Mitsuaki Tabuchi

Genetic variants of NRAMP1 and active tuberculosis in Japanese populations

To the Editor: Tuberculosis is a major public health concern worldwide. Approximately 30% of world’s population is infected with Mycobacterium tuberculosis, and 3000000 people die as a consequence each year. While significant exposure and socioeconomic factors are key elements in the development of tuberculosis, intrinsic host factors, especially genetic susceptibility to this pathogen, are important. Studies of mycobacterial infections in animal models provide compelling evidence for a genetic component of host resistance/susceptibility to this bacillus (1). Early animal experiments identified a single dominant gene, designated Bcg (2). A candidate gene was identified and designated as the natural-resistance-associated-macrophage protein 1 gene (Nramp1) (2). The human homologue of NRAMP1 has been cloned (3, 4) and it is localised on chromosome 2q35 (4). To date, 11 variants including five coding regions, three in the introns, and two in the 3% non-coding region have been identified (5, 6). Early attempts to find a genetic linkage between NRAMP1 and tuberculosis failed (7), but a significant genetic association was found between four variants of NRAMP1 and active M. tuberculosis in west Africans (8). To test whether variants of NRAMP1 relate to active tuberculosis in Japanese populations, we conducted a genetic association study using two independent populations from Tokyo and Osaka. Controls were randomly selected from clients in a commercial medical examination company (9). All patients were newly diagnosed cases of pulmonary tuberculosis, with positive acid-fast bacilli showing in sputum smear specimens; none of them was HIV-positive. We first genotyped 30 patients from each of the two populations for 5% promoter (GT)n (8), INT4 (intron 4) (5), Asn543Asp (5), and 3% untranslated region (UTR) variants (8), previously tested by Bellamy et al. (8); we found that the former two and the latter two were in perfect linkage disequilibrium in our populations, as described (5, 8). We thus focused on genotyping the 5% promoter (GT)n and Asn543Asp variants (5, 8); three alleles, 199bp (designated as allele 1), 201bp (allele 2), and 203bp (allele 3) (10) were identified and imaged by automated sequencer (ABI 310) (5). A weak association was found between Asn543Asp and tuberculosis in the Tokyo population; in contrast, there was a significant association with the 5% promoter (GT)n in both populations (Table 1). Of the four (GT)n alleles, allele 3 has been shown to drive promoter activity after stimulation with lipopoysaccharide, or interferon-g, while alleles 1 and 2 reduce it (10); poor promoter alleles (1 and 2), especially allele 1, thus showed a significantly stronger association with tuberculosis in both populations (Table 1); furthermore, the frequencies of homozygous allele 3 were significantly lower in both tuberculosis populations (Table 1). Since there was no significant difference in genotype frequencies between the two populations, we conducted a combined multivariate analysis on the total of 496 subjects; simple factorial analysis of variance (ANOVA) confirmed that there was no interaction between Asn543 and the 5% promoter (GT)n, and that allele 3 is a significant independent genetic marker for resistance to tuberculosis (f-value=3.24, df=2, p=0.039); by comparison, heterozygosity at the INT4 and 3% UTR loci was the highest risk factor in west Africans (8). In conclusion, we show that variants of NRAMP1 are associated with active tuberculosis in Japanese populations (combined odds ratio= 2.07, 95% confidence interval: 1.32–2.61, p= 0.0003); these findings support the first report in west Africans (8). However, the pattern of allelic association with tuberculosis is different in the

The phosphatidylinositol 4,5-biphosphate and TORC2 binding proteins Slm1 and Slm2 function in sphingolipid regulation.

ABSTRACT The Stt4 phosphatidylinositol 4-kinase has been shown to generate a pool of phosphatidylinositol 4-phosphate (PI4P) at the plasma membrane, critical for actin cytoskeleton organization and cell viability. To further understand the essential role of Stt4-mediated PI4P production, we performed a genetic screen using the stt4ts mutation to identify candidate regulators and effectors of PI4P. From this analysis, we identified several genes that have been previously implicated in lipid metabolism. In particular, we observed synthetic lethality when both sphingolipid and PI4P synthesis were modestly diminished. Consistent with these data, we show that the previously characterized phosphoinositide effectors, Slm1 and Slm2, which regulate actin organization, are also necessary for normal sphingolipid metabolism, at least in part through regulation of the calcium/calmodulin-dependent phosphatase calcineurin, which binds directly to both proteins. Additionally, we identify Isc1, an inositol phosphosphingolipid phospholipase C, as an additional target of Slm1 and Slm2 negative regulation. Together, our data suggest that Slm1 and Slm2 define a molecular link between phosphoinositide and sphingolipid signaling and thereby regulate actin cytoskeleton organization.

Retromer-mediated direct sorting is required for proper endosomal recycling of the mammalian iron transporter DMT1

Endosomal recycling of the mammalian iron transporter DMT1 is assumed to be important for efficient and rapid uptake of iron across the endosomal membrane in the transferrin cycle. Here, we show that the retromer, a complex that mediates retrograde transport of transmembrane cargoes from endosomes to the trans-Golgi network, is required for endosomal recycling of DMT1-II, an alternative splicing isoform of DMT1. Bacterially expressed Vps26-Vsp29-Vsp35 trimer, a retromer cargo recognition complex, specifically binds to the cytoplasmic tail domain of DMT1-II in vitro. In particular, this binding is dependent on a specific hydrophobic motif of DMT1-II, which is required for its endosomal recycling. DMT1-II colocalizes with the Vps35 subunit of the retromer in TfR-positive endosomes. Depletion of the retromer by siRNA against Vps35 leads to mis-sorting of DMT1-II to LAMP2-positive structures, and expression of siRNA-resistant Vps35 can rescue this effect. These findings demonstrate that the retromer recognizes the recycling signal of DMT1-II and ensures its proper endosomal recycling.

VPS35 dysfunction impairs lysosomal degradation of α-synuclein and exacerbates neurotoxicity in a Drosophila model of Parkinson's disease

Mutations in vacuolar protein sorting 35 (VPS35) have been linked to familial Parkinsons disease (PD). VPS35, a component of the retromer, mediates the retrograde transport of cargo from the endosome to the trans-Golgi network. Here we showed that retromer depletion increases the lysosomal turnover of the mannose 6-phosphate receptor, thereby affecting the trafficking of cathepsin D (CTSD), a lysosome protease involved in α-synuclein (αSYN) degradation. VPS35 knockdown perturbed the maturation step of CTSD in parallel with the accumulation of αSYN in the lysosomes. Furthermore, we found that the knockdown of Drosophila VPS35 not only induced the accumulation of the detergent-insoluble αSYN species in the brain but also exacerbated both locomotor impairments and mild compound eye disorganization and interommatidial bristle loss in flies expressing human αSYN. These findings indicate that the retromer may play a crucial role in αSYN degradation by modulating the maturation of CTSD and might thereby contribute to the pathogenesis of the disease.

Complete nucleotide sequence of human NRAMP2 cDNA

The mouse Lsh/Ity/Bcg locus regulates macrophage activation for antimicrobial activity against intracellular pathogens, and the Nramp1 gene was isolated as its candidate. In the present study, a full length cDNA for human NRAMP2 has been isolated and characterized. Nucleotide sequence analysis reveals that the cDNA, 4142 bp in length, coded for a protein of 561 amino acid residues with a molecular weight mass of 61,456. Predicted amino acid sequence analysis of the NRAMP2 molecule indicates that NRAMP1 and NRAMP2 sequences share 64% identical residues overall, whereas only 21% at the NH2-terminal cytoplasmic domain, where the NRAMP1 molecule was associated with microtubules, was found to be identical. This suggests that the NH2-terminal region of the NRAMP2 molecule may have a particular function, different from that of the NRAMP1 molecule.

Heme and non-heme iron transporters in non-polarized and polarized cells

BackgroundHeme and non-heme iron from diet, and recycled iron from hemoglobin are important products of the synthesis of iron-containing molecules. In excess, iron is potentially toxic because it can produce reactive oxygen species through the Fenton reaction. Humans can absorb, transport, store, and recycle iron without an excretory system to remove excess iron. Two candidate heme transporters and two iron transporters have been reported thus far. Heme incorporated into cells is degraded by heme oxygenases (HOs), and the iron product is reutilized by the body. To specify the processes of heme uptake and degradation, and the reutilization of iron, we determined the subcellular localizations of these transporters and HOs.ResultsIn this study, we analyzed the subcellular localizations of 2 isoenzymes of HOs, 4 isoforms of divalent metal transporter 1 (DMT1), and 2 candidate heme transporters--heme carrier protein 1 (HCP1) and heme responsive gene-1 (HRG-1)--in non-polarized and polarized cells. In non-polarized cells, HCP1, HRG-1, and DMT1A-I are located in the plasma membrane. In polarized cells, they show distinct localizations: HCP1 and DMT1A-I are located in the apical membrane, whereas HRG-1 is located in the basolateral membrane and lysosome. 16Leu at DMT1A-I N-terminal cytosolic domain was found to be crucial for plasma membrane localization. HOs are located in smooth endoplasmic reticulum and colocalize with NADPH-cytochrome P450 reductase.ConclusionsHCP1 and DMT1A-I are localized to the apical membrane, and HRG-1 to the basolateral membrane and lysosome. These findings suggest that HCP1 and DMT1A-I have functions in the uptake of dietary heme and non-heme iron. HRG-1 can transport endocytosed heme from the lysosome into the cytosol. These localization studies support a model in which cytosolic heme can be degraded by HOs, and the resulting iron is exported into tissue fluids via the iron transporter ferroportin 1, which is expressed in the basolateral membrane in enterocytes or in the plasma membrane in macrophages. The liberated iron is transported by transferrin and reutilized for hemoglobin synthesis in the erythroid system.

Comparison of outer membrane protein genes omp and pmp in the whole genome sequences of Chlamydia pneumoniae isolates from Japan and the United States.

Chlamydia pneumoniae is a widespread pathogen of the respiratory tract that is also associated with atherosclerosis. The whole genome sequence was determined for a Japanese isolate, C. pneumoniae strain J138. The sequence predicted a variety of genes encoding outer membrane proteins (OMPs) including ompA and porB, another 10 predicted omp genes, and 27 pmp genes. All were detected in the whole genome sequence of strain CWL029, a strain isolated and sequenced in the United States. A comparative study of the OMPs of the two strains revealed a nucleotide sequence identity of 89.6%-100% (deduced amino acid sequence identity, 71.1%-100%). The overall genomic organization and location of genes are identical in both strains. Thus, a few unique sequences of the OMPs may be essential for specific attributes that define the differential biology of two C. pneumoniae strains.

Hepatitis C virus protein and iron overload induce hepatic steatosis through the unfolded protein response in mice

Background/Aim: Hepatic iron overload and steatosis play critical roles in the progression of hepatitis C virus (HCV)‐associated chronic liver disease. However, how these two pathophysiological features affect each other remains unknown. The aim of this study was to investigate how hepatic iron overload contributes to the development of hepatic steatosis in the presence of HCV proteins.

Methyl 3-Hydroxymyristate, a Diffusible Signal Mediating phc Quorum Sensing in Ralstonia solanacearum.

Ralstonia solanacearum, a plant pathogenic bacterium causing “bacterial wilt” on crops, uses a quorum sensing (QS) system consisting of phc regulatory elements to control its virulence. Methyl 3‐hydroxypalmitate (3‐OH PAME) was previously identified as the QS signal in strain AW1. However, 3‐OH PAME has not been reportedly detected from any other strains, and this suggests that they produce another unknown QS signal. Here we identify (R)‐methyl 3‐hydroxymyristate [(R)‐3‐OH MAME] as a new QS signal that regulates the production of virulence factors and secondary metabolites. (R)‐3‐OH MAME was synthesized by the methyltransferase PhcB and sensed by the histidine kinase PhcS. The phylogenetic trees of these proteins from R. solanacearum strains were divided into two groups, according to their QS signal types—(R)‐3‐OH MAME or (R)‐3‐OH PAME. These results demonstrate that (R)‐3‐OH MAME is another crucial QS signal and highlight the unique evolution of QS systems in R. solanacearum.

Functional analysis of the human NRAMP family expressed in fission yeast.

The Bcg/Ity/Lsh locus in the mouse genome regulates macrophage activation for antimicrobial activity against intracellular pathogens, and the positional cloning of this locus identified the Nramp1 (natural resistance-associated macrophage protein) gene. Nramp2 was initially isolated as a homologue of Nramp1. Recently, the rat divalent metal transporter DMT1 was identified electrophysiologically, and was found to be an isoform of Nramp2, a mutation which was subsequently identified in rats suffering from hereditary iron-deficiency anaemia. Despite the 64% amino acid sequence identity of Nramp1 and Nramp2, no divalent metal transport activity has yet been detected from Nramp1, and the function of Nramp1 on the molecular level is still unclear. To investigate the divalent metal transport activity of NRAMP molecules, we constructed four chimeric NRAMP genes by swapping the domains of human NRAMP1 and NRAMP2 with each other. The functional characteristics of wild-type NRAMP1, NRAMP2 and their chimeras were determined by expression in the divalent metal transporter-disrupted strain of fission yeast, pdt1Delta, and we analysed the divalent metal transport activity by complementation of the EGTA- and pH-sensitive phenotype of pdt1Delta. Replacement of the N-terminal cytoplasmic domain of NRAMP2 with the NRAMP1 counterpart resulted in inactive chimeras, indicating that the functional difference between NRAMP1 and NRAMP2 is located in this region. However, results obtained with the reverse construct and other chimeras indicated that these regions are not solely responsible for the differences in EGTA- and pH-sensitivity of NRAMP1 and NRAMP2. These findings indicate that NRAMP1 itself cannot represent the divalent metal transport activity in S. pombe and the additional protein segments of the molecules located elsewhere in NRAMP1 are also functionally distinct from their NRAMP2 counterparts.