A New Inhibitor of the Chymotrypsin-Like Activity of the Multicatalytic Proteinase Complex (20S Proteasome) Induces Accumulation of Ubiquitin-Protein Conjugates in a Neuronal Cell

Abstract: Exposure of HT4 cells (a mouse neuronal cell line) to a new potent permeable peptidyl aldehyde inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (MPC) causes accumulation of ubiquitinylated proteins. In contrast, inhibition of calpain or treatment with a lysosomotropic agent failed to produce detectable ubiquitin-protein conjugates. The appearance of such conjugates is not a nonspecific phenomenon because incubation with the peptidyl alcohol analogue of the inhibitor does not produce accumulation of ubiquitinylated proteins. The MPC inhibitor may therefore be a useful tool for identification and study of physiological pathways involving MPC. Furthermore, the inhibitor may help develop a model for the study of neurodegeneration where accumulation of ubiquitin-protein conjugates is commonly detected in abnormal brain inclusions.     

The Potent Cdc7-Dbf4 (DDK) Kinase Inhibitor XL413 Has Limited Activity in Many Cancer Cell Lines and Discovery of Potential New DDK Inhibitor Scaffolds

Cdc7-Dbf4 kinase or DDK (Dbf4-dependent kinase) is required to initiate DNA replication by phosphorylating and activating the replicative Mcm2-7 DNA helicase. DDK is overexpressed in many tumor cells and is an emerging chemotherapeutic target since DDK inhibition causes apoptosis of diverse cancer cell types but not of normal cells. PHA-767491 and XL413 are among a number of potent DDK inhibitors with low nanomolar IC₅₀ values against the purified kinase. Although XL413 is highly selective for DDK, its activity has not been extensively characterized on cell lines. We measured anti-proliferative and apoptotic effects of XL413 on a panel of tumor cell lines compared to PHA-767491, whose activity is well characterized. Both compounds were effective biochemical DDK inhibitors but surprisingly, their activities in cell lines were highly divergent. Unlike PHA-767491, XL413 had significant anti-proliferative activity against only one of the ten cell lines tested. Since XL413 did not effectively inhibit DDK in multiple cell lines, this compound likely has limited bioavailability. To identify potential leads for additional DDK inhibitors, we also tested the cross-reactivity of ∼400 known kinase inhibitors against DDK using a DDK thermal stability shift assay (TSA). We identified 11 compounds that significantly stabilized DDK. Several inhibited DDK with comparable potency to PHA-767491, including Chk1 and PKR kinase inhibitors, but had divergent chemical scaffolds from known DDK inhibitors. Taken together, these data show that several well-known kinase inhibitors cross-react with DDK and also highlight the opportunity to design additional specific, biologically active DDK inhibitors for use as chemotherapeutic agents.     

组织型纤溶酶原激活剂工程细胞系遗传特性及成瘤性分析

构建了突变体t-PA,使其在CHO-dhfr细胞中表达。获得t-PA工程细胞系。对该细胞系进行了遗传特性分析,在培养基中加入秋水仙素,通过低张、固定处理并将细胞滴片、染色,进行染色体分析,结果表明,该工程细胞系染色体条数为20条,畸变类型有异着丝粒、四倍体、裂隙、断片、微小体,畸变率为15％,属于正常范围。同时进行成瘤性检测,选用4周龄裸鼠作为试验鼠,以人宫颈癌细胞(Hela细胞)为阳性对照,CHO-dhfr细胞为阴性对照,试验表明,t-PA工程细胞及表达产物对裸鼠均无成瘤性。     

The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity

The membrane-anchored serine protease prostasin (CAP1/PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. Prostasin null (Prss8^−/−) mice lack barrier formation and display fatal postnatal dehydration. In sharp contrast, mice homozygous for a point mutation in the Prss8 gene, which causes the substitution of the active site serine within the catalytic histidine-aspartate-serine triad with alanine and renders prostasin catalytically inactive (Prss8^{Cat−/Cat−} mice), develop barrier function and are healthy when followed for up to 20 weeks. This striking difference could not be explained by genetic modifiers or by maternal effects, as these divergent phenotypes were displayed by Prss8^−/− and Prss8^{Cat−/Cat−} mice born within the same litter. Furthermore, Prss8^{Cat−/Cat−} mice were able to regenerate epidermal covering following cutaneous wounding. This study provides the first demonstration that essential in vivo functions of prostasin are executed by a non-enzymatic activity of this unique membrane-anchored serine protease.     

The Chromone Alkaloid,Rohitukine, Affords Anti-Cancer Activity via Modulating Apoptosis Pathways in A549 Cell Line and Yeast Mitogen Activated Protein Kinase (MAPK) Pathway

The field of cancer research and treatment has made significant progress, yet we are far from having completely safe, efficient and specific therapies that target cancer cells and spare the healthy tissues. Natural compounds may reduce the problems related to cancer treatment. Currently, many plant products are being used to treat cancer. In this study, Rohitukine, a natural occurring chromone alkaloid extracted from Dysoxylum binectariferum, was investigated for cytotoxic properties against budding yeast as well as against lung cancer (A549) cells. We endeavored to specifically study Rohitukine in S. cerevisiae in the context of MAPK pathways as yeast probably represents the experimental model where the organization and regulation of MAPK pathways are best understood. MAPK are evolutionarily conserved protein kinases that transfer extracellular signals to the machinery controlling essential cellular processes like growth, migration, differentiation, cell division and apoptosis. We aimed at carrying out hypothesis driven studies towards targeting the important network of cellular communication, a critical process that gets awry in cancer. Employing mutant strains of genetic model system Saccharomyces cerevisiae. S. cerevisiae encodes five MAPKs involved in control of distinct cellular responses such as growth, differentiation, migration and apoptosis. Our study involves gene knockouts of Slt2 and Hog1 which are functional homologs of human ERK5 and mammalian p38 MAPK, respectively. We performed cytotoxicity assay to evaluate the effect of Rohitukine on cell viability and also determined the effects of drug on generation of reactive oxygen species, induction of apoptosis and expression of Slt2 and Hog1 gene at mRNA level in the presence of drug. The results of this study show a differential effect in the activity of drug between the WT, Slt2 and Hog1 gene deletion strain indicating involvement of MAPK pathway. Further, we investigated Rohitukine induced cytotoxic effects in lung cancer cells and stimulated the productions of ROS after exposure for 24 hrs. Results from western blotting suggest that Rohitukine triggered apoptosis in A549 cell line through upregulation of p53, caspase9 and down regulation of Bcl-2 protein. The scope of this study is to understand the mechanism of anticancer activity of Rohitukine to increase the repertoire of anticancer drugs, so that problem created by emergence of resistance towards standard anticancer compounds can be alleviated.     

Protein Kinase D�Cmediated Phosphorylation of Polycystin-2 (TRPP2) Is Essential for Its Effects on Cell Growth and Calcium Channel Activity

PKD2 is mutated in 15% of patients with autosomal dominant polycystic kidney disease. The PKD2 protein, polycystin-2 or TRPP2, is a nonselective Ca²⁺-permeable cation channel that has been shown to function at several locations, including primary cilia, basolateral membrane, and at the endoplasmic reticulum (ER). Nevertheless, the factors that regulate the channel activity of polycystin-2 are not well understood. Polycystin-2 has been shown previously to be regulated by phosphorylation at two serine residues (Ser⁸¹² and Ser⁷⁶) with distinct functional consequences. Here, we report the identification of a previously unrecognized phosphorylation site within the polycystin-2 C terminus (Ser⁸⁰¹), and we demonstrate that it is phosphorylated by protein kinase D. Phosphorylation at this site was significantly increased in response to serum and epidermal growth factor stimulation. In nonciliated Madin-Darby canine kidney I cells, inducible expression of polycystin-2 inhibited cell proliferation compared with wild-type cells. Mutagenesis at Ser⁸⁰¹ abolished these effects and reduced ATP-stimulated Ca²⁺ release from ER stores. Finally, we show that a pathogenic mutation (S804N) within the consensus kinase recognition sequence abolished Ser⁸⁰¹ phosphorylation. These results suggest that growth factor-stimulated, protein kinase D-mediated phosphorylation of polycystin-2 is essential for its ER channel function and links extracellular stimuli to its effects on cell growth and intracellular calcium regulation.     

Differential Ratios of Omega Fatty Acids (AA/EPA+DHA) Modulate Growth,Lipid Peroxidation and Expression of Tumor Regulatory MARBPs in Breast Cancer Cell Lines MCF7 and MDA-MB-231

Omega 3 (n3) and Omega 6 (n6) polyunsaturated fatty acids (PUFAs) have been reported to exhibit opposing roles in cancer progression. Our objective was to determine whether different ratios of n6/n3 (AA/EPA+DHA) FAs could modulate the cell viability, lipid peroxidation, total cellular fatty acid composition and expression of tumor regulatory Matrix Attachment Region binding proteins (MARBPs) in breast cancer cell lines and in non-cancerous, MCF10A cells. Low ratios of n6/n3 (1:2.5, 1:4, 1:5, 1:10) FA decreased the viability and growth of MDA-MB-231 and MCF7 significantly compared to the non-cancerous cells (MCF10A). Contrarily, higher n6/n3 FA (2.5:1, 4:1, 5:1, 10:1) decreased the survival of both the cancerous and non-cancerous cell types. Lower ratios of n6/n3 selectively induced LPO in the breast cancer cells whereas the higher ratios induced in both cancerous and non-cancerous cell types. Interestingly, compared to higher n6/n3 FA ratios, lower ratios increased the expression of tumor suppressor MARBP, SMAR1 and decreased the expression of tumor activator Cux/CDP in both breast cancer and non-cancerous, MCF10A cells. Low n6/n3 FAs significantly increased SMAR1 expression which resulted into activation of p21^WAF1/CIP1 in MDA-MB-231 and MCF7, the increase being ratio dependent in MDA-MB-231. These results suggest that increased intake of n3 fatty acids in our diet could help both in the prevention as well as management of breast cancer.     

The Use of Two Bifidobacterium Strains Enhanced Growth Performance and Nutrient Utilization of Rainbow Trout (Oncorhynchus mykiss) Fry

Probiotics and Antimicrobial Proteins - This study aims to evaluate the effects of Bifidobacterium animalis PTCC-1631 and Bifidobacterium lactis PTCC-1736 on growth, nutrient digestibility, carcass...     

The Mechanisms of Anticancer Activity of Nisin Peptide on Myelogenous Leukemia Cell Line (K562) As a New Treatment: Inducing Apoptosis by Changing in the Expression of Bax and Bcl-2 Genes

International Journal of Peptide Research and Therapeutics - Lactococcus lactis is a gram positive bacteria that produces nisin, a polycyclic peptide, during fermentation process. In recent...     

The Effects of Copper (II) Ions on <Emphasis Type="Italic">Enterococcus hirae</Emphasis> Cell Growth and the Proton-Translocating F<Subscript>o</Subscript>F<Subscript>1</Subscript> ATPase Activity

Enterococcus hirae grow well under anaerobic conditions at alkaline pH (pH 8.0) producing acids by glucose fermentation. Bacterial growth was shown to be accompanied by decrease of redox potential from positive values (~+35 mV) to negative ones (~−220 mV). An oxidizer copper (II) ions (Cu²⁺) affected bacterial growth in a concentration-dependent manner (within the range of 0.05 mM to 1 mM) increasing lag phase duration and decreasing specific growth rate. These effects were observed with the wild-type strain ATCC9790 and the atpD mutant strain MS116 (with absent β subunit of F₁ of the F_oF₁ ATPase) both. Also ATPase activity and proton–potassium ions exchange were assessed with and without N,N′-dicyclohexylcarbodiimide (DCCD), inhibitor of the F_oF₁ ATPase. In both cases (DCCD ±), even low Cu²⁺ concentrations had noticeable effect on ATPase activity, but with less visible concentration-dependent manner. Changes in the number of accessible SH-groups were observed with E. hirae ATCC9790 and MS116 membrane vesicles. In both strains Cu²⁺ markedly decreased the number of SH-groups in the presence of K⁺ ions. The addition of ATP increased the amount of accessible SH-groups in ATCC9790 and decreased this number in MS116; Cu²⁺ blocked ATP-installed increase in SH-groups number in ATCC9790. H⁺–K⁺-exchange of bacteria was markedly inhibited by Cu²⁺, but stronger effects were detected together with DCCD. Moreover, discrimination between Cu²⁺ and other bivalent cation—Ni²⁺ was shown. It is suggested that Cu²⁺ ions inhibit E. hirae cell growth by direct affect on the F_oF₁ ATPase leading to conformational changes in this protein complex and decrease in its activity.