Human Tyrosine Hydroxylase Natural Allelic Variation: Influence on Autonomic Function and Hypertension

The catecholamine biosynthetic pathway consists of several enzymatic steps in series, beginning with the amino acids phenylalanine and tyrosine, and eventuating in the catecholamines norepinephrine (noradrenaline) and epinephrine (adrenaline). Since the enzyme tyrosine hydroxylase (TH; tyrosine 3-mono-oxygenase; EC 1.14.16.2; chromosome 11p15.5) is generally considered to be rate-limiting in this pathway, probed as to whether common genetic variation at the TH gene occurred, and whether such variants contributed to inter-individual alterations in autonomic function, either biochemical or physiological. We began with sequencing a tetranucleotide (TCAT) repeat in the first intron, and found that the two most common versions, (TCAT)6 and (TCAT)10i, predicted heritable autonomic traits in twin pairs. We then conducted systematic polymorphism discovery across the ~8 kbp locus, and discovered numerous variants, principally non-coding. The proximal promoter block contained four common variants, and its haplotypes and SNPs (especially C-824T, rs10770141) predicted catecholamine secretion, environmental stress-induced BP increments, and hypertension. Finally, we found that two of the common promoter variants, C-824T (rs10770141) and A-581G (rs10770140), were functional in that they differentially affected transcriptional activity of the isolated promoter, disrupted recognition motifs for specific transcription factor binding, altered the promoter responses to the co-transfected (exogenous) factors, and bound the endogenous factors in the chromatin fraction of the nucleus. We concluded that common variation in the proximal TH promoter is functional, giving rise to changes in autonomic function and consequently cardiovascular risk

Determinants for chromogranin A sorting into the regulated secretory pathway are also sufficient to generate granule-like structures in non-endocrine cells.

In endocrine cells, prohormones and granins are segregated in the TGN (trans-Golgi network) from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner on specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures that resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of CgA (chromogranin A) is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP (green fluorescent protein) or a short epitope tag was expressed in COS-1 fibroblast cells and analysed by fluorescence and electron microscopy and pulse-chase labelling. Full-length CgA as well as deletion constructs containing the N-terminal 77 residues generated granule-like structures in the cell periphery that co-localized with co-expressed SgII (secretogranin II). These are essentially the same segments of the protein that were previously shown to be required for granule sorting in wild-type PC12 (pheochromocytoma cells) cells and for rescuing a regulated secretory pathway in A35C cells, a variant PC12 line deficient in granule formation. The results support the notion that self-aggregation is at the core of granule formation and sorting into the regulated pathway

The protein architecture of human secretory vesicles reveals differential regulation of signaling molecule secretion by protein kinases.

Secretory vesicles are required for release of chemical messengers to mediate intercellular signaling among human biological systems. It is necessary to define the organization of the protein architecture of the 'human' dense core secretory vesicles (DCSV) to understand mechanisms for secretion of signaling molecules essential for cellular regulatory processes. This study, therefore, conducted extensive quantitative proteomics and systems biology analyses of human DCSV purified from human pheochromocytoma. Over 600 human DCSV proteins were identified with quantitative evaluation of over 300 proteins, revealing that most proteins participate in producing peptide hormones and neurotransmitters, enzymes, and the secretory machinery. Systems biology analyses provided a model of interacting DCSV proteins, generating hypotheses for differential intracellular protein kinases A and C signaling pathways. Activation of cellular PKA and PKC pathways resulted in differential secretion of neuropeptides, catecholamines, and β-amyloid of Alzheimer's disease for mediating cell-cell communication. This is the first study to define a model of the protein architecture of human DCSV for human disease and health

Proteolytic Cleavage of Human Chromogranin A Containing Naturally Occurring Catestatin Variants: Differential Processing at Catestatin Region by Plasmin

The plasma level of chromogranin A (CgA) is elevated in genetic hypertension. Conversely, the plasma level of the CgA peptide catestatin is diminished in individuals with established hypertension and those with a genetic risk of this disease. Resequencing of the human CHGA gene identified three naturally occurring variants of catestatin (Gly364Ser, Pro370Leu, and Arg374Gln) that exhibit different potencies in inhibiting catecholamine secretion. Here, we have examined whether there is any differential processing of the three CHGA variants to catestatin by the endoproteolytic enzyme plasmin. Plasmin digestion of the purified CgA proteins generated a stable biologically active 14-amino acid peptide (human CgA360–373) from the wild-type, Gly364Ser, and Arg374Gln proteins despite the disruption of the dibasic site (Arg373Arg374) in the Arg374Gln variant. Unexpectedly, the action of plasmin in generating the catestatin peptide from the Pro370Leu protein was less efficient. The efficiency of cleavage at the dibasic Arg373↓Arg374 site in synthetic human CgA360–380 was 3- to 4-fold less in Pro370Leu CgA, compared with the wild type. Circular dichroism of the synthetic CgA352–372 suggested a difference in the amount of α-helix and β-sheet between the wild-type and Pro370Leu CgA peptides. Because the Pro370 residue is in the P4 position, the local secondary structure in the vicinity of the cleavage site may enforce the specificity or accessibility to plasmin. The less efficient proteolytic processing of the Pro370Leu protein by plasmin, coupled with the strong association of this variant with ethnicity, suggests that the Pro370Leu CHGA gene variant may contribute to the differential prevalence of cardiovascular disease across ethnic groups