Crystal structure of human DJ-1, a protein associated with early onset Parkinson's disease

We report the crystal structure at 1.8-A resolution of human DJ-1, which has been linked to early onset Parkinson's disease. The monomer of DJ-1 contains the alpha/beta-fold that is conserved among members of the DJ-1/ThiJ/PfpI superfamily. However, the structure also contains an extra helix at the C terminus, which mediates a novel mode of dimerization for the DJ-1 proteins. A putative active site has been identified near the dimer interface, and the residues Cys-106, His-126, and Glu-18 may play important roles in the catalysis by this protein. Studies with the disease-causing L166P mutant suggest that the mutation has disrupted the C-terminal region and the dimerization of the protein. The DJ-1 proteins may function only as dimers. The Lys to Arg mutation at residue 130, the site of sumoylation of DJ-1, has minimal impact on the structure of the protein.     

Vitamin C transport systems of mammalian cells

Vitamin C is essential for many enzymatic reactions and also acts as a free radical scavenger. Specific non-overlapping transport proteins mediate the transport of the oxidized form of vitamin C, dehydroascorbic acid, and the reduced form, L-ascorbic acid, across biological membranes. Dehydroascorbic acid uptake is via the facilitated-diffusion glucose transporters, GLUT 1, 3 and 4, but under physiological conditions these transporters are unlikely to play a major role in the uptake of vitamin C due to the high concentrations of glucose that will effectively block influx. L-ascorbic acid enters cells via Na+-dependent systems, and two isoforms of these transporters (SVCT1 and SVCT2) have recently been cloned from humans and rats. Transport by both isoforms is stereospecific, with a pH optimum of approximately 7.5 and a Na+:ascorbic acid stoichiometry of 2:1. SVCT2 may exhibit a higher affinity for ascorbic acid than SVCT1 but with a lower maximum velocity. SVCT1 and SVCT2 are predicted to have 12 transmembrane domains, but they share no structural homology with other Na+ co-transporters. Potential sites for phosphorylation by protein kinase C exist on the cytoplasmic surface of both proteins, with an additional protein kinase A site in SVCT1. The two isoforms also differ in their tissue distribution: SVCT1 is present in epithelial tissues, whereas SVCT2 is present in most tissues with the exception of lung and skeletal muscle.     

Programmed translational frameshifting is likely required for expressions of genes encoding putative nuclear protein kinases of the ciliate Euplotes octocarinatus

Three macronuclear genes encoding putative nuclear protein kinases of the ciliate Euplotes octocarinatus syngen 1 were isolated and sequenced. All three deduced gene products share significant properties with a group of recently identified nuclear serine/threonine protein kinases named Ndr. The three predicted proteins contain the twelve conserved catalytic subdomains of protein kinases and 22 near universally-conserved amino acids residues that are characteristic of serine/threonine protein kinases. In addition, there is an approximately 30 amino acid-peptide insertion between subdomains VII and VIII that contains a potential nuclear localization signal. Sequence analysis suggests that expression of the Eondr2 gene requires a + 1 programmed translational frameshift for its translation. Comparison of the deduced EoNdr2 with other known Ndr protein kinases implies that a + 1 ribosomal frameshift occurs at the motif AAATAA.     

A functional update of the Escherichia coli K-12 genome

Background  

Since the genome of Escherichia coli K-12 was initially annotated in 1997, additional functional information based on biological characterization and functions of sequence-similar proteins has become available. On the basis of this new information, an updated version of the annotated chromosome has been generated.     

Multicolor fluorescent differential display

Differential display and DNA microarray have emerged as the two most popular methods for gene expression profiling. Here, we developed a multicolor fluorescent differential display (FDD) method that combines the virtues of both differential display in signal amplification and DNA microarray in signal analysis. As in DNA microarray, RNA samples being compared can be labeled with either a red or green fluorescent dye and displayed in a single lane, allowing convenient scoring and quantification of the differentially expressed messages. In addition, the multicolor FDD has a built-in signal proofreading capability that is achieved by labeling each RNA sample from a comparative study with both red and green fluorescent dyes followed by their reciprocal mixings in color. Thus, the multicolor FDD provides a platform upon which a sensitive and accurate gene expression profiling by differential display can be automated and digitally analyzed. It is envisioned that cDNAs generated by the multicolor FDD may also be used directly as probes for DNA microarray, allowing an integration of the two most widely used technologies for comprehensive analysis of gene expression.     

Identification of a CD20-, FcepsilonRIbeta-, and HTm4-related gene family: sixteen new MS4A family members expressed in human and mouse

CD20, high-affinity IgE receptor beta chain (FcepsilonRIbeta), and HTm4 are structurally related cell-surface proteins expressed by hematopoietic cells. In the current study, 16 novel human and mouse genes that encode new members of this nascent protein family were identified. All family members had at least four potential membrane-spanning domains, with N- and C-terminal cytoplasmic domains. This family was therefore named the membrane-spanning 4A gene family, with at least 12 subgroups (MS4A1 through MS4A12) currently representing at least 21 distinct human and mouse proteins. Each family member had unique patterns of expression among hematopoietic cells and nonlymphoid tissues. Four of the 6 human MS4A genes identified in this study mapped to chromosome 11q12-q13.1 along with CD20, FcepsilonRIbeta, and HTm4. Thus, like CD20 and FcepsilonRIbeta, the other MS4A family members are likely to be components of oligomeric cell surface complexes that serve diverse signal transduction functions.     

Tumour necrosis factor-alpha enhances bradykinin-induced signal transduction via activation of Ras/Raf/MEK/MAPK in canine tracheal smooth muscle cells

Inhalation of tumour necrosis factor-alpha (TNF-alpha) induced a bronchial hyperreactivity to contractile agonists. However, the mechanisms of TNF-alpha involved in the pathogenesis of bronchial hyperreactivity were not completely understood. Therefore, we investigated the effect of TNF-alpha on bradykinin (BK)-induced inositol phosphate (IP) accumulation and Ca(2+) mobilization, and up-regulation of BK receptor density in canine cultured tracheal smooth muscle cells (TSMCs). Pretreatment of TSMCs with TNF-alpha potentiated BK-induced IP accumulation and Ca(2+) mobilization. However, there was no effect on the IP response induced by endothelin-1 (ET-1), 5-hydroxytryptamine (5-HT), and carbachol. Pretreatment with PDGF B-chain homodimer (PDGF-BB) also enhanced BK-induced IP response. These enhancements induced by TNF-alpha and PDGF-BB might be due to an increase in BK B(2) receptor density (B(max)), since [3H]BK binding to TSMCs was inhibited by the B(2) selective agonist and antagonist, BK and Hoe 140, but not by the B(1) selective reagents. The enhancing effects of TNF-alpha and PDGF-BB were attenuated by PD98059 (an inhibitor of activation of MAPK kinase, MEK) and cycloheximide (an inhibitor of protein synthesis), suggesting that TNF-alpha may share a common signalling pathway with PDGF-BB via protein(s) synthesis in TSMCs. Furthermore, overexpression of dominant negative mutants, H-Ras-15A and Raf-N4, significantly suppressed p42/p44 mitogen-activated protein kinase (MAPK) activation induced by TNF-alpha and PDGF-BB and attenuated the effect of TNF-alpha on BK-induced IP response, indicating that Ras and Raf may be required for activation of these kinases. These results suggest that the augmentation of BK-induced responses produced by TNF-alpha might be, at least in part, mediated through activation of Ras/Raf/MEK/MAPK pathway in TSMCs.