A fatty acid elongase ELO with novel activity from Dictyostelium discoideum

Fatty acid elongation was examined in the cellular slime mold, Dictyostelium discoideum. Profiling of the total fatty acid content of D. discoideum indicated that fatty acid elongation is active. Orthologs of the fatty acid elongase ELO family were identified in the D. discoideum genome and the cDNA for one, eloA, was cloned and functionally characterized by expression in yeast. EloA is a highly active ELO with strict substrate specificity for monounsaturated fatty acids, in particular 16:1^Δ9 to produce the unusual 18:1^Δ11 fatty acid. This is the first report on fatty acid elongation in a cellular slime mold.     

Protein kinase C-theta-mediated signals enhance CD4+ T cell survival by up-regulating Bcl-xL

Productive engagement of TCR results in delivering signals required for T cell proliferation as well as T cell survival. Blocking TCR-mediated survival signals, T cells undergo apoptosis instead of proliferation upon TCR stimulation. During the activation process, T cells produce IL-2, which acts as an extrinsic survival factor. In addition, TCR stimulation results in up-regulation of Bcl-xL to enhance T cell survival intrinsically. We show in this study that protein kinase C (PKC)-theta is required for enhancing the survival of activated CD4+ T cells by up-regulating Bcl-xL. In response to TCR stimulation, CD4+ PKC-theta-/- T cells failed to up-regulate Bcl-xL, and underwent accelerated apoptosis via a caspase- and mitochondria-dependent pathway. Similar to PKC-theta-deficient primary CD4+ T cells, small interfering RNA-mediated knockdown of PKC-theta in Jurkat cells also resulted in apoptosis upon TCR stimulation. Forced expression of Bcl-xL was sufficient to inhibit apoptosis observed in PKC-theta knockdown cells. Furthermore, ectopic expression of PKC-theta stimulated a reporter gene driven by a mouse Bcl-xL promoter. Whereas an inactive form of PKC-theta or knockdown of endogenous PKC-theta led to inhibition of Bcl-xL reporter. PKC-theta-mediated activation of Bcl-xL reporter was inhibited by dominant-negative IkappaB kinase beta or dominant-negative AP-1. Thus, the PKC-theta-mediated signals may function not only in the initial activation of naive CD4+ T cells, but also in their survival during T cell activation by regulating Bcl-xL levels through NF-kappaB and AP-1 pathways.     

Genetic diversity in two leading Plasmodium vivax malaria vaccine candidates AMA1 and MSP119 at three sites in India

Plasmodium vivax, a major contributor to the malaria burden in India, has the broadest geographic distribution and shows higher genetic diversity than P. falciparum. Here, we investigated the genetic diversity of two leading P. vivax vaccine candidate antigens, at three geographically diverse malaria-endemic regions in India. Pvama1 and Pvmsp1₁₉ partial coding sequences were generated from one hundred P. vivax isolates in India (Chennai n = 28, Nadiad n = 50 and Rourkela n = 22) and ~1100 published sequences from Asia, South America, North America, and Oceania regions included. These data were used to assess the genetic diversity and potential for vaccine candidacy of both antigens on a global scale. A total of 44 single nucleotide polymorphism (SNPs) were identified among 100 Indian Pvama1 sequences, including 10 synonymous and 34 nonsynonymous mutations. Nucleotide diversity was higher in Rourkela and Nadiad as compared to Chennai. Nucleotide diversity measures showed a strong balancing selection in Indian and global population for domain I of Pvama1, which suggests that it is a dominant target of the protective immune response. In contrast, the Pvmsp1₁₉ region showed highly conserved sequences in India and across the Oceania, South America, North America and Asia, demonstrating low genetic diversity in the global population when compared to Pvama1. Results suggest the possibility of including Pvmsp1₁₉ in a multivalent vaccine formulation against P. vivax infections. However, the high genetic diversity seen in Pvama1 would be more challenging for vaccine development.     

Protein chaperones Q8ZP25_SALTY from Salmonella typhimurium and HYAE_ECOLI from Escherichia coli exhibit thioredoxin-like structures despite lack of canonical thioredoxin active site sequence motif

The structure of the 142-residue protein Q8ZP25_SALTY encoded in the genome of Salmonella typhimurium LT2 was determined independently by NMR and X-ray crystallography, and the structure of the 140-residue protein HYAE_ECOLI encoded in the genome of Escherichia coli was determined by NMR. The two proteins belong to Pfam (Finn et al. 34:D247–D251, 2006) PF07449, which currently comprises 50 members, and belongs itself to the ‘thioredoxin-like clan’. However, protein HYAE_ECOLI and the other proteins of Pfam PF07449 do not contain the canonical Cys-X-X-Cys active site sequence motif of thioredoxin. Protein HYAE_ECOLI was previously classified as a [NiFe] hydrogenase-1 specific chaperone interacting with the twin-arginine translocation (Tat) signal peptide. The structures presented here exhibit the expected thioredoxin-like fold and support the view that members of Pfam family PF07449 specifically interact with Tat signal peptides.     

History cleans up messes: The impact of time in driving divergence and introgression in a tropical suture zone

Contact zones provide an excellent arena in which to address questions about how genomic divergence evolves during lineage divergence. They allow us to both infer patterns of genomic divergence in allopatric populations isolated from introgression and to characterize patterns of introgression after lineages meet. Thusly motivated, we analyze genome‐wide introgression data from four contact zones in three genera of lizards endemic to the Australian Wet Tropics. These contact zones all formed between morphologically cryptic lineage‐pairs within morphologically defined species, and the lineage‐pairs meeting in the contact zones diverged anywhere from 3.1 to 5.8 million years ago. By characterizing patterns of molecular divergence across an average of 11K genes and fitting geographic clines to an average of 7.5K variants, we characterize how patterns of genomic differentiation and introgression change through time. Across this range of divergences, we find that genome‐wide differentiation increases but becomes no less heterogeneous. In contrast, we find that introgression heterogeneity decreases dramatically, suggesting that time helps isolated genomes “congeal.” Thus, this work emphasizes the pivotal role that history plays in driving lineage divergence.     

Role of exchange protein directly activated by cAMP (EPAC1) in breast cancer cell migration and apoptosis

Despite the current progress in cancer research and therapy, breast cancer remains the leading cause of mortality among half a million women worldwide. Migration and invasion of cancer cells are associated with prevalent tumor metastasis as well as high mortality. Extensive studies have powerfully established the role of prototypic second messenger cAMP and its two ubiquitously expressed intracellular cAMP receptors namely the classic protein kinaseA/cAMP-dependent protein kinase (PKA) and the more recently discovered exchange protein directly activated by cAMP/cAMP-regulated guanine nucleotide exchange factor (EPAC/cAMP-GEF) in cell migration, cell cycle regulation, and cell death. Herein, we performed the analysis of the Cancer Genome Atlas (TCGA) dataset to evaluate the essential role of cAMP molecular network in breast cancer. We report that EPAC1, PKA, and AKAP9 along with other molecular partners are amplified in breast cancer patients, indicating the importance of this signaling network. To evaluate the functional role of few of these proteins, we used pharmacological modulators and analyzed their effect on cell migration and cell death in breast cancer cells. Hence, we report that inhibition of EPAC1 activity using pharmacological modulators leads to inhibition of cell migration and induces cell death. Additionally, we also observed that the inhibition of EPAC1 resulted in disruption of its association with the microtubule cytoskeleton and delocalization of AKAP9 from the centrosome as analyzed by in vitro imaging. Finally, this study suggests for the first time the mechanistic insights of mode of action of a primary cAMP-dependent sensor, Exchange protein activated by cAMP 1 (EPAC1), via its interaction with A-kinase anchoring protein 9 (AKAP9). This study provides a new cell signaling cAMP–EPAC1–AKAP9 direction to the development of additional biotherapeutics for breast cancer.     

Dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in store-operated calcium influx. Evidence for similarities in store-operated and calcium release-activated calcium channel components

Store-operated calcium entry (SOCE) is a ubiquitous mechanism that is mediated by distinct SOC channels, ranging from the highly selective calcium release-activated Ca2+ (CRAC) channel in rat basophilic leukemia and other hematopoietic cells to relatively Ca2+-selective or non-selective SOC channels in other cells. Although the exact composition of these channels is not yet established, TRPC1 contributes to SOC channels and regulation of physiological function of a variety of cell types. Recently, Orai1 and STIM1 have been suggested to be sufficient for generating CRAC channels. Here we show that Orai1 and STIM1 are also required for TRPC1-SOC channels. Knockdown of TRPC1, Orai1, or STIM1 attenuated, whereas overexpression of TRPC1, but not Orai1 or STIM1, induced an increase in SOC entry and I(SOC) in human salivary gland cells. All three proteins were co-localized in the plasma membrane region of cells, and thapsigargin increased co-immunoprecipitation of TRPC1 with STIM1, and Orai1 in human salivary gland cells as well as dispersed mouse submandibular gland cells. In aggregate, the data presented here reveal that all three proteins are essential for generation of I(SOC) in these cells and that dynamic assembly of TRPC1-STIM1-Orai1 ternary complex is involved in activation of SOC channel in response to internal Ca2+ store depletion. Thus, these data suggest a common molecular basis for SOC and CRAC channels.     

Functional properties of the Drosophila melanogaster inositol 1,4,5-trisphosphate receptor mutants

The inositol (1,4,5)-trisphosphate receptor (InsP(3)R) is an intracellular calcium (Ca(2+)) release channel that plays a crucial role in cell signaling. In Drosophila melanogaster a single InsP(3)R gene (itpr) encodes a protein (DmInsP(3)R) that is approximately 60% conserved with mammalian InsP(3)Rs. A number of itpr mutant alleles have been identified in genetic screens and studied for their effect on development and physiology. However, the functional properties of wild-type or mutant DmInsP(3)Rs have never been described. Here we use the planar lipid bilayer reconstitution technique to describe single-channel properties of embryonic and adult head DmInsP(3)R splice variants. The three mutants chosen in this study reside in each of the three structural domains of the DmInsP(3)R-the amino-terminal ligand binding domain (ug3), the middle-coupling domain (wc703), and the channel-forming region (ka901). We discovered that 1), the major functional properties of DmInsP(3)R (conductance, gating, and sensitivity to InsP(3) and Ca(2+)) are remarkably conserved with the mammalian InsP(3)R1; 2), single-channel conductance of the adult head DmInsP(3)R isoform is 89 pS and the embryonic DmInsP(3)R isoform is 70 pS; 3), ug3 mutation affects sensitivity of the DmInsP(3)Rs to activation by InsP(3), but not their InsP(3)-binding properties; 4), wc703 channels have increased sensitivity to modulation by Ca(2+); and 5), homomeric ka901 channels are not functional. We correlated the results obtained in planar lipid bilayer experiments with measurements of InsP(3)-induced Ca(2+) fluxes in microsomes isolated from wild-type and heterozygous itpr mutants. Our study validates the use of D. melanogaster as an appropriate model for InsP(3)R structure-function studies and provides novel insights into the fundamental mechanisms of the InsP(3)R function.     

Association of AGTR1 (A1166C) and ACE (I/D) Polymorphisms with Breast Cancer Risk in North Indian Population

Renin angiotensin system (RAS) comprising Angiotensin converting enzyme (ACE), Angiotensin II (Ang II) and its receptor Angiotensin II receptor type I (AGTR1), plays a critical role in several diseases including cancer. A single nucleotide polymorphism (SNP) A1166C located in 3′ untranslated region (UTR) of AGTR1 and an insertion/deletion (I/D) polymorphism present in intron 16 of ACE gene have been associated with many diseases, but their association with Breast cancer (BCa) is still debatable. Here, we for the first time investigated the association of these polymorphisms in a North Indian BCa cohort including 161 patients and 152 healthy women. The polymorphisms were evaluated by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) respectively. The association between these polymorphisms and BCa risk was estimated by calculating Odds Ratio (OR) and chi-square (χ²) test. The DD genotype/D allele of ACE (I/D) polymorphism and “AC and CC” genotype/C allele of AGTR1 (A1166C) polymorphism were associated with higher risk of BCa when evaluated independently. Furthermore, interaction analysis of “AC and CC” and DD genotype and combination of “C and D” alleles of both polymorphisms revealed significantly greater BCa risk than that observed independently. Conclusively, women harboring “AC or CC” genotype/C allele for AGTR1 (A1166C) polymorphism and DD genotype/D allele for ACE (I/D) polymorphisms have a predisposition to develop more aggressive disease with advanced staging and larger tumor size. Our study indicates importance of genetic screening based on these polymorphisms for women, who may have higher risk of BCa.