A computational method to characterize the missense mutations in the catalytic domain of GAA protein causing Pompe disease

Pompe disease is an autosomal recessive lysosomal storage disease caused by acid α-glucosidase (GAA) deficiency, resulting in intralysosomal accumulation of glycogen, including cardiac, skeletal, and smooth muscle cells. The GAA gene is located on chromosome 17 (17q25.3), the GAA protein consists of 952 amino acids; of which 378 amino acids (347-726) falls within the catalytic domain of the protein and comprises of active sites (518 and 521) and binding sites (404, 600, 616, and 674). In this study, we used several computational tools to classify the missense mutations in the catalytic domain of GAA for their pathogenicity and stability. Eight missense mutations (R437C, G478R, N573H, Y575S, G605D, V642D, L705P, and L712P) were predicted to be pathogenic and destabilizing to the protein structure. These mutations were further subjected to phenotyping analysis using SNPeffect 4.0 to predict the chaperone binding sites and structural stability of the protein. The mutations R437C and G478R were found to compromise the chaperone-binding activity with GAA. Molecular docking analysis revealed that the G478R mutation to be more significant and hinders binding to the DNJ (Miglustat) compared with the R437C. Further molecular dynamic analysis for the two mutations demonstrated that the G478R mutation was acquired higher deviation, fluctuation, and lower compactness with decreased intramolecular hydrogen bonds compared to the mutant R437C. These data are expected to serve as a platform for drug design against Pompe disease and will serve as an ultimate tool for variant classification and interpretations.     

Chaperone-mediated autophagy and aging: a novel regulatory role of lipids revealed

A wide pool of cytosolic proteins is selectively degraded in lysosomes by chaperonemediated autophagy (CMA). Binding of these proteins to a receptor at the lysosomal membrane is the limiting step in CMA. Levels of this receptor are tightly regulated through changes in its degradation, multimeric organization and dynamic distribution between the lysosomal membrane and lumen. We have now reported that subcompartmentalization of the receptor in discrete lipid microdomains at the lysosomal membrane regulates its engagement in each of these processes-degradation, multimerization and membrane retrieval. Changes in the lipid composition of the membrane thus affect the dynamics of the receptor and, consequently, CMA activity. As an example of CMA dysfunction resulting from perturbation of the lipid composition of the lysosomal membrane, we discuss here a second study in which we analyzed the changes in the dynamics of the receptor during aging. CMA activity decreases with age primarily due to a decrease in the levels of the CMA receptor at the lysosomal membrane. Now we have found that age-related alterations in the lipid composition of the discrete microdomains at the lysosomal membrane are behind the reduced lysosomal levels of the receptor and, consequently, the declined CMA activity that occurs during aging.     

Weighted rank aggregation of cluster validation measures: a Monte Carlo cross-entropy approach

MOTIVATION: Biologists often employ clustering techniques in the explorative phase of microarray data analysis to discover relevant biological groupings. Given the availability of numerous clustering algorithms in the machine-learning literature, an user might want to select one that performs the best for his/her data set or application. While various validation measures have been proposed over the years to judge the quality of clusters produced by a given clustering algorithm including their biological relevance, unfortunately, a given clustering algorithm can perform poorly under one validation measure while outperforming many other algorithms under another validation measure. A manual synthesis of results from multiple validation measures is nearly impossible in practice, especially, when a large number of clustering algorithms are to be compared using several measures. An automated and objective way of reconciling the rankings is needed. RESULTS: Using a Monte Carlo cross-entropy algorithm, we successfully combine the ranks of a set of clustering algorithms under consideration via a weighted aggregation that optimizes a distance criterion. The proposed weighted rank aggregation allows for a far more objective and automated assessment of clustering results than a simple visual inspection. We illustrate our procedure using one simulated as well as three real gene expression data sets from various platforms where we rank a total of eleven clustering algorithms using a combined examination of 10 different validation measures. The aggregate rankings were found for a given number of clusters k and also for an entire range of k. AVAILABILITY: R code for all validation measures and rank aggregation is available from the authors upon request. SUPPLEMENTARY INFORMATION: Supplementary information are available at http://www.somnathdatta.org/Supp/RankCluster/supp.htm.     

Reactive oxygen species induced by shear stress mediate cell death in Bacillus subtilis

Exposure of Bacillus subtilis to a shear rate of 1,482/s leads to a rapid loss of cell viability after 10 h of growth. Biochemical and molecular evidences provided below strongly suggest that cell death under high shear results from an apoptosis-like process similar to that described in eukaryotes, with activation of a caspase-3-like protease (C(3)LP) followed by DNA fragmentation. Shear stress leads to an increase in specific intracellular reactive oxygen species (siROS), possibly through activation of NADH oxidase (NOX). The formation of siROS precedes the activation of C(3)LP and DNA fragmentation, thus establishing siROS as the molecular link between shear stress and apoptosis-like cell death. A model is proposed in which NOX is viewed as being strategically placed on the plasma membrane of B. subtilis that senses and converts a mechanical force arising from shear stress into a chemical signal leading to activation of C(3)LP, DNA fragmentation, and thus, apoptosis-like cell death.     

Alterations of <Emphasis Type="Italic">ROBO1/DUTT1</Emphasis> and <Emphasis Type="Italic">ROBO2</Emphasis> loci in early dysplastic lesions of head and neck: clinical and prognostic implications

Deletion of chromosomal 3p12.3 was suggested to be associated with dysplastic lesions of head and neck. This region harbors two candidate tumor suppressors ROBO1/DUTT1, ROBO2 and two non-coding RNAs (ncRNAs) located at intron 2 of ROBO1/DUTT1. Aim of this study is to understand the role of these genes in development of head and neck squamous cell carcinoma. A collection of 72 dysplastic lesions and 116 HNSCC samples and two oral cancer cell lines were analyzed for ROBO1/DUTT1 and ROBO2 deletion and promoter methylation. ROBO1/DUTT1, ROBO2 and two ncRNAs mRNA expression were analyzed by Q-PCR. Immunohistochemical analysis of ROBO1/DUTT1 and ROBO2 was performed. Alterations of these genes were correlated with different clinicopathological parameters. High frequency of molecular alterations (deletion/methylation) was seen in ROBO1/DUTT1 than ROBO2. In mild dysplastic lesions both of these genes showed high molecular alterations and remained more or less constant in subsequent stages. Q-PCR analysis showed reduced expression of these genes and the two ncRNAs. In vitro demethylation experiment by 5-aza-dC showed upregulation of ROBO1/DUTT1 and ROBO2 while the expression of the ncRNAs remained unchanged. Immunohistochemical analysis of ROBO1/DUTT1 and ROBO2 showed concordance with their mRNA expression and molecular alterations. Poor patients’ outcome was predicted in the cases with alteration of ROBO1/DUTT1 along with tobacco addiction and nodal involvement. Our data suggests (a) ROBO1/DUTT1 and the ncRNAs are transcribed from different promoters, and (b) inactivation of ROBO1/DUTT1 could be used as molecular signature for early detection and prognosis of the head and neck cancer. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A novel salt-tolerant L-myo-inositol-1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka, a halophytic wild rice: molecular cloning, bacterial overexpression, characterization, and functional introgression into tobacco-conferring salt tolerance phenotype

l-myo-Inositol-1-phosphate synthase (EC 5.5.1.4, MIPS), an evolutionarily conserved enzyme protein, catalyzes the synthesis of inositol, which is implicated in a number of metabolic reactions in the biological kingdom. Here we report on the isolation of the gene (PINO1) for a novel salt-tolerant MIPS from the wild halophytic rice, Porteresia coarctata (Roxb.) Tateoka. Identity of the PINO1 gene was confirmed by functional complementation in a yeast inositol auxotrophic strain. Comparison of the nucleotide and deduced amino acid sequences of PINO1 with that of the homologous gene from Oryza sativa L. (RINO1) revealed distinct differences in a stretch of 37 amino acids, between amino acids 174 and 210. Purified bacterially expressed PINO1 protein demonstrated a salt-tolerant character in vitro compared with the salt-sensitive RINO1 protein as with those purified from the native source or an expressed salt-sensitive mutant PINO1 protein wherein amino acids 174-210 have been deleted. Analysis of the salt effect on oligomerization and tryptophan fluorescence of the RINO1 and PINO1 proteins revealed that the structure of PINO1 protein is stable toward salt environment. Furthermore, introgression of PINO1 rendered transgenic tobacco plants capable of growth in 200-300 mm NaCl with retention of approximately 40-80% of the photosynthetic competence with concomitant increased inositol production compared with unstressed control. MIPS protein isolated from PINO1 transgenics showed salt-tolerant property in vitro confirming functional expression in planta of the PINO1 gene. To our knowledge, this is the first report of a salt-tolerant MIPS from any source.     

Disulfide cross-linking reveals a site of stable interaction between C-terminal regulatory domains of the two MalK subunits in the maltose transport complex

Understanding the structure and function of the ATP-binding cassette (ABC) transporters is very important because defects in ABC transporters lie at the root of several serious diseases including cystic fibrosis. MalK, the ATP-binding cassette of the maltose transporter of Escherichia coli, is distinct from most other ATP-binding cassettes in that it contains an additional C-terminal regulatory domain. The published structure of a MalK dimer is elongated with C-terminal domains at opposite poles (Diederichs, K., Diez, J., Greller, G., Muller, C., Breed, J., Schnell, C., Vonrhein, C., Boos, W., and Welte, W. (2000) EMBO J. 19, 5951-5961). Some uncertainty exists as to whether the orientation of MalK in the dimer structure is correct. Superpositioning of the N-terminal domains of MalK onto the ATP-binding domains of an alternate ABC dimer, in which ATP is bound along the dimer interface between Walker A and LSGGQ motifs, places both N- and C-terminal domains of MalK along the dimer interface. Consistent with this model, a cysteine substitution at position 313 in the C-terminal domain of an otherwise cysteine-free MalK triggered disulfide bond formation between two MalK subunits in an intact maltose transporter. Disulfide bond formation did not inhibit the function of the transporter, suggesting that the C-terminal domains of MalK remain in close proximity throughout the transport cycle. Enzyme IIAglc still inhibited the ATPase activity of the disulfide-linked transporter indicating that the mechanism of inducer exclusion was unaffected. These data support a model for ATP hydrolysis in which the C-terminal domains of MalK remain in contact whereas the N-terminal domains of MalK open and close to allow nucleotide binding and dissociation.     

In vitro selection of endosulfan-tolerant strains of Brassica compestris (cv. Brown Sarson)

Endosulfan tolerant lines of mustard (Brassica campestris cv. Brown Sarson) have been developed through tissue culture methods. Cotyledonary expiants excised from eight day old in vitro grown seedlings were used for inducing callus. Fast growing friable callus was then transferred to MS medium containing (0.1–2.0 ugl^–1) endosulfan for selection. Five alternating exposures with and without endosulfan containing medium yielded an endosulfan tolerant cell line (ETL). The plants regenerated from ETL were found to tolerate three fold higher concentrations of endosulfan. Callus induced from randomly selected endosulfan tolerant regenerated plants were also tolerant to 3.0 ugl endosulfan, thereby, suggesting that tolerance has been acquired at the gene level.Biochemical investigation revealed higher levels of total free sugar, free amino acids, protein and activity of peroxidase in the tolerant cell line.Abbreviations MS Murashige and Skoog medium (1962) - NSM non-selective medium - SM selective medium - BAP 6-Benzylaminopurine - NAA -naphthaleneacetic acid - Z zeatin