Site-directed mutagenesis of the proton-pumping pyridine nucleotide transhydrogenase of Escherichia coli

AbstractThe pyridine nucleotide transhydrogenase of Escherichia coli catalyzes the reversible transfer of hydride ion equivalents between NAD+ and NADP+ coupled to the translocation of protons across the cytoplasmic membrane. It is composed of two subunits (α, β) organized as an α2β2 tetramer. This brief review describes the use of site-directed mutagenesis to investigate the structure, mechanism and assembly of the transhydrogenase. This technique has located the binding sites for NAD(H) and NADP(H) in the α and β subunits, respectively. Mutagenesis has shown that the cysteine residues of the enzyme are not essential for its function, and that inhibition of the enzyme by sulfhydryl-specific reagents must be due to perturbation of the three-dimensional structure. The sites of reaction of the inhibitors N,N′-dicyclohexylcarbodiimide and N-(1-pyrene)maleimide have been located. Selective mutation and insertion of cysteine residues followed by cupric o-phenanthrolinate-induced disulfide crosslinking has defined a region of interaction between the α subunits in the holoenzyme. Determination of the accessibility of selectively inserted cysteine residues has been used to determine the folding pattern of the transmembrane helices of the β subunit. Site-directed matagenesis of the transmembrane domain of the β subunit has permitted the identification of histidine, aspartic acid and asparagine residues which are part of the proton-pumping pathway of the transhydrogenase. Site-directed matagenesis and amino acid deletions have shown that the six carboxy terminal residues of the α subunit and the two carboxy terminal residues of the β subunit are necessary for correct assembly of the transhydrogenase in the cytoplasmic membrane

Isolation of a fourth cysteinyl-containing peptide of the α-subunit of the F1 ATPase from Escherichia coli necessitates revision of the DNA sequence

AbstractThe rapid determination of cysteinyl residues by Creighton's method [(1980) Nature 284, 487-489] led to the discovery of a discrepancy between protein and DNA sequence data in the α-subunit of the F1 ATPase from Escherichia coli [(1984) Arch. Biochem. Biophys. 229, 320-328]. We have isolated a cysteinyl-containing decapeptide from the α-subunit with a protein sequence (AGCAMGEYFR) which is only partially recognizable from DNA data. Re-sequencing of DNA in the region coding for the peptide has resulted in two corrections: insertion of a cytosine before position 715 and deletion of a thymine at position 731 of the uncA gene

Climatic and tectonic drivers shaped the tropical distribution of coral reefs

Today, warm-water coral reefs are limited to tropical-to-subtropical latitudes. These diverse ecosystems extended further poleward in the geological past, but the mechanisms driving these past distributions remain uncertain. Here, we test the role of climate and palaeogeography in shaping the distribution of coral reefs over geological timescales. To do so, we combine habitat suitability modelling, Earth System modelling and the ~247-million-year geological record of scleractinian coral reefs. A broader latitudinal distribution of climatically suitable habitat persisted throughout much of the Mesozoic–early Paleogene due to an expanded tropical belt and more equable distribution of shallow marine substrate. The earliest Cretaceous might be an exception, with reduced shallow marine substrate during a ‘cold-snap’ interval. Climatically suitable habitat area became increasingly skewed towards the tropics from the late Paleogene, likely steepening the latitudinal biodiversity gradient of reef-associated taxa. This was driven by global cooling and increases in tropical shallow marine substrate resulting from the tectonic evolution of the Indo-Australian Archipelago. Although our results suggest global warming might permit long-term poleward range expansions, coral reef ecosystems are unlikely to keep pace with the rapid rate of anthropogenic climate change

Phage Encoded H-NS: A Potential Achilles Heel in the Bacterial Defence System

The relationship between phage and their microbial hosts is difficult to elucidate in complex natural ecosystems. Engineered systems performing enhanced biological phosphorus removal (EBPR), offer stable, lower complexity communities for studying phage-host interactions. Here, metagenomic data from an EBPR reactor dominated by Candidatus Accumulibacter phosphatis (CAP), led to the recovery of three complete and six partial phage genomes. Heat-stable nucleoid structuring (H-NS) protein, a global transcriptional repressor in bacteria, was identified in one of the complete phage genomes (EPV1), and was most similar to a homolog in CAP. We infer that EPV1 is a CAP-specific phage and has the potential to repress up to 6% of host genes based on the presence of putative H-NS binding sites in the CAP genome. These genes include CRISPR associated proteins and a Type III restriction-modification system, which are key host defense mechanisms against phage infection. Further, EPV1 was the only member of the phage community found in an EBPR microbial metagenome collected seven months prior. We propose that EPV1 laterally acquired H-NS from CAP providing it with a means to reduce bacterial defenses, a selective advantage over other phage in the EBPR system. Phage encoded H-NS could constitute a previously unrecognized weapon in the phage-host arms race

Genetic drivers of heterogeneity in type 2 diabetes pathophysiology

Type 2 diabetes (T2D) is a heterogeneous disease that develops through diverse pathophysiological processes1,2 and molecular mechanisms that are often specific to cell type3,4. Here, to characterize the genetic contribution to these processes across ancestry groups, we aggregate genome-wide association study data from 2,535,601 individuals (39.7% not of European ancestry), including 428,452 cases of T2D. We identify 1,289 independent association signals at genome-wide significance (P &lt; 5 × 10-8) that map to 611 loci, of which 145 loci are, to our knowledge, previously unreported. We define eight non-overlapping clusters of T2D signals that are characterized by distinct profiles of cardiometabolic trait associations. These clusters are differentially enriched for cell-type-specific regions of open chromatin, including pancreatic islets, adipocytes, endothelial cells and enteroendocrine cells. We build cluster-specific partitioned polygenic scores5 in a further 279,552 individuals of diverse ancestry, including 30,288 cases of T2D, and test their association with T2D-related vascular outcomes. Cluster-specific partitioned polygenic scores are associated with coronary artery disease, peripheral artery disease and end-stage diabetic nephropathy across ancestry groups, highlighting the importance of obesity-related processes in the development of vascular outcomes. Our findings show the value of integrating multi-ancestry genome-wide association study data with single-cell epigenomics to disentangle the aetiological heterogeneity that drives the development and progression of T2D. This might offer a route to optimize global access to genetically informed diabetes care.</p

Association of genetic variation with systolic and diastolic blood pressure among African Americans: the Candidate Gene Association Resource study

The prevalence of hypertension in African Americans (AAs) is higher than in other US groups; yet, few have performed genome-wide association studies (GWASs) in AA. Among people of European descent, GWASs have identified genetic variants at 13 loci that are associated with blood pressure. It is unknown if these variants confer susceptibility in people of African ancestry. Here, we examined genome-wide and candidate gene associations with systolic blood pressure (SBP) and diastolic blood pressure (DBP) using the Candidate Gene Association Resource (CARe) consortium consisting of 8591 AAs. Genotypes included genome-wide single-nucleotide polymorphism (SNP) data utilizing the Affymetrix 6.0 array with imputation to 2.5 million HapMap SNPs and candidate gene SNP data utilizing a 50K cardiovascular gene-centric array (ITMAT-Broad-CARe [IBC] array). For Affymetrix data, the strongest signal for DBP was rs10474346 (P= 3.6 × 10−8) located near GPR98 and ARRDC3. For SBP, the strongest signal was rs2258119 in C21orf91 (P= 4.7 × 10−8). The top IBC association for SBP was rs2012318 (P= 6.4 × 10−6) near SLC25A42 and for DBP was rs2523586 (P= 1.3 × 10−6) near HLA-B. None of the top variants replicated in additional AA (n = 11 882) or European-American (n = 69 899) cohorts. We replicated previously reported European-American blood pressure SNPs in our AA samples (SH2B3, P= 0.009; TBX3-TBX5, P= 0.03; and CSK-ULK3, P= 0.0004). These genetic loci represent the best evidence of genetic influences on SBP and DBP in AAs to date. More broadly, this work supports that notion that blood pressure among AAs is a trait with genetic underpinnings but also with significant complexit

Genetic variants in novel pathways influence blood pressure and cardiovascular disease risk.

Blood pressure is a heritable trait influenced by several biological pathways and responsive to environmental stimuli. Over one billion people worldwide have hypertension (≥140 mm Hg systolic blood pressure or  ≥90 mm Hg diastolic blood pressure). Even small increments in blood pressure are associated with an increased risk of cardiovascular events. This genome-wide association study of systolic and diastolic blood pressure, which used a multi-stage design in 200,000 individuals of European descent, identified sixteen novel loci: six of these loci contain genes previously known or suspected to regulate blood pressure (GUCY1A3-GUCY1B3, NPR3-C5orf23, ADM, FURIN-FES, GOSR2, GNAS-EDN3); the other ten provide new clues to blood pressure physiology. A genetic risk score based on 29 genome-wide significant variants was associated with hypertension, left ventricular wall thickness, stroke and coronary artery disease, but not kidney disease or kidney function. We also observed associations with blood pressure in East Asian, South Asian and African ancestry individuals. Our findings provide new insights into the genetics and biology of blood pressure, and suggest potential novel therapeutic pathways for cardiovascular disease prevention

Causal effect of plasminogen activator inhibitor type 1 on coronary heart disease

Background--Plasminogen activator inhibitor type 1 (PAI-1) plays an essential role in the fibrinolysis system and thrombosis. Population studies have reported that blood PAI-1 levels are associated with increased risk of coronary heart disease (CHD). However, it is unclear whether the association reflects a causal influence of PAI-1 on CHD risk. Methods and Results--To evaluate the association between PAI-1 and CHD, we applied a 3-step strategy. First, we investigated the observational association between PAI-1 and CHD incidence using a systematic review based on a literature search for PAI-1 and CHD studies. Second, we explored the causal association between PAI-1 and CHD using a Mendelian randomization approach using summary statistics from large genome-wide association studies. Finally, we explored the causal effect of PAI-1 on cardiovascular risk factors including metabolic and subclinical atherosclerosis measures. In the systematic meta-analysis, the highest quantile of blood PAI-1 level was associated with higher CHD risk comparing with the lowest quantile (odds ratio=2.17; 95% CI: 1.53, 3.07) in an age- and sex-adjusted model. The effect size was reduced in studies using a multivariable-adjusted model (odds ratio=1.46; 95% CI: 1.13, 1.88). The Mendelian randomization analyses suggested a causal effect of increased PAI-1 level on CHD risk (odds ratio=1.22 per unit increase of log-transformed PAI-1; 95% CI: 1.01, 1.47). In addition, we also detected a causal effect of PAI-1 on elevating blood glucose and high-density lipoprotein cholesterol. Conclusions--Our study indicates a causal effect of elevated PAI-1 level on CHD risk, which may be mediated by glucose dysfunction

A Meta-analysis of Gene Expression Signatures of Blood Pressure and Hypertension

Genome-wide association studies (GWAS) have uncovered numerous genetic variants (SNPs) that are associated with blood pressure (BP). Genetic variants may lead to BP changes by acting on intermediate molecular phenotypes such as coded protein sequence or gene expression, which in turn affect BP variability. Therefore, characterizing genes whose expression is associated with BP may reveal cellular processes involved in BP regulation and uncover how transcripts mediate genetic and environmental effects on BP variability. A meta-analysis of results from six studies of global gene expression profiles of BP and hypertension in whole blood was performed in 7017 individuals who were not receiving antihypertensive drug treatment. We identified 34 genes that were differentially expressed in relation to BP (Bonferroni-corrected p&lt;0.05). Among these genes, FOS and PTGS2 have been previously reported to be involved in BP-related processes; the others are novel. The top BP signature genes in aggregate explain 5%–9% of inter-individual variance in BP. Of note, rs3184504 in SH2B3, which was also reported in GWAS to be associated with BP, was found to be a trans regulator of the expression of 6 of the transcripts we found to be associated with BP (FOS, MYADM, PP1R15A, TAGAP, S100A10, and FGBP2). Gene set enrichment analysis suggested that the BP-related global gene expression changes include genes involved in inflammatory response and apoptosis pathways. Our study provides new insights into molecular mechanisms underlying BP regulation, and suggests novel transcriptomic markers for the treatment and prevention of hypertension

Association of genetic variation with systolic and diastolic blood pressure among African Americans: the Candidate Gene Association Resource study.

The prevalence of hypertension in African Americans (AAs) is higher than in other US groups; yet, few have performed genome-wide association studies (GWASs) in AA. Among people of European descent, GWASs have identified genetic variants at 13 loci that are associated with blood pressure. It is unknown if these variants confer susceptibility in people of African ancestry. Here, we examined genome-wide and candidate gene associations with systolic blood pressure (SBP) and diastolic blood pressure (DBP) using the Candidate Gene Association Resource (CARe) consortium consisting of 8591 AAs. Genotypes included genome-wide single-nucleotide polymorphism (SNP) data utilizing the Affymetrix 6.0 array with imputation to 2.5 million HapMap SNPs and candidate gene SNP data utilizing a 50K cardiovascular gene-centric array (ITMAT-Broad-CARe [IBC] array). For Affymetrix data, the strongest signal for DBP was rs10474346 (P= 3.6 × 10(-8)) located near GPR98 and ARRDC3. For SBP, the strongest signal was rs2258119 in C21orf91 (P= 4.7 × 10(-8)). The top IBC association for SBP was rs2012318 (P= 6.4 × 10(-6)) near SLC25A42 and for DBP was rs2523586 (P= 1.3 × 10(-6)) near HLA-B. None of the top variants replicated in additional AA (n = 11 882) or European-American (n = 69 899) cohorts. We replicated previously reported European-American blood pressure SNPs in our AA samples (SH2B3, P= 0.009; TBX3-TBX5, P= 0.03; and CSK-ULK3, P= 0.0004). These genetic loci represent the best evidence of genetic influences on SBP and DBP in AAs to date. More broadly, this work supports that notion that blood pressure among AAs is a trait with genetic underpinnings but also with significant complexity