Diversity in Functional Organization of Class I and Class II Biotin Protein Ligase

The cell envelope of Mycobacterium tuberculosis (M. tuberculosis) is composed of a variety of lipids including mycolic acids, sulpholipids, lipoarabinomannans, etc., which impart rigidity crucial for its survival and pathogenesis. Acyl CoA carboxylase (ACC) provides malonyl-CoA and methylmalonyl-CoA, committed precursors for fatty acid and essential for mycolic acid synthesis respectively. Biotin Protein Ligase (BPL/BirA) activates apo-biotin carboxyl carrier protein (BCCP) by biotinylating it to an active holo-BCCP. A minimal peptide (Schatz), an efficient substrate for Escherichia coli BirA, failed to serve as substrate for M. tuberculosis Biotin Protein Ligase (MtBPL). MtBPL specifically biotinylates homologous BCCP domain, MtBCCP87, but not EcBCCP87. This is a unique feature of MtBPL as EcBirA lacks such a stringent substrate specificity. This feature is also reflected in the lack of self/promiscuous biotinylation by MtBPL. The N-terminus/HTH domain of EcBirA has the selfbiotinable lysine residue that is inhibited in the presence of Schatz peptide, a peptide designed to act as a universal acceptor for EcBirA. This suggests that when biotin is limiting, EcBirA preferentially catalyzes, biotinylation of BCCP over selfbiotinylation. R118G mutant of EcBirA showed enhanced self and promiscuous biotinylation but its homologue, R69A MtBPL did not exhibit these properties. The catalytic domain of MtBPL was characterized further by limited proteolysis. Holo-MtBPL is protected from proteolysis by biotinyl-59 AMP, an intermediate of MtBPL catalyzed reaction. In contrast, apo-MtBPL is completely digested by trypsin within 20 min of co-incubation. Substrate selectivity and inability to promote self biotinylation are exquisite features of MtBPL and are a consequence of the unique molecular mechanism of an enzyme adapted for the high turnover of fatty acid biosynthesis.

Identification of Local Conformational Similarity in Structurally Variable Regions of Homologous Proteins Using Protein Blocks

Structure comparison tools can be used to align related protein structures to identify structurally conserved and variable regions and to infer functional and evolutionary relationships. While the conserved regions often superimpose well, the variable regions appear non superimposable. Differences in homologous protein structures are thought to be due to evolutionary plasticity to accommodate diverged sequences during evolution. One of the kinds of differences between 3-D structures of homologous proteins is rigid body displacement. A glaring example is not well superimposed equivalent regions of homologous proteins corresponding to a-helical conformation with different spatial orientations. In a rigid body superimposition, these regions would appear variable although they may contain local similarity. Also, due to high spatial deviation in the variable region, one-to-one correspondence at the residue level cannot be determined accurately. Another kind of difference is conformational variability and the most common example is topologically equivalent loops of two homologues but with different conformations. In the current study, we present a refined view of the ``structurally variable'' regions which may contain local similarity obscured in global alignment of homologous protein structures. As structural alphabet is able to describe local structures of proteins precisely through Protein Blocks approach, conformational similarity has been identified in a substantial number of `variable' regions in a large data set of protein structural alignments; optimal residue-residue equivalences could be achieved on the basis of Protein Blocks which led to improved local alignments. Also, through an example, we have demonstrated how the additional information on local backbone structures through protein blocks can aid in comparative modeling of a loop region. In addition, understanding on sequence-structure relationships can be enhanced through our approach. This has been illustrated through examples where the equivalent regions in homologous protein structures share sequence similarity to varied extent but do not preserve local structure.

Calcium/Calmodulin Dependent Protein Kinase II Bound to NMDA Receptor 2B Subunit Exhibits Increased ATP Affinity and Attenuated Dephosphorylation

Calcium/calmodulin dependent protein kinase II (CaMKII) is implicated to play a key role in learning and memory. NR2B subunit of N-methyl-D-aspartate receptor (NMDAR) is a high affinity binding partner of CaMKII at the postsynaptic membrane. NR2B binds to the T-site of CaMKII and modulates its catalysis. By direct measurement using isothermal titration calorimetry (ITC), we show that NR2B binding causes about 11 fold increase in the affinity of CaMKII for ATP gamma S, an analogue of ATP. ITC data is also consistent with an ordered binding mechanism for CaMKII with ATP binding the catalytic site first followed by peptide substrate. We also show that dephosphorylation of phospho-Thr(286)-alpha-CaMKII is attenuated when NR2B is bound to CaMKII. This favors the persistence of Thr(286) autophosphorylated state of CaMKII in a CaMKII/phosphatase conjugate system in vitro. Overall our data indicate that the NR2B- bound state of CaMKII attains unique biochemical properties which could help in the efficient functioning of the proposed molecular switch supporting synaptic memory.

Scroll-Wave Dynamics in Human Cardiac Tissue: Lessons from a Mathematical Model with Inhomogeneities and Fiber Architecture

Cardiac arrhythmias, such as ventricular tachycardia (VT) and ventricular fibrillation (VF), are among the leading causes of death in the industrialized world. These are associated with the formation of spiral and scroll waves of electrical activation in cardiac tissue; single spiral and scroll waves are believed to be associated with VT whereas their turbulent analogs are associated with VF. Thus, the study of these waves is an important biophysical problem. We present a systematic study of the combined effects of muscle-fiber rotation and inhomogeneities on scroll-wave dynamics in the TNNP (ten Tusscher Noble Noble Panfilov) model for human cardiac tissue. In particular, we use the three-dimensional TNNP model with fiber rotation and consider both conduction and ionic inhomogeneities. We find that, in addition to displaying a sensitive dependence on the positions, sizes, and types of inhomogeneities, scroll-wave dynamics also depends delicately upon the degree of fiber rotation. We find that the tendency of scroll waves to anchor to cylindrical conduction inhomogeneities increases with the radius of the inhomogeneity. Furthermore, the filament of the scroll wave can exhibit drift or meandering, transmural bending, twisting, and break-up. If the scroll-wave filament exhibits weak meandering, then there is a fine balance between the anchoring of this wave at the inhomogeneity and a disruption of wave-pinning by fiber rotation. If this filament displays strong meandering, then again the anchoring is suppressed by fiber rotation; also, the scroll wave can be eliminated from most of the layers only to be regenerated by a seed wave. Ionic inhomogeneities can also lead to an anchoring of the scroll wave; scroll waves can now enter the region inside an ionic inhomogeneity and can display a coexistence of spatiotemporal chaos and quasi-periodic behavior in different parts of the simulation domain. We discuss the experimental implications of our study.

A Ten-microRNA Expression Signature Predicts Survival in Glioblastoma

Glioblastoma (GBM) is the most common and aggressive primary brain tumor with very poor patient median survival. To identify a microRNA (miRNA) expression signature that can predict GBM patient survival, we analyzed the miRNA expression data of GBM patients (n = 222) derived from The Cancer Genome Atlas (TCGA) dataset. We divided the patients randomly into training and testing sets with equal number in each group. We identified 10 significant miRNAs using Cox regression analysis on the training set and formulated a risk score based on the expression signature of these miRNAs that segregated the patients into high and low risk groups with significantly different survival times (hazard ratio HR] = 2.4; 95% CI = 1.4-3.8; p < 0.0001). Of these 10 miRNAs, 7 were found to be risky miRNAs and 3 were found to be protective. This signature was independently validated in the testing set (HR = 1.7; 95% CI = 1.1-2.8; p = 0.002). GBM patients with high risk scores had overall poor survival compared to the patients with low risk scores. Overall survival among the entire patient set was 35.0% at 2 years, 21.5% at 3 years, 18.5% at 4 years and 11.8% at 5 years in the low risk group, versus 11.0%, 5.5%, 0.0 and 0.0% respectively in the high risk group (HR = 2.0; 95% CI = 1.4-2.8; p < 0.0001). Cox multivariate analysis with patient age as a covariate on the entire patient set identified risk score based on the 10 miRNA expression signature to be an independent predictor of patient survival (HR = 1.120; 95% CI = 1.04-1.20; p = 0.003). Thus we have identified a miRNA expression signature that can predict GBM patient survival. These findings may have implications in the understanding of gliomagenesis, development of targeted therapy and selection of high risk cancer patients for adjuvant therapy.

Angelman Syndrome Protein UBE3A Interacts with Primary Microcephaly Protein ASPM, Localizes to Centrosomes and Regulates Chromosome Segregation

Many proteins associated with the phenotype microcephaly have been localized to the centrosome or linked to it functionally. All the seven autosomal recessive primary microcephaly (MCPH) proteins localize at the centrosome. Microcephalic osteodysplastic primordial dwarfism type II protein PCNT and Seckel syndrome (also characterized by severe microcephaly) protein ATR are also centrosomal proteins. All of the above findings show the importance of centrosomal proteins as the key players in neurogenesis and brain development. However, the exact mechanism as to how the loss-of-function of these proteins leads to microcephaly remains to be elucidated. To gain insight into the function of the most commonly mutated MCPH gene ASPM, we used the yeast two-hybrid technique to screen a human fetal brain cDNA library with an ASPM bait. The analysis identified Angelman syndrome gene product UBE3A as an ASPM interactor. Like ASPM, UBE3A also localizes to the centrosome. The identification of UBE3A as an ASPM interactor is not surprising as more than 80% of Angelman syndrome patients have microcephaly. However, unlike in MCPH, microcephaly is postnatal in Angelman syndrome patients. Our results show that UBE3A is a cell cycle regulated protein and its level peaks in mitosis. The shRNA knockdown of UBE3A in HEK293 cells led to many mitotic abnormalities including chromosome missegregation, abnormal cytokinesis and apoptosis. Thus our study links Angelman syndrome protein UBE3A to ASPM, centrosome and mitosis for the first time. We suggest that a defective chromosome segregation mechanism is responsible for the development of microcephaly in Angelman syndrome.

Estimating the Threshold Surface Density of Gp120-CCR5 Complexes Necessary for HIV-1 Envelope-Mediated Cell-Cell Fusion

Reduced expression of CCR5 on target CD4(+) cells lowers their susceptibility to infection by R5-tropic HIV-1, potentially preventing transmission of infection and delaying disease progression. Binding of the HIV-1 envelope (Env) protein gp120 with CCR5 is essential for the entry of R5 viruses into target cells. The threshold surface density of gp120-CCR5 complexes that enables HIV-1 entry remains poorly estimated. We constructed a mathematical model that mimics Env-mediated cell-cell fusion assays, where target CD4(+)CCR5(+) cells are exposed to effector cells expressing Env in the presence of a coreceptor antagonist and the fraction of target cells fused with effector cells is measured. Our model employs a reaction network-based approach to describe protein interactions that precede viral entry coupled with the ternary complex model to quantify the allosteric interactions of the coreceptor antagonist and predicts the fraction of target cells fused. By fitting model predictions to published data of cell-cell fusion in the presence of the CCR5 antagonist vicriviroc, we estimated the threshold surface density of gp120-CCR5 complexes for cell-cell fusion as similar to 20 mu m(-2). Model predictions with this threshold captured data from independent cell-cell fusion assays in the presence of vicriviroc and rapamycin, a drug that modulates CCR5 expression, as well as assays in the presence of maraviroc, another CCR5 antagonist, using sixteen different Env clones derived from transmitted or early founder viruses. Our estimate of the threshold surface density of gp120-CCR5 complexes necessary for HIV-1 entry thus appears robust and may have implications for optimizing treatment with coreceptor antagonists, understanding the non-pathogenic infection of non-human primates, and designing vaccines that suppress the availability of target CD4(+)CCR5(+) cells.

Gene Expression Profiling of Preovulatory Follicle in the Buffalo Cow: Effects of Increased IGF-I Concentration on Periovulatory Events

The preovulatory follicle in response to gonadotropin surge undergoes dramatic biochemical, and morphological changes orchestrated by expression changes in hundreds of genes. Employing well characterized bovine preovulatory follicle model, granulosa cells (GCs) and follicle wall were collected from the preovulatory follicle before, 1, 10 and 22 h post peak LH surge. Microarray analysis performed on GCs revealed that 450 and 111 genes were differentially expressed at 1 and 22 h post peak LH surge, respectively. For validation, qPCR and immunocytochemistry analyses were carried out for some of the differentially expressed genes. Expression analysis of many of these genes showed distinct expression patterns in GCs and the follicle wall. To study molecular functions and genetic networks, microarray data was analyzed using Ingenuity Pathway Analysis which revealed majority of the differentially expressed genes to cluster within processes like steroidogenesis, cell survival and cell differentiation. In the ovarian follicle, IGF-I is established to be an important regulator of the above mentioned molecular functions. Thus, further experiments were conducted to verify the effects of increased intrafollicular IGF-I levels on the expression of genes associated with the above mentioned processes. For this purpose, buffalo cows were administered with exogenous bGH to transiently increase circulating and intrafollicular concentrations of IGF-I. The results indicated that increased intrafollicular concentrations of IGF-I caused changes in expression of genes associated with steroidogenesis (StAR, SRF) and apoptosis (BCL-2, FKHR, PAWR). These results taken together suggest that onset of gonadotropin surge triggers activation of various biological pathways and that the effects of growth factors and peptides on gonadotropin actions could be examined during preovulatory follicle development.

Insights into the Regulatory Characteristics of the Mycobacterial Dephosphocoenzyme A Kinase: Implications for the Universal CoA Biosynthesis Pathway

Being vastly different from the human counterpart, we suggest that the last enzyme of the Mycobacterium tuberculosis Coenzyme A biosynthetic pathway, dephosphocoenzyme A kinase (CoaE) could be a good anti-tubercular target. Here we describe detailed investigations into the regulatory features of the enzyme, affected via two mechanisms. Enzymatic activity is regulated by CTP which strongly binds the enzyme at a site overlapping that of the leading substrate, dephosphocoenzyme A (DCoA), thereby obscuring the binding site and limiting catalysis. The organism has evolved a second layer of regulation by employing a dynamic equilibrium between the trimeric and monomeric forms of CoaE as a means of regulating the effective concentration of active enzyme. We show that the monomer is the active form of the enzyme and the interplay between the regulator, CTP and the substrate, DCoA, affects enzymatic activity. Detailed kinetic data have been corroborated by size exclusion chromatography, dynamic light scattering, glutaraldehyde crosslinking, limited proteolysis and fluorescence investigations on the enzyme all of which corroborate the effects of the ligands on the enzyme oligomeric status and activity. Cysteine mutagenesis and the effects of reducing agents on mycobacterial CoaE oligomerization further validate that the latter is not cysteine-mediated or reduction-sensitive. These studies thus shed light on the novel regulatory features employed to regulate metabolite flow through the last step of a critical biosynthetic pathway by keeping the latter catalytically dormant till the need arises, the transition to the active form affected by a delicate crosstalk between an essential cellular metabolite (CTP) and the precursor to the pathway end-product (DCoA).

A Transcript Cleavage Factor of Mycobacterium tuberculosis Important for Its Survival

After initiation of transcription, a number of proteins participate during elongation and termination modifying the properties of the RNA polymerase (RNAP). Gre factors are one such group conserved across bacteria. They regulate transcription by projecting their N-terminal coiled-coil domain into the active center of RNAP through the secondary channel and stimulating hydrolysis of the newly synthesized RNA in backtracked elongation complexes. Rv1080c is a putative gre factor (MtbGre) in the genome of Mycobacterium tuberculosis. The protein enhanced the efficiency of promoter clearance by lowering abortive transcription and also rescued arrested and paused elongation complexes on the GC rich mycobacterial template. Although MtbGre is similar in domain organization and shares key residues for catalysis and RNAP interaction with the Gre factors of Escherichia coli, it could not complement an E. coli gre deficient strain. Moreover, MtbGre failed to rescue E. coli RNAP stalled elongation complexes, indicating the importance of specific protein-protein interactions for transcript cleavage. Decrease in the level of MtbGre reduced the bacterial survival by several fold indicating its essential role in mycobacteria. Another Gre homolog, Rv3788 was not functional in transcript cleavage activity indicating that a single Gre is sufficient for efficient transcription of the M. tuberculosis genome.

DNA analysis indicates that Asian elephants are native to Borneo and are therefore a high priority for conservation

The origin of Borneo's elephants is controversial. Two competing hypotheses argue that they are either indigenous, tracing back to the Pleistocene, or were introduced, descending from elephants imported in the 16th-18th centuries. Taxonomically, they have either been classified as a unique subspecies or placed under the Indian or Sumatran subspecies. If shown to be a unique indigenous population, this would extend the natural species range of the Asian elephant by 1300 km, and therefore Borneo elephants would have much greater conservation importance than if they were a feral population. We compared DNA of Borneo elephants to that of elephants from across the range of the Asian elephant, using a fragment of mitochondrial DNA, including part of the hypervariable d-loop, and five autosomal microsatellite loci. We find that Borneo's elephants are genetically distinct, with molecular divergence indicative of a Pleistocene colonisation of Borneo and subsequent isolation. We reject the hypothesis that Borneo's elephants were introduced. The genetic divergence of Borneo elephants warrants their recognition as a separate evolutionary significant unit. Thus, interbreeding Borneo elephants with those from other populations would be contraindicated in ex situ conservation, and their genetic distinctiveness makes them one of the highest priority populations for Asian elephant conservation.

Recombination Drives Genetic Diversification of Streptococcus dysgalactiae Subspecies equisimilis in a Region of Streptococcal Endemicity

Infection of the skin or throat by Streptococcus dysgalactiae subspecies equisimilis (SDSE) may result in a number of human diseases. To understand mechanisms that give rise to new genetic variants in this species, we used multi-locus sequence typing (MLST) to characterise relationships in the SDSE population from India, a country where streptococcal disease is endemic. The study revealed Indian SDSE isolates have sequence types (STs) predominantly different to those reported from other regions of the world. Emm-ST combinations in India are also largely unique. Split decomposition analysis, the presence of emm-types in unrelated clonal complexes, and analysis of phylogenetic trees based on concatenated sequences all reveal an extensive history of recombination within the population. The ratio of recombination to mutation (r/m) events (11:1) and per site r/m ratio (41:1) in this population is twice as high as reported for SDSE from non-endemic regions. Recombination involving the emm-gene is also more frequent than recombination involving housekeeping genes, consistent with diversification of M proteins offering selective advantages to the pathogen. Our data demonstrate that genetic recombination in endemic regions is more frequent than non-endemic regions, and gives rise to novel local SDSE variants, some of which may have increased fitness or pathogenic potential.

Nature's Swiss Army Knives: Ovipositor Structure Mirrors Ecology in a Multitrophic Fig Wasp Community

Background: Resource partitioning is facilitated by adaptations along niche dimensions that range from morphology to behaviour. The exploitation of hidden resources may require specially adapted morphological or sensory tools for resource location and utilisation. Differences in tool diversity and complexity can determine not only how many species can utilize these hidden resources but also how they do so. Methodology and Principal Findings: The sclerotisation, gross morphology and ultrastructure of the ovipositors of a seven-member community of parasitic wasps comprising of gallers and parasitoids developing within the globular syconia (closed inflorescences) of Ficus racemosa (Moraceae) was investigated. These wasps also differ in their parasitism mode (external versus internal oviposition) and their timing of oviposition into the expanding syconium during its development. The number and diversity of sensilla, as well as ovipositor teeth, increased from internally ovipositing to externally ovipositing species and from gallers to parasitoids. The extent of sclerotisation of the ovipositor tip matched the force required to penetrate the syconium at the time of oviposition of each species. The internally ovipositing pollinator had only one type of sensillum and a single notch on the ovipositor tip. Externally ovipositing species had multiple sensilla types and teeth on their ovipositors. Chemosensilla were most concentrated at ovipositor tips while mechanoreceptors were more widely distributed, facilitating the precise location of hidden hosts in these wasps which lack larval host-seeking behaviour. Ovipositor traits of one parasitoid differed from those of its syntopic galler congeners and clustered with those of parasitoids within a different wasp subfamily. Thus ovipositor tools can show lability based on adaptive necessity, and are not constrained by phylogeny. Conclusions/Significance: Ovipositor structure mirrored the increasingly complex trophic ecology and requirements for host accessibility in this parasite community. Ovipositor structure could be a useful surrogate for predicting the biology of parasites in other communities.

Modulation of Catalytic Activity in Multi-Domain Protein Tyrosine Phosphatases

Signaling mechanisms involving protein tyrosine phosphatases govern several cellular and developmental processes. These enzymes are regulated by several mechanisms which include variation in the catalytic turnover rate based on redox stimuli, subcellular localization or protein-protein interactions. In the case of Receptor Protein Tyrosine Phosphatases (RPTPs) containing two PTP domains, phosphatase activity is localized in their membrane-proximal (D1) domains, while the membrane-distal (D2) domain is believed to play a modulatory role. Here we report our analysis of the influence of the D2 domain on the catalytic activity and substrate specificity of the D1 domain using two Drosophila melanogaster RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of Drosophila Leukocyte antigen Related (DLAR) and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A). While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A reveal a conformational rationale for the experimental observations. These studies reveal that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain.

Clinical Proteomics of the Neglected Human Malarial Parasite Plasmodium vivax

Recent reports highlight the severity and the morbidity of disease caused by the long neglected malaria parasite Plasmodium vivax. Due to inherent difficulties in the laboratory-propagation of P. vivax, the biology of this parasite has not been adequately explored. While the proteome of P. falciparum, the causative agent of cerebral malaria, has been extensively explored from several sources, there is limited information on the proteome of P. vivax. We have, for the first time, examined the proteome of P. vivax isolated directly from patients without adaptation to laboratory conditions. We have identified 153 proteins from clinical P. vivax, majority of which do not show homology to any previously known gene products. We also report 29 new proteins that were found to be expressed in P. vivax for the first time. In addition, several proteins previously implicated as anti-malarial targets, were also found in our analysis. Most importantly, we found several unique proteins expressed by P. vivax. This study is an important step in providing insight into physiology of the parasite under clinical settings.