X-ray structure of human acid-beta-glucosidase covalently bound to conduritol-B-epoxide. Implications for Gaucher disease

Gaucher disease is an inherited metabolic disorder caused by mutations in the lysosomal enzyme acid-beta-glucosidase (GlcCerase). We recently determined the x-ray structure of GlcCerase to 2.0 A resolution (Dvir, H., Harel, M., McCarthy, A. A., Toker, L., Silman, I., Futerman, A. H., and Sussman, J. L. (2003) EMBO Rep.4, 704-709) and have now solved the structure of Glc-Cerase conjugated with an irreversible inhibitor, conduritol-B-epoxide (CBE). The crystal structure reveals that binding of CBE to the active site does not induce a global conformational change in GlcCerase and confirms that Glu340 is the catalytic nucleophile. However, only one of two alternative conformations of a pair of flexible loops (residues 345-349 and 394-399) located at the entrance to the active site in native GlcCerase is observed in the GlcCerase-CBE structure, a conformation in which the active site is accessible to CBE. Analysis of the dynamics of these two alternative conformations suggests that the two loops act as a lid at the entrance to the active site. This possibility is supported by a cluster of mutations in loop 394-399 that cause Gaucher disease by reducing catalytic activity. Moreover, in silico mutational analysis demonstrates that all these mutations stabilize the conformation that limits access to the active site, thus providing a mechanistic explanation of how mutations in this loop result in Gaucher disease.     

JLP associates with kinesin light chain 1 through a novel leucine zipper-like domain

Scaffolding proteins exist in eukaryotes to properly assemble signaling proteins into specific multimeric functional complexes. JLP is a novel leucine zipper protein belonging to a family of scaffolding proteins that assemble JNK signaling modules. JLP is a proline-rich protein that contains two leucine zipper domains and a highly conserved C-terminal domain. We have identified kinesin light chain 1 (KLC1) as a binding partner for the second leucine zipper domain of JLP using yeast two-hybrid screening. The interaction domain of KLC1 was mapped to its tetratripeptide repeat, which contains a novel leucine zipper-like domain that is crucial for the interaction with JLP. Mutations of Leu-280, Leu-287, Val-294, and Leu-301 within this domain of KLC1 disrupted its ability to associate with JLP. Immunofluorescence studies showed that JLP and KLC1 co-localized in the cytoplasm and that the localization of JLP was dependent on its second leucine zipper. Ectopic expression of a dominant negative form of KLC1 resulted in the mislocalization of endogenous JLP. Moreover, the association between JLP and KLC1 occurred in vivo and was important in the formation of ternary complex with JNK1. These results identify a novel protein-protein interaction between KLC1 and JLP that involves leucine zipper-like domains and support the role of motor proteins in the spatial regulation of signaling modules.     

E infα supk transgene in B10 mice suppresses the development of myasthenia gravis

Mice bearing the H-2 ^bhaplotype are susceptible to the development of experimental autoimmune myasthenia gravis (EAMG), induced by acetylcholine receptor (AChR) autoimmunity. One of the genes influencing EAMG susceptibility has been mapped to the A ^blocus of the major histocompatibility complex, and the A chain has been implicated in the pathogenesis. Mice of the H-2 ^bhaplotype, including C57BL/10 (B10), have a genomic deletion of the E gene and therefore fail to express the E molecule on their cell surface. To test the hypothesis that failure to express the cell surface E molecule in B10 mice contributes to EAMG pathogenesis, E _inf ^supk transgenic B10 mice expressing the T molecule were examined. Expression of the E molecule in E _inf ^supk transgenic B10 mice partially prevented the development of EAMG.     

Cooperativity of Cdk4R24C and Ras in melanoma development

The importance of the Cdk4 protein in human cancer became evident following the identification of a germ line mutation in the Cdk4 locus that predisposes humans to melanoma. This mutation results in substitution of argininefirst with cysteine at position 24 (R24C). In an earlier study, we introduced the R24C mutation into the Cdk4 locus of mice using Cre-loxp-mediated “knock-in” technology and observed a very low incidence of spontaneous melanomas in Cdk4^R24C/R24C mice. This suggested that additional oncogenic mutations might be required for development of melanomas. Here we report an increased incidence of spontaneous cutaneous melanoma in mice expressing the oncogene HRAS(G12V) in melanocytes on a Cdk4^R24C background. Treatment of Tyr-HRas:Cdk4^R24C/R24C mice with the carcinogen, DMBA/TPA resulted in a further increase in the number of nevi and melanomas developed when compared with Tyr-HRas:Cdk4^+/+ mice. In summary, in Tyr-HRas:Cdk4^R24C/R24C mice, we observed that activated Cdk4 cooperates with the oncogenic HRAS(G12V) protein to increase the susceptibility of melanoma development in vivo.Key words: Cdk4^R24C, ras, melanoma, skin, carcinogen     

A major difference between the divergence patterns within the lines-1 families in mice and voles

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.      

Binpairs: Utilization of Illumina Paired-End Information for Improving Efficiency of Taxonomic Binning of Metagenomic Sequences

Motivation

Paired-end sequencing protocols, offered by next generation sequencing (NGS) platforms like Illumia, generate a pair of reads for every DNA fragment in a sample. Although this protocol has been utilized for several metagenomics studies, most taxonomic binning approaches classify each of the reads (forming a pair), independently. The present work explores some simple but effective strategies of utilizing pairing-information of Illumina short reads for improving the accuracy of taxonomic binning of metagenomic datasets. The strategies proposed can be used in conjunction with all genres of existing binning methods.

Results

Validation results suggest that employment of these “Binpairs” strategies can provide significant improvements in the binning outcome. The quality of the taxonomic assignments thus obtained are often comparable to those that can only be achieved with relatively longer reads obtained using other NGS platforms (such as Roche).

Availability

An implementation of the proposed strategies of utilizing pairing information is freely available for academic users at https://metagenomics.atc.tcs.com/binning/binpairs.     

Diabetic Peripheral Neuropathy: Role of Reactive Oxygen and Nitrogen Species

The prevalence of diabetes has reached epidemic proportions. There are two forms of diabetes: type 1 diabetes mellitus is due to auto-immune-mediated destruction of pancreatic β-cells resulting in absolute insulin deficiency and type 2 diabetes mellitus is due to reduced insulin secretion and or insulin resistance. Both forms of diabetes are characterized by chronic hyperglycemia, leading to the development of diabetic peripheral neuropathy (DPN) and microvascular pathology. DPN is characterized by enhanced or reduced thermal, chemical, and mechanical pain sensitivities. In the long-term, DPN results in peripheral nerve damage and accounts for a substantial number of non-traumatic lower-limb amputations. This review will address the mechanisms, especially the role of reactive oxygen and nitrogen species in the development and progression of DPN.     

Inositol monophosphate phosphatase genes of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background  

Mycobacteria use inositol in phosphatidylinositol, for anchoring lipoarabinomannan (LAM), lipomannan (LM) and phosphatidylinosotol mannosides (PIMs) in the cell envelope, and for the production of mycothiol, which maintains the redox balance of the cell. Inositol is synthesized by conversion of glucose-6-phosphate to inositol-1-phosphate, followed by dephosphorylation by inositol monophosphate phosphatases (IMPases) to form myo-inositol. To gain insight into how Mycobacterium tuberculosis synthesises inositol we carried out genetic analysis of the four IMPase homologues that are present in the Mycobacterium tuberculosis genome.