Volume 387, Issue 12

Endothelial NO synthase (eNOS) is the predominant enzyme responsible for vascular NO synthesis. A functional eNOS transfers electrons from NADPH to its heme center, where L-arginine is oxidized to L-citrulline and NO. Common conditions predisposing to atherosclerosis, such as hypertension, hypercholesterolemia, diabetes mellitus and smoking, are associated with enhanced production of reactive oxygen species (ROS) and reduced amounts of bioactive NO in the vessel wall. NADPH oxidases represent major sources of ROS in cardiovascular pathophysiology. NADPH oxidase-derived superoxide avidly interacts with eNOS-derived NO to form peroxynitrite (ONOO – ), which oxidizes the essential NOS cofactor (6R-)5,6,7,8-tetrahydrobiopterin (BH 4 ). As a consequence, oxygen reduction uncouples from NO synthesis, thereby rendering NOS to a superoxide-producing pro-atherosclerotic enzyme. Supplementation with BH 4 corrects eNOS dysfunction in several animal models and in patients. Administration of high local doses of the antioxidant L-ascorbic acid (vitamin C) improves endothelial function, whereas large-scale clinical trials do not support a strong role for oral vitamin C and/or E in reducing cardiovascular disease. Statins, angiotensin-converting enzyme inhibitors and AT1 receptor blockers have the potential of reducing vascular oxidative stress. Finally, novel approaches are being tested to block pathways leading to oxidative stress (e.g. protein kinase C) or to upregulate antioxidant enzymes.

Leucine aminopeptidases (LAPs) are metallopeptidases that cleave N-terminal residues from proteins and peptides. While hydrolyzing Leu substrates, LAPs often have a broader specificity. LAPs are members of the M1 or M17 peptidase families, and therefore the LAP nomenclature is complex. LAPs are often viewed as cell maintenance enzymes with critical roles in turnover of peptides. In mammals, the M17 and M1 enzymes with LAP activity contribute to processing peptides for MHC I antigen presentation, processing of bioactive peptides (oxytocin, vasopressin, enkephalins), and vesicle trafficking to the plasma membrane. In microbes, the M17 LAPs have a role in proteolysis and have also acquired the ability to bind DNA. This property enables LAPs to serve as transcriptional repressors to control pyrimidine, alginate and cholera toxin biosynthesis, as well as mediate site-specific recombination events in plasmids and phages. In plants the roles of the M17 LAPs and the peptidases related to M1 LAPs are being elucidated. Roles in defense, membrane transport of auxin receptors, and meiosis have been implicated.

There have been new developments in the elucidation of the biological functions of the inter-α-inhibitor (IαI) family. The anti-proteolytic activity of the IαI family originates from bikunin (also known as urinary trypsin inhibitor). Growing evidence indicates that bikunin is not just an anti-proteolytic agent, but can also be considered an anti-inflammatory agent that suppresses lipopolysaccharide (LPS)-induced cytokine synthesis. Bikunin functions to inhibit calcium influx and extracellular signal-regulated kinase (ERK) signaling via LPS receptors and/or as yet unidentified bikunin signaling receptors. By signaling via the LPS receptor, LPS increases calcium influx and yields phosphorylated ERK, which activates multiple transcription factors, such as nuclear factor κB (NF-κB) or early growth response-1 (Egr-1), which in turn promote cytokine expression. Deficits in the signaling cascades caused by free or cell-bound bikunin are predicted to down-regulate cytokine expression, render macrophages/neutrophils more inactive, and impair inflammatory processes. This brief review largely focuses on our current understanding of the apparent functions of bikunin, its ligands, the effector molecules with which it interacts, and its regulation.

It is now appreciated that mitochondria form tubular networks that adapt to the requirements of the cell by undergoing changes in their shape through fission and fusion. Proper mitochondrial distribution also appears to be required for ATP delivery and calcium regulation, and, in some cases, for cell development. While we now realise the great importance of mitochondria for the cell, we are only beginning to work out how these organelles undergo the drastic morphological changes that are essential for cellular function. Of the few known components involved in shaping mitochondria, some have been found to be essential to life and their gene mutations are linked to neurological disorders, while others appear to be recruited in the activation of cell death pathways. Here we review our current understanding of the functions of the main players involved in mitochondrial fission, fusion and distribution in mammalian cells.

Clitocypin from the basidiomycete Clitocybe nebularis is the first fungal protein cysteine protease inhibitor to be characterised in detail, yet no information on its molecular genetics is available. Owing to its unique characteristics, it was assigned as the only member of a new family of cysteine protease inhibitors in the MEROPS inhibitor classification. Here we describe the full-length sequence of the clitocypin gene. A BLAST search confirmed its lack of significant sequence similarity to any other gene. The gene is composed of four exons and three short introns and belongs to a small family of closely related genes with more than 90% identity. Sequence variability is evenly distributed in introns and exons and deduced amino acid substitutions are distributed throughout the protein sequence. Basidiocarps collected at two distant locations were examined and the level of heterogeneity found in one basidiocarp is similar to that between the two. Sequencing of the ribosomal DNA spacers from the two basidiocarps confirmed that the heterogeneity observed in the clitocypin gene is not due to evolutionary divergence of the two specimens caused by geographic separation. Clitocypin is expressed in different parts of the basidiocarp and in cultured mycelia in a manner suggesting regulation by developmental and/or environmental factors.

Class IA phosphoinositide 3-kinases (PI3Ks) represent a group of heterodimeric lipid kinases with important functions in cellular signal transduction. The regulatory p85 subunit constitutively binds to the catalytic p110 subunit and mediates the recruitment of the heterodimer to various membrane-localized proteins upon activation by a vast array of stimuli. The functional characterization of protein domains that mediate p85 function has been hampered by a lack of structural data. Therefore, we investigated a 35-aa region in the inter-SH2 domain of p85, reported to be necessary for binding of p110, by site-directed mutagenesis and evaluated the importance of individual amino acids for PI3K heterodimer formation. This approach led to the identification of an 11-aa region required for p110 binding in vitro and mesoderm induction during early Xenopus development in vivo . Further analyses revealed two pairs of hydrophobic amino acids within this region, which are particularly important for high-affinity intersubunit interaction. Thus, our data provide further insight into the molecular mechanisms of PI3K intersubunit interaction and led to the identification of new p85 mutant proteins with varying degrees of dominant-negative effects that will be helpful for future PI3K-related research.

Bacillus anthracis , which causes anthrax, has attracted attention because of its potential use as a biological weapon. The risk of multidrug resistance against B. anthracis increases the need for antibiotics with new molecular targets. Nucleoside analogs are well-known antiviral and anticancer prodrugs, and thymidine kinase catalyzes the rate-limiting step in the activation of pyrimidine nucleoside analogs used in chemotherapy. The thymidine kinase gene from B. anthracis Sterne strain (34F2) ( Ba -TK) was cloned and expressed in E. coli , and the product was purified and characterized regarding its substrate specificity. Ba -TK phosphorylated pyrimidine nucleosides and all natural nucleoside triphosphates served as phosphate donors. Size exclusion chromatography indicated a dimeric form of Ba -TK, regardless of the presence of ATP. Thymidine was the most efficient substrate with a low K m value (0.6 μM) and a V max of 3.3 μmol dTMP mg -1 min -1 , but deoxyuridine ( K m =4.2 μM, V max =4.1 μmol dUMP mg -1 min -1 ) was also a good substrate. Several pyrimidine analogs were also tested and analogs with 5-position modifications showed higher activities compared to analogs with 3′- and N3-position modifications. Deoxyuridine analogs were the most potent inhibitors of B. anthracis growth in vitro . These results may be used to guide future development of nucleoside analogs against B. anthracis .

Vitamin B 1 is an essential cofactor for key enzymes such as 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase. Plants, bacteria and fungi, as well as Plasmodium falciparum , are capable of synthesising vitamin B 1 de novo , whereas mammals have to take up this cofactor from their diet. Thiamine, a B 1 vitamer, has to be pyrophosphorylated by thiamine pyrophosphokinase (TPK) to the active form. The human malaria parasite P. falciparum expresses an N-terminally extended pyrophosphokinase throughout the entire erythrocytic life cycle, which was analysed by Northern and Western blotting. The recombinant enzyme shows a specific activity of 27 nmol min -1 mg -1 protein and specificity for thiamine with a K m value of 73 μM, while thiamine monophosphate is not accepted. Mutational analysis of the N-terminal extension of the plasmodial TPK showed that it influences thiamine binding as well as metal dependence, which suggests N-terminal participation in the conformation of the active site. Protein sequences of various plasmodial TPKs were analysed for their phylogeny, which classified the Plasmodium TPKs to a group distinct from the mammalian TPKs. To verify the location of the parasite TPK within the cell, immunofluorescence analyses were performed. Co-staining of Pf TPK with a GFP marker visualised its cytosolic localisation.

The HspBP1 homolog Fes1p was recently identified as a nucleotide exchange factor (NEF) of Ssa1p, a canonical Hsp70 molecular chaperone in the cytosol of Saccharomyces cerevisiae . Besides the Ssa-type Hsp70s, the yeast cytosol contains three additional classes of Hsp70, termed Ssb, Sse and Ssz. Here, we show that Fes1p also functions as NEF for the ribosome-bound Ssb Hsp70s. Sequence analysis indicated that residues important for interaction with Fes1p are highly conserved in Ssa1p and Ssb1p, but not in Sse1p and Ssz1p. Indeed, Fes1p interacts with Ssa1p and Ssb1p with similar affinity, but does not form a complex with Sse1p. Functional analysis showed that Fes1p accelerates the release of the nucleotide analog MABA-ADP from Ssb1p by a factor of 35. In contrast to the interaction between mammalian HspBP1 and Hsp70, however, addition of ATP only moderately decreases the affinity of Fes1p for Ssb1p. Point mutations in Fes1p abolishing complex formation with Ssa1p also prevent the interaction with Ssb1p. The ATPase activity of Ssb1p is stimulated by the ribosome-associated complex of Zuotin and Ssz1p (RAC). Interestingly, Fes1p inhibits the stimulation of Ssb1p ATPase by RAC, suggesting a complex regulatory role of Fes1p in modulating the function of Ssb Hsp70s in co-translational protein folding.

MIA (melanoma inhibitory activity) has been identified as a small protein secreted by malignant melanoma cells that interacts with extracellular matrix proteins including fibronectin. These findings suggest that MIA may play a role in tumor progression and the spread of malignant melanomas by mediating detachment of cells from extracellular matrix molecules. Here, we present a detailed study on functionally important MIA domains. Using site-directed mutagenesis, amino acids important for MIA structure and/or function were determined. Amino acids conserved in SH3 domains were shown to be important for structural integrity. In addition, amino acid residues necessary for MIA function were identified. Interestingly, not all of them are conserved with respect to other members of the MIA protein family. In summary, our results lead to a better understanding of MIA function. Regulating MIA functions in vivo may provide a novel therapeutic strategy for metastatic melanoma disease.

Recent evidence suggests that many tissue kallikreins are implicated in carcinogenesis. Kallikrein 8 ( KLK8 ) plays a role in the physiology of the central nervous system. Kallikrein 7 ( KLK7 ) takes part in skin desquamation. Both show altered expression in ovarian and breast cancer. In this study, we examined the level of mRNA expression of the KLK7 and KLK8 genes in 73 intracranial tumors using qualitative RT-PCR. The results were correlated with clinical and histomorphological variables and patient outcome. The expression of both genes was also examined in the brain cancer cell lines U-251 MG, D54 and SH-SY5Y and the invasive capacity of glioblastoma cells U-251 MG overexpressing hK7 or hK8 was also investigated in an in vitro Matrigel assay. Follow-up analysis revealed that expression of KLK7 mRNA was associated with shorter overall survival (OS) compared to patients with no KLK7 expression, as determined by Cox proportional hazard regression analysis. Overexpression of hK7 protein by cultivated brain tumor cells significantly enhanced the invasive potential in the Matrigel invasion assay, in contrast to cells overexpressing hK8 protein. Our data suggest that hK7 protein overexpression is associated with a more aggressive phenotype in brain cancer cells.

SR-A1 is a human high-molecular-weight SR-related CTD-associated factor that links the machineries of transcription and mRNA splicing. In this study we examined the prognostic value of SR-A1 gene expression using a semi-quantitative RT-PCR method. High SR-A1 expression was observed in 31/81 (38.3%) breast cancer tissues and was found to be more frequent in patients with tumors of large size ( p =0.027), as well as in lymph node-positive patients ( p =0.035). Follow-up analysis revealed that low SR-A1 expression increases the probability of both overall and disease-free survival of patients. Our results suggest that SR-A1 may possibly be characterized as a new marker of unfavorable prognosis for breast cancer.

Endotoxin-induced cytokine production is an important mechanism in the development of several types of liver damage. Methionine, some of its precursors and metabolites were reported to have protective effects against such injury. The aim of this study was to investigate whether methionine, its precursors or metabolites [phosphatidylcholine, choline, betaine, S -adenosylmethionine (SAM)] have a modulating effect on tumor necrosis factor α (TNF-α) production by endotoxin-stimulated human mononuclear leukocytes and whether SAM-dependent polyamines (spermidine, spermine) are mediators of SAM-induced inhibition of TNF-α synthesis. Methionine and betaine had a moderate stimulatory effect on TNF-α production, whereas phosphatidylcholine (ID 50 5.4 mM), SAM (ID 50 131 μM), spermidine (ID 50 4.5 μM) and spermine (ID 50 3.9 μM) had a predominantly inhibitory effect. Putrescine did not alter TNF-α release. Inhibitors of polyamine synthesis that blocked either putrescine (difluoromethylornithine) or spermine (CGP48664A) production did not affect TNF-α synthesis. Endotoxin stimulation of leukocytes did not alter the intracellular levels of polyamines. In addition, supplementation with SAM did not change the intracellular concentration of either polyamine measured. We conclude that phosphatidylcholine-induced immunosuppression is not caused by methionine and polyamines are not involved in SAM-induced inhibition of TNF-α production. The limitation of TNF-α release by spermidine is specific and is not due to its conversion into spermine.

The BCL2 family of genes (B-cell CLL/lymphoma 2; Bcl-2) plays a pivotal role in the highly regulated process of apoptosis. We have recently cloned a newly identified member of this family, BCL2L12 , which was found to be differentially expressed in many tumors. It is known that topotecan and methotrexate act through induction of apoptosis in cancer cells. In the present study we investigated the expression profile of the novel apoptotic gene BCL2L12 in relation to other apoptotic genes in the human leukemic cell line HL-60, after treatment with topotecan or methotrexate. The kinetics of apoptosis induction and cell toxicity were investigated by DNA laddering and the MTT method, respectively. Gene expression levels were analyzed by RT-PCR using gene-specific primers. Downregulation of BCL2L12 , BCL2 and FAS was observed after treatment of HL-60 cells with topotecan, while treatment with methotrexate led to downregulation of BCL2 and FAS , with no change in BCL2L12 expression. Our results support the significance of mRNA modulations in the expression of apoptosis-related genes during treatment of human leukemic cells with anticancer drugs.

Two genes coding for cysteine peptidase inhibitors of the cystatin family (Om-cystatin 1 and 2) were isolated from a gut-specific cDNA library of the soft tick Ornithodoros moubata . Both cystatins were clearly down-regulated after a blood meal. Om-cystatin 1 is mainly expressed in the tick gut, while Om-cystatin 2 mRNA was also found in other tick tissues. Authentic Om-cystatin 2 was significantly more abundant than Om-cystatin 1 in the gut contents of fasting ticks and was associated with hemosome-derived residual bodies accumulated in the gut lumen. Om-cystatin 2 was also expressed by type 2 secretory cells in the salivary glands of unfed ticks. The inhibitory specificity of recombinant Om-cystatins 1 and 2 was tested with mammalian cysteine peptidases, as well as endogenous cysteine peptidases present in the tick gut. Both cystatins efficiently inhibited papain-like peptidases, including cathepsin B and H, but differed significantly in their affinity towards cathepsin C and failed to block asparaginyl endopeptidase. Our results suggest that the secreted cystatin isoinhibitors are involved in the regulation of multiple proteolytic targets in the tick digestive system and tick-host interaction.