Catestatin Gly364Ser Variant Alters Systemic Blood Pressure and the Risk for Hypertension in Human Populations via Endothelial Nitric Oxide Pathway

Granin-derived peptides.

Abstract: The granin family comprises altogether 7 different proteins originating from the diffuse neuroendocrine system and elements of the central and peripheral nervous systems. The family is dominated by three uniquely acidic members, namely chromogranin A (CgA), chromogranin B (CgB) and secretogranin II (SgII). Since the late 1980s it has become evident that these proteins are proteolytically processed, intragranularly and/or extracellularly into a range of biologically active peptides; a number of them with regulatory properties of physiological and/or pathophysiological significance. The aim of this comprehensive overview is to provide an up-to-date insight into the distribution and properties of the well established granin-derived peptides and their putative roles in homeostatic regulations. Hence, focus is directed to peptides derived from the three main granins, e.g. to the chromogranin A derived vasostatins, betagranins, pancreastatin and catestatins, the chromogranin B-derived secretolytin and the secretogranin II-derived secretoneurin (SN). In addition, the distribution and properties of the chromogranin A-derived peptides prochromacin, chromofungin, WE14, parastatin, GE-25 and serpinins, the CgB-peptide PE-11 and the SgII-peptides EM66 and manserin will also be commented on. Finally, the opposing effects of the CgA-derived vasostatin-I and catestatin and the SgII-derived peptide SN on the integrity of the vasculature, myocardial contractility, angiogenesis in wound healing, inflammatory conditions and tumors will be discussed.

Chromogranin A and its fragments in cardiovascular, immunometabolic, and cancer regulation.

Abstract: Chromogranin A (CgA)-the index member of the chromogranin/secretogranin secretory protein family-is ubiquitously distributed in endocrine, neuroendocrine, and immune cells. Elevated levels of CgA-related polypeptides, consisting of full-length molecules and fragments, are detected in the blood of patients suffering from neuroendocrine tumors, heart failure, renal failure, hypertension, rheumatoid arthritis, and inflammatory bowel disease. Full-length CgA and various CgA-derived peptides, including vasostatin-1, pancreastatin, catestatin, and serpinin, are expressed at different relative levels in normal and pathological conditions and exert diverse, and sometime opposite, biological functions. For example, CgA is overexpressed in genetic hypertension, whereas catestatin is diminished. In rodents, the administration of catestatin decreases hypertension, cardiac contractility, obesity, atherosclerosis, and inflammation, and it improves insulin sensitivity. By contrast, pancreastatin is elevated in diabetic patients, and the administration of this peptide to obese mice decreases insulin sensitivity and increases inflammation. CgA and the N-terminal fragment of vasostatin-1 can enhance the endothelial barrier function, exert antiangiogenic effects, and inhibit tumor growth in animal models, whereas CgA fragments lacking the CgA C-terminal region promote angiogenesis and tumor growth. Overall, the CgA system, consisting of full-length CgA and its fragments, is emerging as an important and complex player in cardiovascular, immunometabolic, and cancer regulation.

Circulating chromogranin A and its fragments as diagnostic and prognostic disease markers.

Abstract: Chromogranin A (CgA), a secretory protein released in the blood by neuroendocrine cells and neurons, is the precursor of various bioactive fragments involved in the regulation of the cardiovascular system, metabolism, innate immunity, angiogenesis, and tissue repair. After the original demonstration that circulating CgA can serve as a biomarker for a wide range of neuroendocrine tumors, several studies have shown that increased levels of CgA can be present also in the blood of patients with cardiovascular, gastrointestinal, and inflammatory diseases with, in certain cases, important diagnostic and prognostic implications. Considering the high structural and functional heterogeneity of the CgA system, comprising precursor and fragments, it is not surprising that the different immunoassays used in these studies led, in some cases, to discrepant results. Here, we review these notions and we discuss the importance of measuring total-CgA, full-length CgA, specific fragments, and their relative levels for a more thorough assessment of the pathophysiological function and diagnostic/prognostic value of the CgA system.

Sympathetic nervous system activation and heart failure: Current state of evidence and the pathophysiology in the light of novel biomarkers

Abstract: Heart failure (HF) is a complex clinical syndrome characterized by the activation of at least several neurohumoral pathways that have a common role in maintaining cardiac output and adequate perfusion pressure of target organs and tissues. The sympathetic nervous system (SNS) is upregulated in HF as evident in dysfunctional baroreceptor and chemoreceptor reflexes, circulating and neuronal catecholamine spillover, attenuated parasympathetic response, and augmented sympathetic outflow to the heart, kidneys and skeletal muscles. When these sympathoexcitatory effects on the cardiovascular system are sustained chronically they initiate the vicious circle of HF progression and become associated with cardiomyocyte apoptosis, maladaptive ventricular and vascular remodeling, arrhythmogenesis, and poor prognosis in patients with HF. These detrimental effects of SNS activity on outcomes in HF warrant adequate diagnostic and treatment modalities. Therefore, this review summarizes basic physiological concepts about the interaction of SNS with the cardiovascular system and highlights key pathophysiological mechanisms of SNS derangement in HF. Finally, special emphasis in this review is placed on the integrative and up-to-date overview of diagnostic modalities such as SNS imaging methods and novel laboratory biomarkers that could aid in the assessment of the degree of SNS activation and provide reliable prognostic information among patients with HF.

Catestatin reverses the hypertrophic effects of norepinephrine in H9c2 cardiac myoblasts by modulating the adrenergic signaling.

Abstract: Catestatin (CST) is a catecholamine release-inhibitory peptide secreted from the adrenergic neurons and the adrenal glands. It regulates the cardiovascular functions and it is associated with cardiovascular diseases. Though its mechanisms of actions are not known, there are evidences of cross-talk between the adrenergic and CST signaling. We hypothesized that CST moderates the adrenergic overdrive and studied its effects on norepinephrine-mediated hypertrophic responses in H9c2 cardiac myoblasts. CST alone regulated the expression of a number of fetal genes that are induced during hypertrophy. When cells were pre-treated CST, it blunted the modulation of those genes by norepinephrine. Norepinephrine (2 µM) treatment also increased cell size and enhanced the level of Troponin T in the sarcomere. These effects were attenuated by the treatment with CST. CST attenuated the immediate generation of ROS and the increase in glutathione peroxidase activity induced by norepinephrine treatment. Expression of fosB and AP-1 promoter–reporter constructs was used as the endpoint readout for the interaction between the CST and adrenergic signals at the gene level. It showed that CST largely attenuates the stimulatory effects of norepinephrine and other mitogenic signals through the modulation of the gene regulatory modules in a characteristic manner. Depending upon the dose, the signaling by CST appears to be disparate, and at 10–25 nM doses, it primarily moderated the signaling by the β1/2-adrenoceptors. This study, for the first time, provides insights into the modulation of adrenergic signaling in the heart by CST.

VMD: Visual molecular dynamics

Abstract: VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.

Haploview: analysis and visualization of LD and haplotype maps

Abstract: Summary: Research over the last few years has revealed significant haplotype structure in the human genome. The characterization of these patterns, particularly in the context of medical genetic association studies, is becoming a routine research activity. Haploview is a software package that provides computation of linkage disequilibrium statistics and population haplotype patterns from primary genotype data in a visually appealing and interactive interface. Availability: http://www.broad.mit.edu/mpg/haploview/ Contact: jcbarret@broad.mit.edu

AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility

Abstract: We describe the testing and release of AutoDock4 and the accompanying graphical user interface AutoDockTools. AutoDock4 incorporates limited flexibility in the receptor. Several tests are reported here, including a redocking experiment with 188 diverse ligand-protein complexes and a cross-docking experiment using flexible sidechains in 87 HIV protease complexes. We also report its utility in analysis of covalently bound ligands, using both a grid-based docking method and a modification of the flexible sidechain technique.

A method and server for predicting damaging missense mutations.

Abstract: To the Editor: Applications of rapidly advancing sequencing technologies exacerbate the need to interpret individual sequence variants. Sequencing of phenotyped clinical subjects will soon become a method of choice in studies of the genetic causes of Mendelian and complex diseases. New exon capture techniques will direct sequencing efforts towards the most informative and easily interpretable protein-coding fraction of the genome. Thus, the demand for computational predictions of the impact of protein sequence variants will continue to grow. Here we present a new method and the corresponding software tool, PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), which is different from the early tool PolyPhen1 in the set of predictive features, alignment pipeline, and the method of classification (Fig. 1a). PolyPhen-2 uses eight sequence-based and three structure-based predictive features (Supplementary Table 1) which were selected automatically by an iterative greedy algorithm (Supplementary Methods). Majority of these features involve comparison of a property of the wild-type (ancestral, normal) allele and the corresponding property of the mutant (derived, disease-causing) allele, which together define an amino acid replacement. Most informative features characterize how well the two human alleles fit into the pattern of amino acid replacements within the multiple sequence alignment of homologous proteins, how distant the protein harboring the first deviation from the human wild-type allele is from the human protein, and whether the mutant allele originated at a hypermutable site2. The alignment pipeline selects the set of homologous sequences for the analysis using a clustering algorithm and then constructs and refines their multiple alignment (Supplementary Fig. 1). The functional significance of an allele replacement is predicted from its individual features (Supplementary Figs. 2–4) by Naive Bayes classifier (Supplementary Methods). Figure 1 PolyPhen-2 pipeline and prediction accuracy. (a) Overview of the algorithm. (b) Receiver operating characteristic (ROC) curves for predictions made by PolyPhen-2 using five-fold cross-validation on HumDiv (red) and HumVar3 (light green). UniRef100 (solid ... We used two pairs of datasets to train and test PolyPhen-2. We compiled the first pair, HumDiv, from all 3,155 damaging alleles with known effects on the molecular function causing human Mendelian diseases, present in the UniProt database, together with 6,321 differences between human proteins and their closely related mammalian homologs, assumed to be non-damaging (Supplementary Methods). The second pair, HumVar3, consists of all the 13,032 human disease-causing mutations from UniProt, together with 8,946 human nsSNPs without annotated involvement in disease, which were treated as non-damaging. We found that PolyPhen-2 performance, as presented by its receiver operating characteristic curves, was consistently superior compared to PolyPhen (Fig. 1b) and it also compared favorably with the three other popular prediction tools4–6 (Fig. 1c). For a false positive rate of 20%, PolyPhen-2 achieves the rate of true positive predictions of 92% and 73% on HumDiv and HumVar, respectively (Supplementary Table 2). One reason for a lower accuracy of predictions on HumVar is that nsSNPs assumed to be non-damaging in HumVar contain a sizable fraction of mildly deleterious alleles. In contrast, most of amino acid replacements assumed non-damaging in HumDiv must be close to selective neutrality. Because alleles that are even mildly but unconditionally deleterious cannot be fixed in the evolving lineage, no method based on comparative sequence analysis is ideal for discriminating between drastically and mildly deleterious mutations, which are assigned to the opposite categories in HumVar. Another reason is that HumDiv uses an extra criterion to avoid possible erroneous annotations of damaging mutations. For a mutation, PolyPhen-2 calculates Naive Bayes posterior probability that this mutation is damaging and reports estimates of false positive (the chance that the mutation is classified as damaging when it is in fact non-damaging) and true positive (the chance that the mutation is classified as damaging when it is indeed damaging) rates. A mutation is also appraised qualitatively, as benign, possibly damaging, or probably damaging (Supplementary Methods). The user can choose between HumDiv- and HumVar-trained PolyPhen-2. Diagnostics of Mendelian diseases requires distinguishing mutations with drastic effects from all the remaining human variation, including abundant mildly deleterious alleles. Thus, HumVar-trained PolyPhen-2 should be used for this task. In contrast, HumDiv-trained PolyPhen-2 should be used for evaluating rare alleles at loci potentially involved in complex phenotypes, dense mapping of regions identified by genome-wide association studies, and analysis of natural selection from sequence data, where even mildly deleterious alleles must be treated as damaging.

Comparative Protein Structure Modeling Using MODELLER

Abstract: Functional characterization of a protein sequence is a common goal in biology, and is usually facilitated by having an accurate three-dimensional (3-D) structure of the studied protein. In the absence of an experimentally determined structure, comparative or homology modeling can sometimes provide a useful 3-D model for a protein that is related to at least one known protein structure. Comparative modeling predicts the 3-D structure of a given protein sequence (target) based primarily on its alignment to one or more proteins of known structure (templates). The prediction process consists of fold assignment, target-template alignment, model building, and model evaluation. This unit describes how to calculate comparative models using the program MODELLER and discusses all four steps of comparative modeling, frequently observed errors, and some applications. Modeling lactate dehydrogenase from Trichomonas vaginalis (TvLDH) is described as an example. The download and installation of the MODELLER software is also described.