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

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

Steven J Bark et al. PLoS One. 2012.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Functional categories of human DCSV soluble and membrane proteins.

Pie charts illustrate the relative portion of proteins in each functional category for the soluble (panel A) and membrane (panel B) fractions of human DCSV. Each functional category, with name and percent of the total number of DCSV proteins, of the pie chart is shown as a distinct color. The proteins comprising each functional category are listed in Table S3, and proteomics identification of soluble and membrane proteins of human DCSV are listed in Tables S1 and S2.

Figure 2
Figure 2. Quantification of protein categories of human dense core secretory vesicles.

Relative quantification of proteins in the main functional categories were assessed by normalized spectral abundance factors (NSAF), as described in Experimental Procedures S1. Bar graphs illustrate average NSAF (sum) for each protein group with s.e.m. NSAF values for human DCSV proteins are provided in Table S4.

Figure 3
Figure 3. Cytoscape systems biology analyses of the human DCSV proteome.

Components of the DCSV proteomics data were analyzed by the Cytoscape systems biology program for predicting protein interaction networks. The functional protein categories are illustrated on the right hand side. Based on quantitative NASF data of the proteins, individual proteins are indicated as predominantly soluble (green circles), predominantly membrane (red circles), or present in both soluble and membrane at similar levels (yellow) (Figure 3). These color-coded protein symbols are those which were quantitated by NSAF. Proteins illustrated by grey circles are those which were identified, but not quantitated since they did not meet the criteria for quantitation in at least 3 out of 4 nano-LC-MS/MS runs.

Figure 4
Figure 4. Systems biology analyses distinguishes protein kinase A (PKA) and protein kinase C (PKC) pathways in human DCSV.

Human DCSV proteins interacting with PKA or PKC are illustrated in green or red circles, respectively. Proteins interacting with both PKA and PKC are shown in yellow.

Figure 5
Figure 5. Differential regulation of neuropeptides, catecholamines, and β-amyloid secretion by activation of PKA and PKC in neuronal-like chromaffin cells.

Adrenal medullary chromaffin cells in primary culture (bovine) were treated with forskolin that activates protein kinase A (PKA) that activates PKA via adenylyl cyclase stimulation and cAMP production, or with PMA (phorbol myristate acetate) that directly activates PKC. In time-course studies for treatment with forskolin or PMA for 15 minutes to 6 hours, the media was collected for measurement of secreted (Met)enkephalin and galanin neuropeptides (panels a and b, respectively), beta-amyloid peptide (Aβ(1–40), panel c), and the catecholamines dopamine, norepinephrine, and epinephrine (panels d, e, and f, respectively). Data for Control untreated cells (○), forskolin-treated cells (•), and PMA-treated cells (X) are plotted. Data for each time point represents the mean ± s.e.m. (n = 6); error bars are illustrated, and they are often smaller than the symbol.

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