Sree V. Chintapalli

The BRCA1 tumor suppressor binds to inositol 1,4,5-trisphosphate receptors to stimulate apoptotic calcium release

Background: The non-nuclear BRCA1 tumor suppressor can stimulate cell death, but the mechanisms are unknown. Results: BRCA1 binds to the inositol 1,4,5-trisphophate receptor (IP3R) calcium channel at the endoplasmic reticulum to stimulate apoptotic calcium release. Conclusion: BRCA1 tumor suppressor activity includes direct stimulation of apoptotic cell death via increased IP3R activity. Significance: We identify a novel role for the tumor suppressor BRCA1. The inositol 1,4,5-trisphosphate receptor (IP3R) is a ubiquitously expressed endoplasmic reticulum (ER)-resident calcium channel. Calcium release mediated by IP3Rs influences many signaling pathways, including those regulating apoptosis. IP3R activity is regulated by protein-protein interactions, including binding to proto-oncogenes and tumor suppressors to regulate cell death. Here we show that the IP3R binds to the tumor suppressor BRCA1. BRCA1 binding directly sensitizes the IP3R to its ligand, IP3. BRCA1 is recruited to the ER during apoptosis in an IP3R-dependent manner, and, in addition, a pool of BRCA1 protein is constitutively associated with the ER under non-apoptotic conditions. This is likely mediated by a novel lipid binding activity of the first BRCA1 C terminus domain of BRCA1. These findings provide a mechanistic explanation by which BRCA1 can act as a proapoptotic protein.

Conserved GXXXG- and S/T-Like Motifs in the Transmembrane Domains of NS4B Protein Are Required for Hepatitis C Virus Replication

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is an integral membrane protein, which plays an important role in the organization and function of the HCV replication complex (RC). Although much is understood about its amphipathic N-terminal and C-terminal domains, we know very little about the role of the transmembrane domains (TMDs) in NS4B function. We hypothesized that in addition to anchoring NS4B into host membranes, the TMDs are engaged in intra- and intermolecular interactions required for NS4B structure/function. To test this hypothesis, we have engineered a chimeric JFH1 genome containing the Con1 NS4B TMD region. The resulting virus titers were greatly reduced from those of JFH1, and further analysis indicated a defect in genome replication. We have mapped this incompatibility to NS4B TMD1 and TMD2 sequences, and we have defined putative TMD dimerization motifs (GXXXG in TMD2 and TMD3; the S/T cluster in TMD1) as key structural/functional determinants. Mutations in each of the putative motifs led to significant decreases in JFH1 replication. Like most of the NS4B chimeras, mutant proteins had no negative impact on NS4B membrane association. However, some mutations led to disruption of NS4B foci, implying that the TMDs play a role in HCV RC formation. Further examination indicated that the loss of NS4B foci correlates with the destabilization of NS4B protein. Finally, we have identified an adaptive mutation in the NS4B TMD2 sequence that has compensatory effects on JFH1 chimera replication. Taken together, these data underscore the functional importance of NS4B TMDs in the HCV life cycle.

Enhanced offspring predisposition to steatohepatitis with maternal high-fat diet is associated with epigenetic and microbiome alterations

Objective Non-alcoholic fatty liver disease (NAFLD) is an important co-morbidity associated with obesity and a precursor to steatohepatitis. However, the contributions of gestational and early life influences on development of NAFLD and NASH remain poorly appreciated. Methods Two independent studies were performed to examine whether maternal over-nutrition via exposure to high fat diet (HFD) leads to exacerbated hepatic responses to post-natal HFD and methionine choline deficient (MCD) diets in the offspring. Offspring of both control diet- and HFD-fed dams were weaned onto control and HFD, creating four groups. Results When compared to their control diet-fed littermates, offspring of HF-dams weaned onto HFD gained greater body weight; had increased relative liver weight and showed hepatic steatosis and inflammation. Similarly, this group revealed significantly greater immune response and pro-fibrogenic gene expression via RNA-seq. In parallel, 7–8 week old offspring were challenged with either control or MCD diets for 3 weeks. Responses to MCD diets were also exacerbated due to maternal HFD as seen by gene expression of classical pro-fibrogenic genes. Quantitative genome-scale DNA methylation analysis of over 1 million CpGs showed persistent epigenetic changes in key genes in tissue development and metabolism (Fgf21, Ppargc1β) with maternal HFD and in cell adhesion and communication (VWF, Ephb2) in the combination of maternal HFD and offspring MCD diets. Maternal HFD also influenced gut microbiome profiles in offspring leading to a decrease in α-diversity. Linear regression analysis revealed association between serum ALT levels and Coprococcus, Coriobacteriacae, Helicobacterioceae and Allobaculum. Conclusion Our findings indicate that maternal HFD detrimentally alters epigenetic and gut microbiome pathways to favor development of fatty liver disease and its progressive sequelae.

Reevaluation of the evolutionary events within recA/RAD51 phylogeny

BackgroundThe recA/RAD51 gene family encodes a diverse set of recombinase proteins that affect homologous recombination, DNA-repair, and genome stability. The recA gene family is expressed across all three domains of life - Eubacteria, Archaea, and Eukaryotes - and even in some viruses. To date, efforts to resolve the deep evolutionary origins of this ancient protein family have been hindered by the high sequence divergence between paralogous groups (i.e. ~30% average pairwise identity).ResultsThrough large taxon sampling and the use of a phylogenetic algorithm designed for inferring evolutionary events in highly divergent paralogs, we obtained a robust, parsimonious and more refined phylogenetic history of the recA/RAD51 superfamily.ConclusionsIn summary, our model for the evolution of recA/RAD51 family provides a better understanding of the ancient origin of recA proteins and the multiple events that lead to the diversification of recA homologs in eukaryotes, including the discovery of additional RAD51 sub-families.

PHYRN: A Robust Method for Phylogenetic Analysis of Highly Divergent Sequences

Both multiple sequence alignment and phylogenetic analysis are problematic in the “twilight zone” of sequence similarity (≤25% amino acid identity). Herein we explore the accuracy of phylogenetic inference at extreme sequence divergence using a variety of simulated data sets. We evaluate four leading multiple sequence alignment (MSA) methods (MAFFT, T-COFFEE, CLUSTAL, and MUSCLE) and six commonly used programs of tree estimation (Distance-based: Neighbor-Joining; Character-based: PhyML, RAxML, GARLI, Maximum Parsimony, and Bayesian) against a novel MSA-independent method (PHYRN) described here. Strikingly, at “midnight zone” genetic distances (∼7% pairwise identity and 4.0 gaps per position), PHYRN returns high-resolution phylogenies that outperform traditional approaches. We reason this is due to PHRYNs capability to amplify informative positions, even at the most extreme levels of sequence divergence. We also assess the applicability of the PHYRN algorithm for inferring deep evolutionary relationships in the divergent DANGER protein superfamily, for which PHYRN infers a more robust tree compared to MSA-based approaches. Taken together, these results demonstrate that PHYRN represents a powerful mechanism for mapping uncharted frontiers in highly divergent protein sequence data sets.

Bimodal regulation of an Elk subfamily K+ channel by phosphatidylinositol 4,5-bisphosphate.

PIP2 mediates the bimodal regulation of the EAG family K+ channel ELK1 to produce an overall inhibitory effect.

TRPV channel-mediated calcium transients in nociceptor neurons are dispensable for avoidance behaviour

Animals need to sense and react to potentially dangerous environments. TRP ion channels participate in nociception, presumably via Ca2+ influx, in most animal species. However, the relationship between ion permeation and animals’ nocifensive behaviour is unknown. Here we use an invertebrate animal model with relevance for mammalian pain. We analyse the putative selectivity filter of OSM-9, a TRPV channel, in osmotic avoidance behaviour of Caenorhabditis elegans. Using mutagenized OSM-9 expressed in the head nociceptor neuron, ASH, we study nocifensive behaviour and Ca2+ influx. Within the selectivity filter, M601-F609, Y604G strongly reduces avoidance behaviour and eliminates Ca2+ transients. Y604F also abolishes Ca2+ transients in ASH, while sustaining avoidance behaviour, yet it disrupts behavioral plasticity. Homology modelling of the OSM-9 pore suggests that Y604 may assume a scaffolding role. Thus, aromatic residues in the OSM-9 selectivity filter are critical for pain behaviour and ion permeation. These findings have relevance for understanding evolutionary roots of mammalian nociception.

Formula diet driven microbiota shifts tryptophan metabolism from serotonin to tryptamine in neonatal porcine colon

BackgroundThe gut microbiota of breast-fed and formula-fed infants differ significantly, as do the risks for allergies, gut dysfunction, and upper respiratory tract infections. The connections between breast milk, various formulas, and the profiles of gut bacteria to these childhood illnesses, as well as the mechanisms underlying the effects, are not well understood.MethodsWe investigated distal colon microbiota by 16S RNA amplicon sequencing, morphology by histomorphometry, immune response by cytokine expression, and tryptophan metabolism in a pig model in which piglets were sow-fed, or fed soy or dairy milk-based formula from postnatal day (PND) 2 to 21.ResultsFormula feeding significantly (p < 0.05) altered the colon microbiota relative to the sow feeding. A significant reduction in microbial diversity was noted with formula groups in comparison to sow-fed. Streptococcus, Blautia, Citrobacter, Butrycimonas, Parabacteroides, Lactococcus genera were increased with formula feeding relative to sow feeding. In addition, relative to sow feeding, Anaerotruncus, Akkermansia, Enterococcus, Acinetobacter, Christensenella, and Holdemania were increased in milk-fed piglets, and Biliophila, Ruminococcus, Clostridium were increased in soy-fed piglets. No significant gut morphological changes were noted. However, higher cytokine mRNA expression (BMP4, CCL11, CCL21) was observed in the distal colon of formula groups. Formula feeding reduced enterochromaffin cell number and serotonin, but increased tryptamine levels relative to sow feeding.ConclusionOur data confirm that formula diet alters the colon microbiota and appears to shift tryptophan metabolism from serotonin to tryptamine, which may lead to greater histamine levels and risk of allergies in infants.

Early Postnatal Diets Affect the Bioregional Small Intestine Microbiome and Ileal Metabolome in Neonatal Pigs

Background: Breastfeeding is known to be protective against gastrointestinal disorders and may modify gut development. Although the gut microbiome has been implicated, little is known about how early diet affects the small intestine microbiome.Objective: We hypothesized that disparate early diets would promote unique microbial profiles in the small intestines of neonatal pigs.Methods: Male and female 2-d-old White Dutch Landrace pigs were either sow fed or provided dairy (Similac Advance powder; Ross Products Abbott Laboratories) or soy (Enfamil Prosobee Lipil powder; Mead Johnson Nutritionals) infant formulas until day 21. Bacterial ecology was assessed in the contents of the small intestine through the use of 16S ribosomal RNA sequencing. α-Diversity, β-diversity, and differential abundances of operational taxonomic units were assessed by ANOVA, permutational ANOVA, and negative binomial regression, respectively. Ileum tissue metabolomics were measured by LC-mass spectrometry and assessed by weighted correlation network analysis.Results: Greater α-diversity was observed in the duodena of sow-fed compared with formula-fed neonatal pigs (P < 0.05). No differences were observed in the ilea. Firmicutes represented the most abundant phylum across all diets in duodena (78.8%, 80.1%, and 53.4% relative abundance in sow, dairy, and soy groups, respectively), followed by Proteobacteria in sow (12.2%) and dairy (12.4%) groups and Cyanobacteria in soy-fed (36.2%) pigs. In contrast to those in the duodenum, Proteobacteria was the dominant phylum in the ileum, with >60% relative abundance in all of the groups. In the duodenum, 77 genera were altered by diet, followed by 48 in the jejunum and 19 in the ileum. Metabolomics analyses revealed associations between ileum tissue metabolites (e.g., acylcarnitines, 3-aminoisobutyric acid) and diet-responsive microbial genera.Conclusions: These results indicate that the neonatal diet has regional effects on the small intestine microbiome in pigs, with the most pronounced effects occurring in the duodena. Regional effects may be important factors when considering gut tissue metabolism and development in the postnatal period.

Gut microbiota are linked to increased susceptibility to hepatic steatosis in low-aerobic-capacity rats fed an acute high-fat diet

Poor aerobic fitness is linked to nonalcoholic fatty liver disease and increased all-cause mortality. We previously found that rats with a low capacity for running (LCR) that were fed an acute high-fat diet (HFD; 45% kcal from fat) for 3 days resulted in positive energy balance and increased hepatic steatosis compared with rats that were highly aerobically fit with a high capacity for running (HCR). Here, we tested the hypothesis that poor physiological outcomes in LCR rats following acute HFD feeding are associated with alterations in cecal microbiota. LCR rats exhibited greater body weight, feeding efficiency, 3 days of body weight change, and liver triglycerides after acute HFD feeding compared with HCR rats. Furthermore, compared with HCR rats, LCR rats exhibited reduced expression of intestinal tight junction proteins. Cecal bacterial 16S rDNA revealed that LCR rats had reduced cecal Proteobacteria compared with HCR rats. Microbiota of HCR rats consisted of greater relative abundance of Desulfovibrionaceae and unassigned genera within this family, suggesting increased reduction of endogenous mucins and proteins. Although feeding rats an acute HFD led to reduced Firmicutes in both strains, short-chain fatty acid-producing Phascolarctobacterium was reduced in LCR rats. In addition, Ruminococcae and Ruminococcus were negatively correlated with energy intake in the LCR/HFD rats. Predicted metagenomic function suggested that LCR rats had a greater capacity to metabolize carbohydrate and energy compared with HCR rats. Overall, these data suggest that the populations and metabolic capacity of the microbiota in low-aerobically fit LCR rats may contribute to their susceptibility to acute HFD-induced hepatic steatosis and poor physiologic outcomes.