Genome-wide survey of prokaryotic O-protein phosphatases

Complex and diverse signal transduction circuits are responsible for the efficient functioning of cellular network. Protein kinases and O-protein phosphatases are primarily responsible for propagating such stimuli within a eukaryotic cell. However, there is limited understanding of O-protein phosphatases in the prokaryotic genomes. The availability of complete genome sequence information for several prokaryotes permits a genome-wide survey of O-protein phosphatases. The distribution of the various protein phosphatase families has been observed to be mosaic, with the exception of the members of the phospho protein family P (PPP), which is consistent with previous studies. The PPP family is ubiquitous in the prokaryotic world and undergoes the highest sequence divergence within a genome amongst phosphatases studied. The co-occurrence of low molecular mass tyrosine phosphatase (LMWPc) and PPP domain in a single polypeptide suggests that the protein present in Archaeoglobus fulgidus might represent the progenitor for all protein phosphatases. The curation of data on prokaryotic protein phosphatases provides a convenient framework for the analysis of domain architectures and for characterising structural and functional properties of this important family of signalling proteins.     

Identification and characterization of membralin, a novel tumor-associated gene, in ovarian carcinoma

Identification of new genes in cancer is the key to understand the molecular basis of tumor development as well as provide potential diagnostic markers and therapeutic targets. A novel gene, membralin (GeneBank accession number: DQ005958), was cloned from a human ovarian cancer cell line. Human membralin is unique and does not share significant sequence homology with other human genes, only membralins of other species. The gene contains 11 exons which encode at least two spliced variants in human cancer. The long form of membralin (membralin-1) comprises all 11 exons, encoding a protein of 620-amino acids long and the short form of membralin (membralin-3) contains all exons except for exon 10, encoding a protein of 408 amino acids. Expression of different membralin isoforms depends on tissue type. The long form, membralin-1, is expressed in ovarian and colorectal carcinomas but not in breast or pancreatic carcinomas, which express only the short splice form, membralin-3. Membralin-1-GFP fusion protein demonstrates exclusive cytoplasmic localization. Based on quantitative real-time PCR, in situ hybridization and Western blot analysis, membralin was highly expressed in ovarian serous carcinomas as compared to ovarian surface epithelium (P<0.001). Ovarian carcinomas in effusions demonstrated a significantly higher level of membralin expression than in solid tumors (P<0.001). In conclusion, these findings represent the first characterization of human membralin and suggest that membralin is a novel tumor-associated marker in ovarian serous carcinomas.     

SAM recognition and conformational switching mechanism in the Bacillus subtilis yitJ S box/SAM-I riboswitch

S-box (SAM-I) riboswitches are a widespread class of riboswitches involved in the regulation of sulfur metabolism in Gram-positive bacteria. We report here the 3.0-Å crystal structure of the aptamer domain of the Bacillus subtilis yitJ S-box (SAM-I) riboswitch bound to S-adenosyl-l-methionine (SAM). The RNA folds into two sets of helical stacks spatially arranged by tertiary interactions including a K-turn and a pseudoknot at a four-way junction. The tertiary structure is further stabilized by metal coordination, extensive ribose zipper interactions, and SAM-mediated tertiary interactions. Despite structural differences in the peripheral regions, the SAM-binding core of the B. subtilis yitJ riboswitch is virtually superimposable with the previously determined Thermoanaerobacter tengcongensis yitJ riboswitch structure, suggesting that a highly conserved ligand-recognition mechanism is utilized by all S-box riboswitches. SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing analysis further revealed that the alternative base-pairing element in the expression platform controls the conformational switching process. In the absence of SAM, the apo yitJ aptamer domain folds predominantly into a pre-binding conformation that resembles, but is not identical with, the SAM-bound state. We propose that SAM enters the ligand-binding site through the “J1/2-J3/4” gate and “locks” down the SAM-bound conformation through an induced-fit mechanism. Temperature-dependent SHAPE revealed that the tertiary interaction-stabilized SAM-binding core is extremely stable, likely due to the cooperative RNA folding behavior. Mutational studies revealed that certain modifications in the SAM-binding region result in loss of SAM binding and constitutive termination, which suggests that these mutations lock the RNA into a form that resembles the SAM-bound form in the absence of SAM.     

Domain architectural census of eukaryotic gene products containing O-protein phosphatases

Intricate molecular signalling within cellular environment is manifested through phosphorylation of proteins. Regulation of the phosphorylation state is executed through complex networking among kinases and their biochemical antagonists, the protein phosphatases. Protein dephosphorylation in eukaryotic systems is largely performed through four structurally distinct Ser/Thr and Tyr O-protein phosphatase superfamilies. 555 O-protein phosphatases, belonging to the four distinct families, could be identified using sensitive sequence search techniques across five eukaryotic model organisms (yeast, fly, worm, mouse and humans). These phosphatases could be grouped into 49 subfamilies associated with distinct domain architecture and discrete biochemical function. Only five of the architectures are shared across the five eukaryotic genomes. Interestingly, the number of occurrence of tyrosine phosphatases is correlated to the complexity of the genome. Analysis of domain architectures suggests amenability of the tyrosine phosphatases to occur in complex architectures unlike Ser/Thr phosphatases. Domain duplication and shuffling is shown as the customary mechanism for the evolution of the phosphatases. Several architectures are common between humans and other genomes, which are probably non-linearly inherited in humans or specifically lost in several others.     

Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida

The highly enantioselective arylacetonitrilase of Pseudomonas putida was purified to homogeneity using a combination of (NH₄)₂SO₄ fractionation and different chromatographic techniques. The enzyme has a molecular weight of 412 kDa and consisted of approximately nine to ten identical subunits (43 kDa). The purified enzyme exhibited a pH optimum of 7.0 and temperature optimum of 40°C. The nitrilase was highly susceptible to thiol-specific reagents and metal ions and also required a reducing environment for its activity. These reflected the presence of a catalytically essential thiol group for enzyme activity which is in accordance with the proposed mechanism for nitrilase-catalyzed reaction. The enzyme was highly specific for arylacetonitriles with phenylacetonitrile and its derivatives being the most preferred substrates. Higher specificity constant (k _cat/K _m) values for phenylacetonitrile compared to mandelonitrile also revealed the same. Faster reaction rate achieved with this nitrilase for mandelonitrile hydrolysis was possibly due to the low activation energy required by the protein. Incorporation of low concentration (<5%) of organic solvent increased the enzyme activity by increasing the availability of the substrate. Higher stability of the enzyme at slightly alkaline pH and ambient temperature provides an excellent opportunity to establish a dynamic kinetic resolution process for the production of (R)-(−)-mandelic acid from readily available mandelonitrile.     

sll1981, an acetolactate synthase homologue of Synechocystis sp. PCC6803, functions as l-myo-inositol 1-phosphate synthase

l-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes the first rate limiting conversion of d-glucose 6-phosphate to l-myo-inositol 1-phosphate in the inositol biosynthetic pathway. In an earlier communication we have reported two forms of MIPS in Synechocystis sp. PCC6803 (Chatterjee et al. in Planta 218:989–998, 2004). One of the forms with a ~50 kDa subunit has been found to be coded by an as yet unassigned ORF, sll1722. In the present study we have purified the second isoform of MIPS as a ~65 kDa protein from the crude extract of Synechocystis sp. PCC6803 to apparent homogeneity and biochemically characterized. MALDI-TOF analysis of the 65 kDa protein led to its identification as acetolactate synthase large subunit (EC 2.2.1.6; ALS), the putatively assigned ORF sll1981 of Synechocystis sp. PCC6803. The PCR amplified ~1.6 kb product of sll1981 was found to functionally complement the yeast inositol auxotroph, FY250 and could be expressed as an immunoreactive ~65 kDa MIPS protein in the natural inositol auxotroph, Schizosaccharomyces pombe. In vitro MIPS activity and cross reactivity against MIPS antibody of purified recombinant sll1981 further consolidated its identity as the second probable MIPS gene in Synechocystis sp. PCC6803. Sequence comparison along with available crystal structure analysis of the yeast MIPS reveals conservation of several amino acids in sll1981 essential for substrate and co-factor binding. Comparison with other prokaryotic and eukaryotic MIPS sequences and phylogenetic analysis, however, revealed that like sll1722, sll1981 is quite divergent from others. It is probable that sll1981 may code for a bifunctional enzyme protein having conserved domains for both MIPS and acetolactate synthase (ALS) activities.Anirban Chatterjee and Krishnarup Ghosh Dastidar contributed equally.     

Metastases suppressor NME2 associates with telomere ends and telomerase and reduces telomerase activity within cells

Analysis of chromatin-immunoprecipitation followed by sequencing (ChIP-seq) usually disregards sequence reads that do not map within binding positions (peaks). Using an unbiased approach, we analysed all reads, both that mapped and ones that were not included as part of peaks. ChIP-seq experiments were performed in human lung adenocarcinoma and fibrosarcoma cells for the metastasis suppressor non-metastatic 2 (NME2). Surprisingly, we identified sequence reads that uniquely represented human telomere ends in both cases. In vivo presence of NME2 at telomere ends was validated using independent methods and as further evidence we found intranuclear association of NME2 and the telomere repeat binding factor 2. Most remarkably, results demonstrate that NME2 associates with telomerase and reduces telomerase activity in vitro and in vivo, and sustained NME2 expression resulted in reduced telomere length in aggressive human cancer cells. Anti-metastatic function of NME2 has been demonstrated in human cancers, however, mechanisms are poorly understood. Together, findings reported here suggest a novel role for NME2 as a telomere binding protein that can alter telomerase function and telomere length. This presents an opportunity to investigate telomere-related interactions in metastasis suppression.     

Dissecting the protein-RNA interface: the role of protein surface shapes and RNA secondary structures in protein-RNA recognition

Protein-RNA interactions are essential for many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. Here, we analyzed the protein surface shape (dented, intermediate or protruded) and the RNA base pairing properties (paired or unpaired nucleotides) at the interfaces of 91 protein-RNA complexes derived from the Protein Data Bank. Dented protein surfaces prefer unpaired nucleotides to paired ones at the interface, and hydrogen bonds frequently occur between the protein backbone and RNA bases. In contrast, protruded protein surfaces do not show such a preference, rather, electrostatic interactions initiate the formation of hydrogen bonds between positively charged amino acids and RNA phosphate groups. Interestingly, in many protein-RNA complexes that interact via an RNA loop, an aspartic acid is favored at the interface. Moreover, in most of these complexes, nucleotide bases in the RNA loop are flipped out and form hydrogen bonds with the protein, which suggests that aspartic acid is important for RNA loop recognition through a base-flipping process. This study provides fundamental insights into the role of the shape of the protein surface and RNA secondary structures in mediating protein-RNA interactions.     

Role of SNAP25 Explored in Eastern Indian Attention Deficit Hyperactivity Disorder Probands

Synaptosomal-associated protein 25 (SNAP25) is an essential component for synaptic vesicle mediated release of neurotransmitters. Deficiencies or abnormal structure or function of SNAP25 protein, possibly arising through genetic variations in the relevant DNA code, has been suggested to play role in the pathology of several neurobehavioural disorders including Attention deficit Hyperactivity Disorder (ADHD) and a number of polymorphisms in the SNAP25 gene has been studied for association with the disorder. In the present investigation, for the first time association between ADHD and six SNAP25 polymorphisms, rs1889189, rs362569, rs362988, rs3746544, rs1051312, and rs8636 was explored in eastern Indian population. Subjects were recruited following the Diagnostic and Statistical Manual for Mental Disorders-IV. Genomic DNA isolated from peripheral blood leukocytes of ADHD probands (n = 150), their parents (n = 272) and ethnically matched controls (n = 100) was used for amplifying target sites. Data obtained were subjected to population- as well as family-based analyses. While case–control analysis revealed lack of any significant difference for alleles, family-based studies revealed a mild over transmission rs3746544 ‘T’ and rs8636 ‘C’ alleles (P = 0.05 and 0.03 respectively). Haplotypes formed between rs362569 “T”, 362988 “G”, rs3746544 “T”, rs1051312 “T” and rs8636 “C” in different combinations showed statistically significant transmission to ADHD probands. Excepting rs3746544 and rs8636, all the tested sites showed very low linkage disequilibrium between them. Data obtained in this preliminary study indicates that rs3746544 ‘T’ allele may have some role in the disease etiology in the studied Indian population.