Interaktionsstudien der GvpACNO Proteine und Implikationen für die Gasvesikelbildung von Halobacterium salinarum

Die Gasvesikelbildung in Hbt. salinarum PHH1 wird durch die Expression der p-vac-Region gesteuert. Die p-vac Region besteht aus 14 gvp-Genen, die in zwei entgegengesetzten Gengruppen angeordnet sind, gvpACNO und gvpDEFGHIJKLM. Die Gene gvpFGHIJKLM exprimieren die gleichnamigen Gvp-Proteine, die zu Beginn der Gasvesikelbildung gebildet werden und wahrscheinlich einen Initialkomplex bilden, an dem die Gasvesikelbildung startet. GvpD und GvpE sind regulatorische Proteine, die als Transkriptionsaktivator (GvpE) bzw. -repressor (GvpD) dienen. Aufgebaut wird das Gasvesikel hauptsächlich aus GvpA, dem Hauptstrukturprotein der Gasvesikelhülle, und GvpC, das diese Hülle von außen stabilisiert. Im Rahmen dieser Arbeit wurden erstmals die Interaktionen der ACNO-Proteine untersucht. Dazu wurden zwei verschiedene Untersuchungsmethoden angewandt; die split-GFP-Methode und ein Pulldown-Assay mit der Cellulose-Bindedomäne. Dabei standen vor allem die beiden Strukturproteine GvpA und GvpC im Vordergrund, welche auch durch Fragmentierungen näher untersucht wurden. Dabei zeigte sich, dass besonders der N-terminale Teil von GvpA und der C-terminale Teil von GvpC viele Bindungen mit anderen Gasvesikelproteinen eingehen können. Insgesamt ist die Fragmentierung von Proteinen eine gute Möglichkeit, um die Interaktionsstellen in Proteinen näher zu definieren und einzugrenzen. Zusätzlich wurden diese durch Mutationsstudien untersucht, durch die sowohl in GvpA als auch in GvpJ wichtige Aminosäuren bzw. Motive gefunden wurden, die an der Interaktion von GvpA/GvpF bzw. GvpJ/GvpL beteiligt sind oder diese vermitteln. Dabei nehmen die sehr ähnlichen Proteine GvpA, GvpJ und GvpM im Interaktionsnetzwerk eigenständige Rollen ein. Sie sind nicht gegeneinander austauschbar, sondern interagieren mit verschiedenen Proteinen und über unterschiedliche Interaktionsstellen. Durch die untersuchten Interaktionen zwischen den akzessorischen Proteinen und den ACNO-Proteinen konnte ein vollständiges Interaktionsnetzwerk etabliert werden, an dem alle Gvp-Proteine (außer GvpD und GvpE) beteiligt sind. GvpL weist dabei die meisten und stärksten Interaktionen zu anderen Proteinen auf und nimmt so eine zentrale Rolle in der Gasvesikelbildung und der frühen Komplexbildung zu Beginn der Gasvesikelsynthese ein. Dieser Komplex oder Teilkomplexe aus mehreren akzessorischen Proteinen konnten erstmals über ein bait-Protein isoliert werden. Dazu wurde CBDA verwendet, das nicht in der Lage ist, alle akzessorischen Proteine in Einzelinteraktionen zu binden. Wenn jedoch GvpF bis GvpM mit CBDA co-synthetisiert werden, können alle Proteine durch Western-Analysen detektiert werden. Dazu müssen die akzessorischen Proteine untereinander Interaktionen eingehen, die darauf hindeuten, dass diese einen oder mehrere Komplexe zu Beginn der Gasvesikelsynthese bilden. Der Einbau von GvpA geschieht höchstwahrscheinlich mit GvpF als Bindungspartner, wobei die alpha-Helix 1 von GvpA eine entscheidende Rolle spielt. Um die Dimerisierung von GvpA und damit den Aufbau des Gasvesikels näher zu untersuchen, wurde zusätzlich das bereits etablierte split-GFP-System durch zwei Vektoren erweitert, die eine Interaktionsstudie zweier Proteine in Anwesenheit von weiteren Proteinen ermöglicht. Hier konnte gezeigt werden, dass die GvpA-Dimerisierung erst durch GvpC, GvpN und GvpO ermöglicht wird. GvpN kommt dabei wahrscheinlich eine entscheidende Aufgabe zu, da ein Fehlen von GvpN zu sehr kleinen Gasvesikeln führt, die nicht vergrößert werden können. Besonders das NTP-bindende Motiv (p-loop-Motiv) in GvpN erwies sich als wichtig für die Ausbildung von Gasvesikeln

Interaction of the gas vesicle proteins GvpA, GvpC, GvpN, and GvpO of Halobacterium salinarum

The interactions of the four gas vesicle proteins GvpA, C, N, and O were investigated by split-GFP and pulldown assays. GvpA forms the ribs of the gas vesicle shell, whereas GvpC is attached to the exterior surface and stabilizes the gas vesicle structure. The AAA-ATPase GvpN as well as GvpO is found in much lower amounts. GvpN and GvpO formed homodimers and also the GvpN/GvpO heterodimer; both interacted with the C-terminal domain of GvpC when tested by split-GFP. When analyzed by pulldown assays, GvpN and GvpO also selected GvpA. The N-and C-terminal fragments of GvpC dimerized as Cterm/Cterm and Cterm/Nterm, but not as Nterm/Nterm. These interactions at both termini might lead to a network of GvpC molecules at the gas vesicle surface. However, a GvpA/GvpC interaction was not detectable, suggesting that the contact of both proteins is either mediated by another Gvp, or requires different structures that might form when GvpA is aggregated in the gas vesicle shell. Interactions of GvpA, C, N, and O were also studied with the accessory proteins GvpF through GvpM by split-GFP. GvpN bound GvpL only, whereas GvpO interacted with GvpF, I, and L, and the C-terminal domain of GvpC contacted GvpF, H, I, and L. GvpA/GvpA interactions were difficult to detect by split-GFP, but GvpA selected except for GvpI, K, and L all other accessory Gvp in pulldown assays. We will discuss the implications of these findings on gas-vesicle assembly

Effect of Mutations in GvpJ and GvpM on Gas Vesicle Formation of Halobacterium salinarum

The two haloarchaeal proteins, GvpM and GvpJ, are homologous to GvpA, the major gas vesicle structural protein. All three are hydrophobic and essential for gas vesicle formation. The effect of mutations in GvpJ and GvpM was studied in Haloferax volcanii transformants by complementing the respective mutated gene with the remaining gvp genes and inspecting the cells for the presence of gas vesicles (Vac⁺). In case of GvpJ, 56 of 66 substitutions analyzed yielded Vac⁻ ΔJ + Jmut transformants, indicating that GvpJ is very sensitive to alterations, whereas ten of the 38 GvpM variants resulted in Vac⁻ ΔM + Mmut transformants. The variants were also tested by split-GFP for their ability to interact with their partner protein GvpL. Some of the alterations leading to a Vac⁻ phenotype affected the J/L or M/L interaction. Also, the interactions J/A and J/M were studied using fragments to exclude an unspecific aggregation of these hydrophobic proteins. Both fragments of GvpJ interacted with the M1–25 and M60–84 fragments of GvpM, and fragment J1–56 of GvpJ interacted with the N-terminal fragment A1–22 of GvpA. A comparison of the results on the three homologous proteins indicates that despite their relatedness, GvpA, GvpJ, and GvpM have unique features and cannot substitute each other

Multi-ancestry study of blood lipid levels identifies four loci interacting with physical activity.

Many genetic loci affect circulating lipid levels, but it remains unknown whether lifestyle factors, such as physical activity, modify these genetic effects. To identify lipid loci interacting with physical activity, we performed genome-wide analyses of circulating HDL cholesterol, LDL cholesterol, and triglyceride levels in up to 120,979 individuals of European, African, Asian, Hispanic, and Brazilian ancestry, with follow-up of suggestive associations in an additional 131,012 individuals. We find four loci, in/near CLASP1, LHX1, SNTA1, and CNTNAP2, that are associated with circulating lipid levels through interaction with physical activity; higher levels of physical activity enhance the HDL cholesterol-increasing effects of the CLASP1, LHX1, and SNTA1 loci and attenuate the LDL cholesterol-increasing effect of the CNTNAP2 locus. The CLASP1, LHX1, and SNTA1 regions harbor genes linked to muscle function and lipid metabolism. Our results elucidate the role of physical activity interactions in the genetic contribution to blood lipid levels

Multi-ancestry genome-wide association study accounting for gene-psychosocial factor interactions identifies novel loci for blood pressure traits

Psychological and social factors are known to influence blood pressure (BP) and risk of hypertension and associated cardiovascular diseases. To identify novel BP loci, we carried out genome-wide association meta-analyses of systolic, diastolic, pulse, and mean arterial BP, taking into account the interaction effects of genetic variants with three psychosocial factors: depressive symptoms, anxiety symptoms, and social support. Analyses were performed using a two-stage design in a sample of up to 128,894 adults from five ancestry groups. In the combined meta-analyses of stages 1 and 2, we identified 59 loci (p value < 5e−8), including nine novel BP loci. The novel associations were observed mostly with pulse pressure, with fewer observed with mean arterial pressure. Five novel loci were identified in African ancestry, and all but one showed patterns of interaction with at least one psychosocial factor. Functional annotation of the novel loci supports a major role for genes implicated in the immune response (PLCL2), synaptic function and neurotransmission (LIN7A and PFIA2), as well as genes previously implicated in neuropsychiatric or stress-related disorders (FSTL5 and CHODL). These findings underscore the importance of considering psychological and social factors in gene discovery for BP, especially in non-European populations

Gene-educational attainment interactions in a multi-population genome-wide meta-analysis identify novel lipid loci

Introduction: Educational attainment, widely used in epidemiologic studies as a surrogate for socioeconomic status, is a predictor of cardiovascular health outcomes. Methods: A two-stage genome-wide meta-analysis of low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), and triglyceride (TG) levels was performed while accounting for gene-educational attainment interactions in up to 226,315 individuals from five population groups. We considered two educational attainment variables: “Some College” (yes/no, for any education beyond high school) and “Graduated College” (yes/no, for completing a 4-year college degree). Genome-wide significant (p &lt; 5 × 10−8) and suggestive (p &lt; 1 × 10−6) variants were identified in Stage 1 (in up to 108,784 individuals) through genome-wide analysis, and those variants were followed up in Stage 2 studies (in up to 117,531 individuals). Results: In combined analysis of Stages 1 and 2, we identified 18 novel lipid loci (nine for LDL, seven for HDL, and two for TG) by two degree-of-freedom (2 DF) joint tests of main and interaction effects. Four loci showed significant interaction with educational attainment. Two loci were significant only in cross-population analyses. Several loci include genes with known or suggested roles in adipose (FOXP1, MBOAT4, SKP2, STIM1, STX4), brain (BRI3, FILIP1, FOXP1, LINC00290, LMTK2, MBOAT4, MYO6, SENP6, SRGAP3, STIM1, TMEM167A, TMEM30A), and liver (BRI3, FOXP1) biology, highlighting the potential importance of brain-adipose-liver communication in the regulation of lipid metabolism. An investigation of the potential druggability of genes in identified loci resulted in five gene targets shown to interact with drugs approved by the Food and Drug Administration, including genes with roles in adipose and brain tissue. Discussion: Genome-wide interaction analysis of educational attainment identified novel lipid loci not previously detected by analyses limited to main genetic effects.</p

Multi-ancestry study of blood lipid levels identifies four loci interacting with physical activity.

Many genetic loci affect circulating lipid levels, but it remains unknown whether lifestyle factors, such as physical activity, modify these genetic effects. To identify lipid loci interacting with physical activity, we performed genome-wide analyses of circulating HDL cholesterol, LDL cholesterol, and triglyceride levels in up to 120,979 individuals of European, African, Asian, Hispanic, and Brazilian ancestry, with follow-up of suggestive associations in an additional 131,012 individuals. We find four loci, in/near CLASP1, LHX1, SNTA1, and CNTNAP2, that are associated with circulating lipid levels through interaction with physical activity; higher levels of physical activity enhance the HDL cholesterol-increasing effects of the CLASP1, LHX1, and SNTA1 loci and attenuate the LDL cholesterol-increasing effect of the CNTNAP2 locus. The CLASP1, LHX1, and SNTA1 regions harbor genes linked to muscle function and lipid metabolism. Our results elucidate the role of physical activity interactions in the genetic contribution to blood lipid levels

Multi-ancestry study of blood lipid levels identifies four loci interacting with physical activity

The present work was largely supported by a grant from the US National Heart, Lung, and Blood Institute (NHLBI) of the National Institutes of Health (R01HL118305). The full list of acknowledgments appears in the Supplementary Notes 3 and 4.Peer reviewedPublisher PD

Multiancestry Genome-Wide Association Study of Lipid Levels Incorporating Gene-Alcohol Interactions

A person's lipid profile is influenced by genetic variants and alcohol consumption, but the contribution of interactions between these exposures has not been studied. We therefore incorporated gene-alcohol interactions into a multiancestry genome-wide association study of levels of high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides. We included 45 studies in stage 1 (genome-wide discovery) and 66 studies in stage 2 (focused follow-up), for a total of 394,584 individuals from 5 ancestry groups. Analyses covered the period July 2014-November 2017. Genetic main effects and interaction effects were jointly assessed by means of a 2-degrees-of-freedom (df) test, and a 1-df test was used to assess the interaction effects alone. Variants at 495 loci were at least suggestively associated (P <1 x 10(-6)) with lipid levels in stage 1 and were evaluated in stage 2, followed by combined analyses of stage 1 and stage 2. In the combined analysis of stages 1 and 2, a total of 147 independent loci were associated with lipid levels at P <5 x 10(-8) using 2-df tests, of which 18 were novel. No genome-wide-significant associations were found testing the interaction effect alone. The novel loci included several genes (proprotein convertase subtilisin/kexin type 5 (PCSK5), vascular endothelial growth factor B (VEGFB), and apolipoprotein B mRNA editing enzyme, catalytic polypeptide 1 (APOBEC1) complementation factor (A1CF)) that have a putative role in lipid metabolism on the basis of existing evidence from cellular and experimental models.Peer reviewe

A multi-ancestry genome-wide study incorporating gene-smoking interactions identifies multiple new loci for pulse pressure and mean arterial pressure

Elevated blood pressure (BP), a leading cause of global morbidity and mortality, is influenced by both genetic and lifestyle factors. Cigarette smoking is one such lifestyle factor. Across five ancestries, we performed a genome-wide gene-smoking interaction study of mean arterial pressure (MAP) and pulse pressure (PP) in 129 913 individuals in stage 1 and follow-up analysis in 480 178 additional individuals in stage 2. We report here 136 loci significantly associated with MAP and/or PP. Of these, 61 were previously published through main-effect analysis of BP traits, 37 were recently reported by us for systolic BP and/or diastolic BP through gene-smoking interaction analysis and 38 were newly identified (P <5 x 10(-8), false discovery rate <0.05). We also identified nine new signals near known loci. Of the 136 loci, 8 showed significant interaction with smoking status. They include CSMD1 previously reported for insulin resistance and BP in the spontaneously hypertensive rats. Many of the 38 new loci show biologic plausibility for a role in BP regulation. SLC26A7 encodes a chloride/bicarbonate exchanger expressed in the renal outer medullary collecting duct. AVPR1A is widely expressed, including in vascular smooth muscle cells, kidney, myocardium and brain. FHAD1 is a long non-coding RNA overexpressed in heart failure. TMEM51 was associated with contractile function in cardiomyocytes. CASP9 plays a central role in cardiomyocyte apoptosis. Identified only in African ancestry were 30 novel loci. Our findings highlight the value of multi-ancestry investigations, particularly in studies of interaction with lifestyle factors, where genomic and lifestyle differences may contribute to novel findings.Peer reviewe