCN genes. CCN3 deserves special attention since its mRNA expression is decreased by all the triggers studied.

Kallikrein-Related Peptidase 6 Overexpression Promotes Non-Small Cell Lung Cancer Cell Proliferation and is associated with poor patient outcome

Introduction The human kallikrein-related peptidases (KLK) are a family of serine proteases that are often aberrantly expressed in common human malignancies and contribute to neoplastic progression through multifaceted roles. Methods We evaluated KLK6 expression in the tumoral and normal adjacent lung tissue of 56 patients with Non-Small Cell Lung Cancer (NSCLC) by real-time RT-PCR and immunohistochemistry. To determine the impact of KLK6 overexpression on the growth of lung cancer cells, we integrated the cDNA encoding the complete sequence of KLK6, through homolog recombination, in a NSCLC line (A549 Flp-In) and determined the growth rate of two independent clones. Progression of the KLK6- and parental cells inside the cell cycle was assessed by flow cytometry following synchronization of cells at the end of the G1 phase with starvation and hydroxyurea treatments. Key regulator proteins of the cell cycle were analyzed by Western blot in synchronized and unsynchronized cells. Results We found KLK6 transcript up-regulation in tumor tissues from patients with NSCLC and association of KLK6 status with low patient survival. KLK6 immunoreactivity was restricted to epithelial cells of normal bronchi and detected in most of cancer samples, in which KLK6 signal intensity correlated with well differentiated tumors. Ectopic expression of KLK6 dramatically enhanced NSCLC cell growth. Analysis of cell cycle progression revealed that promotion of cell growth caused by KLK6 results from an acceleration of cell cycle progression through G1/S transition, which was accompanied with a marked increase of cyclin E and repression of p21. In addition, expression of KLK6 in NSCLC cells was associated with an increase of c-Myc that is well-know to promote cell-cycle progression via regulation of cyclin D/E activation and down-regulation of p21. Conclusion ectopic expression of KLK6 facilitates cell cycle progression, certainly through alteration of c-Myc and downstream key regulators, and thus promotes cell proliferation. Moreover, KLK6 is overexpressed in NSCLC and associated with poor prognosis. Altogether, those findings suggest that KLK6 might play a central role in NSCLC development and progression.