Proteomics of Neuroendocrine Secretory Vesicles Reveal Distinct

Proteomics of Neuroendocrine Secretory Vesicles Reveal Distinct Functional Systems for Biosynthesis and Exocytosis of Peptide Hormones and Neurotransmitters Jill Wegrzyn,†,+ Jean Lee,‡,+ John M. Neveu,§ William S. Lane,§ and Vivian Hook*,†,‡ Skaggs School of Pharmacy and Pharmaceutical Sciences, Departments of Pharmacology, Neuroscience, and Medicine, School of Medicine, University of California, San Diego, La Jolla, California 92093, and Microchemistry and Proteomics Analysis Facility, Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138 Received September 25, 2006

Regulated secretory vesicles produce, store, and secrete active peptide hormones and neurotransmitters that function in cell-cell communication. To gain knowledge of the protein systems involved in such secretory vesicle functions, we analyzed proteins in the soluble and membrane fractions of dense core secretory vesicles purified from neuroendocrine chromaffin cells. Soluble and membrane fractions of these vesicles were subjected to SDS-PAGE separation, and proteins from systematically sectioned gel lanes were identified by microcapillary LC-MS/MS (µLC-MS/MS) of tryptic peptides. The identified proteins revealed functional categories of prohormones, proteases, catecholamine neurotransmitter metabolism, protein folding, redox regulation, ATPases, calcium regulation, signaling components, exocytotic mechanisms, and related functions. Several novel secretory vesicle components involved in proteolysis were identified consisting of cathepsin B, cathepsin D, cystatin C, ubiquitin, and TIMP, as well carboxypeptidase E/H and proprotein convertases that are known to participate in prohormone processing. Significantly, the membrane fraction exclusively contained an extensive number of GTP nucleotide-binding proteins related to Rab, Rho, and Ras signaling molecules, together with SNARErelated proteins and annexins that are involved in trafficking and exocytosis of secretory vesicle components. Membranes also preferentially contained ATPases that regulate proton translocation. These results implicate membrane-specific functions for signaling and exocytosis that allow these secretory vesicles to produce, store, and secrete active peptide hormones and neurotransmitters released from adrenal medulla for the control of physiological functions in health and disease. In summary, this proteomic study illustrates secretory vesicle protein systems utilized for the production and secretion of regulatory factors that control neuroendocrine functions. Keywords: secretory vesicles • proteome • proteases • prohormones • mass spectrometry • neuroendocrine

Introduction Secretory vesicles of the regulated secretory pathway are responsible for the biosynthesis, storage, and secretion of peptide hormones and neurotransmitters.1-6 Adrenal medullary chromaffin cells secrete these neurohumoral agents in response to stress via cholinergic receptor activation to regulate physiological systems.4,5 The dense core secretory vesicles of chromaffin cells, known as chromaffin granules, are known to provide an excellent model for investigating secretory vesicle * To whom correspondence should be addressed: Dr. Vivian Hook, Skaggs School of Pharmacy and Pharmaceutical Sciences, Univ. of Calif., San Diego, 9500 Gilman Dr., MC 0744, La Jolla, CA 92093-0744. Phone, (858) 822-6682; fax, (858) 822-6681; e-mail, [email protected]. + These two authors contributed equally to this study. † Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California. ‡ Departments of Pharmacology, Neuroscience, and Medicine, School of Medicine, University of California. § Harvard University.

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mechanisms that participate in producing peptide hormones and neurotransmitters in neuroendocrine systems.1-6 Knowledge of the protein components of the chromaffin secretory vesicle is required to understand the biochemical systems that participate in peptide hormone production and secretion, the key biological function of these secretory vesicles. Previous studies have largely investigated individual components, rather than the entire protein composition of this organelle.1-6 It is important to investigate the proteome of these secretory vesicles to understand the different protein systems that may be utilized for peptide hormone biosynthesis in this organelle, as well as for secretion of potent peptide hormones. Therefore, this proteomic study analyzed proteins of soluble and membrane components of purified chromaffin secretory vesicles. Using a GeLC-MS/MS strategy, we analyzed vesicle proteins by one-dimensional SDS-PAGE separation and tryptic digestion of systematically sectioned gel lanes, followed by mi10.1021/pr060503p CCC: $37.00

 2007 American Chemical Society

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crocapillary HPLC tandem mass spectrometry. Identified proteins were then organized according to functional categories. Comparisons of soluble and membrane protein components revealed distinct and similar proteins in the two subcompartments of chromaffin secretory vesicles in several categories. The main functional categories of proteins represented those for production of peptide hormones and neurotransmitters, regulation of internal vesicle conditions, and trafficking of secretory vesicle components for exocytosis. Notably, membranes exclusively contained an extensive number of GTPbinding proteins that may be involved in organelle trafficking. These data demonstrate the complexity of protein functions in dense core secretory vesicles of neuroendocrine chromaffin cells for the biosynthesis, storage, and secretion of peptide hormone and neurotransmitter molecules for cell-cell communication required for control of physiological functions.

µm spray tip (New Objective, Inc., Woburn, MA) packed with 10 cm of YMC C18 media. The reversed-phase HPLC used was a Hewlett-Packard 1100 HPLC system equipped with a FAMOS (Dionex) autosampler. The HPLC was connected to a threeway tee where flow was split ∼300:1 prior to the injection port of the autosample, with ∼350 nL/min flow directed to the analytical column. HPLC solvents were 0.29% acetic acid in water (A buffer) and acetonitrile (B buffer) with a linear gradient from 1% B to 51% B over 50 min, followed by column wash and reequilibration. Mass spectrometry was performred on a Thermo DECA XP Plus ion trap operating in a data-dependent mode (with the four most-intense ions selected for MS/MS fragmentation from each full MS scan) with dynamic exclusion enabled. Acquired MS/MS spectra were correlated with known sequences using SEQUEST, and the results were manually confirmed for fidelity. An in-house neural network was used to produce a normalized single ScoreFinal (Sf) from the five normal SEQUEST scores plus charge state, peptide length, and database size. Only protein identifications whose peptide spectra summed to an Sf of >1.10 were accepted. Bioinformatic Analysis of Identified Proteins. Protein identifications were based on a minimum of two unique peptide assignments. When ortholog proteins were identified, the choice was made to select those from species Bos taurus, when possible, or from nearest species. Importantly, tryptic peptides shared between several proteins are only counted for the protein that has, over all, the most matching, unique peptides. Batch Entrez13,14 (http://ncbi.nlm.nih.gov/entrez/ batchentrez.cgi?db)Protein) was used to generate FASTA formatted protein sequence databases for each GenInfo Identifier (GI) number for proteins identified in the MS experiment. BLASTCLUST was used to perform pairwise comparisons followed by single-linkage clustering of the statistically significant matches (>95% homology over 90% of the sequence length) (http://www.ncbi.nlm.nih.gov/blast/). The protein list is thus the smallest set of proteins explaining the identified protein present. Following this analysis, an annotated, nonredundant table of soluble and membrane proteins was compiled (Tables 1 and 2). The functional categories of identified proteins were defined by the gene ontology resource (http://www.geneontology.org).15 Further information on the function of proteins was obtained using the KEGG pathway databases, as well as through the MEROPS database for proteases.16 A series of GO terms in each category was acquired using text searching of relevant keywords relating to function and localization. In addition to gene ontologies, both identified and unidentified protein sequences were queried against the TIGRfam (http://www. tigr.org/TIGRFAMs/) database17 to assess protein family. Several peptide sequences were identified that were not functionally annotated in initial database searches. On the basis of the identified tryptic peptide sequences, predicted mouse and human sequences were aligned back to bovine sequences using the TIGR gene indices (http://tigrblast.tigr.org/tgi/). Proteins that demonstrated strong homology to existing bovine sequences were included in the nonredundant assembly of identified chromaffin granule proteins. Proteins that appeared in multiple gel slices were examined in terms of proteolytic processing. Proteins with tryptic peptides in more than two gel slices and with peptides representative of the C- and N-terminus of the identified protein were recorded (Figure 5).

Experimental Procedures SDS-PAGE of Soluble and Membrane Fractions from Purified Chromaffin Secretory Vesicles. Dense core secretory vesicles from sympathoadrenal chromaffin cells (known as chromaffin granules) were purified from fresh bovine adrenal glands by sucrose density centrifugation as described previously.7,8 The density gradient method results in highly purified chromaffin granules, and this method has been established in previous studies, demonstrating the lack of biochemical markers for other cellular organelles including lysosomes, cytoplasm, mitochondria, perioxisomes, and endoplasmic reticulum.9-11 The morphology and homogeneity of these isolated vesicles were examined by electron microscopy, performed as described previously.8 Results demonstrated the integrity and purity of chromaffin granules for this project, indicating that highly purified chromaffin granules comparable to previous studies8 were obtained for this project. For proteomic studies, purified chromaffin granules were lysed and separated into soluble and membrane components under conditions of isotonic buffer conditions (150 mM NaCl in buffer) to represent isotonic salt conditions within the vesicle, as we have described previously.7 During preparation of soluble and membrane fractions, a cocktail of protease inhibitors was included which consisted of 10 µM pepstatin A, 10 µM leupeptin, 10 µM chymostatin, 10 µM E64c, and 1 mM AEBSF. Soluble and membrane fractions were each subjected to one-dimensional SDS-PAGE (12% polyacrylamide, NuPAGE gels), and fractionated proteins were collected as gel slices (10 slices per gel lane) that represented low-to-high molecular weight proteins. Identification of Proteins by Mass Spectrometry. Gel slices were each subjected to tryptic digestion, as described previously12 for identification of proteins by µLC-MS/MS. The gel slices were washed with 50% acetonitrile (aqueous), and then dried completely in a SpeedVac. They were then reduced and alkylated using dithiothreitol (DTT) and iodoacetamide, washed with alternating aliquots of 100 mM ammonium bicarbonate and neat acetonitrile twice, dried as before, and rehydrated with the addition of trypsin and 50 mM ammonium bicarbonate. The samples were incubated with shaking at 37 °C for 16 h during digestion. Afterward, the free liquid was removed and pooled with extractions from the gel of 20 mM ammonium bicarbonate and 5% formic acid in acetonitrile, then dried to a final volume of 20 µL. Nanocapillary reversed-phase HPLC was carried out using a 75 µm inner diameter (i.d.) glass capillary column with 15

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Table 1. Identification of Soluble Chromaffin Vesicle Proteins Separated by SDS-PAGEa

a The soluble fraction was subjected to one-dimensional SDS-PAGE, and 10 gel slices of different molecular weight ranges were excised, subjected to tryptic digestion, and identified by LC-MS/MS. Identification was based on bovine sequences, as well as those of other mammalian species with references to the Genbank number utilized in database searches. The table shows a nonredundant list of the proteins (shown alphabetically) and their presence in the different gel slices, as indicated by the number of tryptic peptides subjected to analysis by tandem MS/MS and by the percentage of amino acid coverage of the identified protein. Gel slices of S1 to S10 represent high to low molecular weights.

Results Identification of Proteins in Purified Secretory Vesicles of Chromaffin Cells. Proteomic studies were utilized to gain knowledge of protein systems involved in secretory vesicle production and secretion of peptide hormones and neurotransmitters. This study obtained purified dense core secre1654

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tory vesicles by sucrose density gradient centrifugation. The integrity and purity of homogeneous secretory vesicles were demonstrated by electron microscopy (Figure 1). These vesicles were lysed using freeze-thawing under isotonic buffer conditions at pH of 6.0 that represents the internal milieu of secretory vesicles.7,18 Soluble and membrane fractions were obtained by

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Proteomics of Secretory Vesicles for Neuropeptide Production Table 2. Identification of Membrane Chromaffin Vesicle Proteins Separated by SDS-PAGEa

a The membane fraction was subjected to one-dimensional SDS-PAGE, and 10 gel slices of different molecular weight ranges were excised, subjected to tryptic digestion, and identified by LC-MS/MS. Identification was based on bovine sequences, as well as those of other mammalian species with reference to the Genbank number utilized in database searches. The table shows a nonredundant list of the proteins (shown alphabetically) and their presence in the different gel slices, as indicated by the number of tryptic peptides subjected to analysis by tandem MS/MS and by the percentage of amino acid coverage of the identified protein. Gel slices of M1 to M10 represent high to low molecular weights.


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Figure 3. Distinct and common proteins in the soluble and membrane fractions of chromaffin secretory vesicles. The Venn diagram illustrates the number of proteins identified in the soluble (blue) and membrane fractions (green) of chromaffin secretory vesicles. The overlapping region of the diagram (purple) illustrates the group of proteins present in both soluble and membrane fractions of the chromaffin secretory vesicles. Figure 1. Examination of purified chromaffin secretory vesicles by electron microscopy. Dense core secretory vesicles were purified using sucrose gradient centrifugation from adrenomedullary chromaffin cells. The integrity and homogeneity of these isolated secretory vesicles were demonstrated here using electron microscopy.

Figure 2. Soluble and membrane proteins separated by onedimensional SDS-PAGE gels. Soluble and membrane proteins from lysed chromaffin secretory vesicles were separated by centrifugation (100 000g). Soluble and membrane protein fractions were each subjected to one-dimensional SDS-PAGE gels and stained with Coomassie blue, and each lane was divided into 10 gel slices for tryptic digestion and identification of eluted peptides by tandem mass spectrometry, as described in the Experimental Procedures.

centrifugation. Isotonic salt conditions at in vivo pH conditions provided separation of soluble and membrane proteins that represent their subvesicular location. Soluble and membrane fractions were subjected to one-dimensional SDS-PAGE gels (Figure 2) which separated proteins of approximately 5-150 kDa, with the majority of proteins in the range of 5-100 kDa. Each gel lane was divided into 10 slices and subjected to tryptic digestion, and tryptic peptides were identified using µLC-MS/MS. Proteins identified from soluble and membrane fractions are shown in Tables 1 and 2, respectively. These tables illustrate the nonredundant lists of identified proteins from each gel slice, along with the percent amino acid coverage. 1656


After clustering and functional categorization of the identified proteins, 63 proteins were identified in the soluble fraction and 80 proteins were identified in the membrane fraction (Tables 1 and 2). Among these proteins, 20 (20%) were present in only the soluble fraction, and 39 (38%) were present in only the membrane fraction. Soluble and membrane fractions shared 43 proteins (42%) that were present in both subcompartments of secretory vesicles (Figure 3). Functional Categories of Soluble and Membrane Proteins of Secretory Vesicles: Similarities and Differences. Comparison of soluble and membrane protein components demonstrated similarities and differences in functional categories consisting of prohormones and neurotransmitter factors, protease components, neurotransmitter metabolizing enzymes, redox regulation, ATPases, protein folding, nucleotide binding and signal transduction, exocytosis, structural proteins, related categories, as well as several unidentified proteins (Figure 4). One-third of soluble proteins and approximately one-fourth of membrane proteins consisted of prohormones and proteases, representing the largest portions of identified proteins. Significantly, one-fourth of membrane proteins were represented by signal transduction functions that were not detected in the soluble fraction. Prohormones and Neurotransmitter Factors. Similar prohormones and neurotransmitter molecules were present in soluble and membrane fractions (Table 3). Chromogranin A, B, and C represent highly abundant proteins17 of 60-75 kDa in both soluble and membrane fractions. The related granins secretogranin III12 and NESP-55 (neuroendocrine secretory protein 55)19,20 were also present. Following the chromogranins, the enkephalins and neuropeptide Y21,22 were also identified. In addition, the prohormone adrenomedullin was present, a potent hypotensive peptide.23,24 Cathelicidins are newly identified peptides in these secretory vesicles; these peptides possess anti-microbial activity against fungi and both Gram-positive and Gram-negative bacteria.25,26 Growth factors were also identified which consisted of VGF nerve growth factor (inducible), vascular endothelial growth factor, and transforming growth factor-beta binding protein.27-29 Decorin was also found in these vesicles; decorin is known to undergo proteolysis by matrix metalloproteinases30 and promotes axon growth and attenuates scar formation in brain.31,32 Results also suggested that several prohormone precursors undergo proteolysis within the secretory vesicle, based on their presence in several gel slices of different apparent molecular weights (Figure 5).


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Figure 4. Diagram of functional protein categories in soluble and membrane domains of chromaffin secretory vesicles. (A) Soluble proteins: relative portion of proteins in different functional categories. The percentages of identified proteins in the soluble fraction are illustrated according to the indicated functional categories. (B) Membrane proteins: relative portion of proteins in different functional categories. The percentages of identified proteins in the membrane fractions are illustrated according to the indicated functional categories. Functional protein categories are Prohormones (blue), Protease Systems (bright yellow), Neurotransmitters (blue-gray), Redox (green), ATPases (pink), Protein Folding (light blue), Carbohydrate Functions (brown), Lipid Functions (light purple), GTP-binding (maroon), Exocytosis (orange), Calcium Regulating (dark turquoise), Structural (light yellow), Cell Adhesion (dark purple), and Miscellaneous (gray).

Interestingly, low molecular weight forms of enkephalin neuropeptides less than 12 kDa derived from proenkephalin of 33-35 kDa33 were present in the soluble fraction but not the membrane fraction of chromaffin granules. These data indicate that a multitude of peptide hormones and neurotransmitter factors are present in these secretory vesicles. Protease Systems. A majority of the identified prohormones undergo proteolytic processing within the secretory vesicle to generate active neuropeptides and neurohumoural factors. This study (Table 3) identified several proteases of different mechanistic classes, which included the subtilisin-like prohormone convertases 1 and 2, along with the metalloprotease carboxy-

peptidase E (CPE), which participate in prohormone processing.1,34,35 CPE also functions as a prohormone sorting receptor.36 Regulators of PC1 and PC2 were identified consisting of proSAAS37 and 7B2,38 respectively. Interestingly, cathepsins B and D were identified, indicating a novel location for these previously known lysosomal proteases. The localization of cathepsin B in these secretory vesicles has been confirmed by immunoelectron microscopy.39 Cystatin C, identified in the membrane and soluble components, is the most effective inhibitor of the cystatin superfamily40 against cathepsin B.41 Ubiquitin was identified, which is a highly conserved 76 amino acid protein that is covalently linked to proteins targeted for Journal of Proteome Research • Vol. 6, No. 5, 2007 1657

research articles Table 3. Functional Categories of Proteins in Soluble and Membrane Fractions of Chromaffin Secretory Vesiclesa

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Table 3 (Continued)

a This table compares soluble (blue) and membrane (green) protein components that have been organized into functional categories, illlustrating differences and similarities in relative distribution of identified proteins in these regions of chromaffin secretory vesicles.


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Figure 5. Proteolysis of secretory vesicle proteins indicated by SDS-PAGE gels. Identification of prohormones and proproteins in gel slices of different apparent molecular weight ranges is illustrated. The presence of a particular proprotein in several gel slices of different molecular weight ranges provides evidence for proteolysis of the protein within chromaffin secretory vesicles. Among all identified proteins in these secretory vesicles, approximately 10% of these proteins were present as a variety of proteolytic fragments, based on evidence for different molecular weight forms of the proprotein (as illustrated in this figure).

degradation by the ubiquitin-proteosome system.42,43 These findings suggest that ubiquitin-targeted protein degradation by proteosomes may occur in secretory vesicles. TIMP, tissue inhibitor of metalloproteinase, was also present. These identified protease system components were mostly present in both soluble and membrane fractions. Neurotransmitter Enzymes and Transporters. Secretory vesicles contain enzymes involved in catecholamine neurotransmitter synthesis,4-6 consisting of tyrosine 3-hydroxylase (TH), dopamine beta-monooxygenase, and phenylethanolamine N-methyltransferase (PNMT4-6) (Table 3). The synaptic vesicle monoamine transporter was also identified, which is involved in the vesicular localization of catecholamines.44,45 Redox Regulation. Soluble and membrane proteins that participate in regulation of redox (reduction-oxidation) condi1660


tions (Table 3) were represented by cytochrome b561 and cytochrome C. Cytochrome b561 donates electrons back and forth with cytochrome C and acts as an electron conductor for dopamine beta-monooxygenase.46 The reducing agent glutathione47 and glutathione peroxidase, a glutathione metabolizing enzyme, were also found. In addition, cytochrome P450 was identified, which is known to metabolize small molecules such as arachidonic acid, as well as drug molecules.48,49 ATPases for Proton Regulation. Multiple ATPases were present in secretory vesicles, consisting of H+ transporting ATPase, aminophospholipid transporter ATPase, V-ATPase for vacuolar-type ATPases, TER ATPase (transitional endoplasmic reticulum ATPase), and related ATPases.50-53 These ATPases participate in proton transport and related functions that can maintain the acidic internal pH of secretory vesicles.


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Protein Folding. The presence of the chaperones Hsp27, Hsp40, and chaperonin 10, as well as peptidylprolyl isomerase B,54-56 suggests their roles for regulating protein folding in secretory vesicles. Appropriate protein functions within secretory vesicles may utilize chaperonins for protein folding that may be required for activities of vesicular proteins.

tified with lipid functions were N-acylsphingosine amidohydrolase and SAP-1 (cerebroside sulfate activator).80-82 Cell Adhesion Proteins. Several proteins involved in cell adhesion were identified which consisted of neural cadherin and contactin 1. Cadherins regulate cell adhesion in a calciumdependent manner83 and modulate axon-spine contact of neurons.84 Contactin (also known as F3) is known as a neuronal cell adhesion molecule for regulating neurite outgrowth.85

Rab and Nucleotide-Binding Proteins for Signal Transduction. The membrane domain exclusively contained numerous proteins of the Rab family critical to intracellular trafficking and transport. Multiple Rab proteins consisting of 4 different members were present in the membrane fraction of secretory vesicles, whereas only one of these isoforms was present in the soluble fraction. This collection of GTP-binding Rab proteins in the membranes of these vesicles has not previously been described, with the exception of Rab3A that enhances regulated secretion.57 The Rab proteins function in the tethering/docking of vesicles on target membranes, as well as in vesicle exocytosis.58-60 Membranes of these vesicles also possess several Rho GTP-binding proteins that were not identified in the soluble fraction. These data implicate Rho and Ras proteins, members of the RAS superfamily of GTP hydrolysis-coupled signal transduction relay proteins, in secretory vesicle dynamics.59-62 Synaptotagmin and Synaptophysin Proteins. Synaptophysins are vesicle-associated membrane proteins that bind with synaptobrevin to inhibit binding of other SNARE proteins in the regulation of exocytosis.63,64 Synaptogamins 1 and 7 in the membrane fraction are known as calcium sensors to regulate fast exocytosis.65,66 Ca2+ Regulation. Several annexin proteins were identified in chromaffin secretory vesicles as membrane and soluble proteins. Annexins can function in calcium-dependent phospholipid binding during secretory vesicle exocytosis.67-69 Four annexin proteins were identified consisting of annexins A2, A4, A11, and VI. Members of the annexin family mediate binding of Ca2+ ions for the regulation of Ca2+ currents across membranes or Ca2+ concentrations within cellular compartments.67-71 The Ca2+-binding protein calnuc was also identified in the membrane fraction of secretory vesicles. Calnuc is known to be stored and processed in the Golgi and secreted in the constitutive secretory pathway.72 Its localization within dense core secretory vesicles implicates calnuc in the regulated secretory pathway for release of hormone and neurotransmitter molecules. Structural Proteins. Structural proteins including talin, tubulin, and elastin microfibril interlacer were identified in the soluble fraction of secretory vesicles. These proteins are likely to serve as structural components of the granule itself, as well as mediators of communication between cells with the assistance of Rab and related proteins. Talin 1 participates in the biology of periactive zones of synapses by regulating actin and clathrin coat dynamics.73 These structural components may contribute to vesicle trafficking mediated by components of the cytoskeleton such as actin, tubulin, and intermediate filaments, as well as by motors and accessory proteins that function with each of the cytoskeletal components.74,75 Carbohydrate and Lipid Functions. Carbohydrate and lipid functions within the chromaffin secretory vesicles were suggested by the presence of proteins that function in carbohydrate and lipid metabolism. Carbohydrate metabolizing proteins identified consisted of beta-mannosidase, cell surface glycoprotein A15, glucosidase II, golgi sialoglycoprotein MG160, and N-acetylglucosaminyltransferase V.76-79 Proteins iden-

Discussion Analysis of the proteome of dense core secretory vesicles of neuroendocrine chromaffin cells in this study has identified new and existing proteins of distinct functional categories that provide the systems for secretory vesicle production and release of peptide hormones and neurotransmitters. Analysis of the identified proteins yielded 14 different functional categories consisting of prohormones, protease systems, catecholamine metabolism, protein folding, redox functions, proton regulation, signaling components for secretory vesicle exocytosis mechanisms, and related functions. These categories can be combined into three main secretory vesicle systems for (1) production of peptide hormones, neurotransmitters, and neurohumoral factors; (2) maintenance of internal vesicular conditions; and (3) vesicular trafficking for exocytosis of secretory vesicles contents (illustrated in Figure 6). These distinct yet complementary groups of proteins provide the functional systems used for secretory vesicle production, storage, and secretion of cellcell communication agents that regulate physiological systems. Significantly, with respect to vesicular trafficking (Figure 6), the study identified numerous Rab and related nucleotidebinding proteins not previously known to be present in these chromaffin secretory vesicles. Numerous Rab protein isoforms were identified (Table 3) consisting of Rab3, Rab7, Rab14, Rab21, and Rab35. More than 60 Rabs are present in the human species. While these secretory vesicles were isolated from bovine adrenal medulla, identification of multiple forms of Rabs in these vesicles demonstrates the complexity of membrane Rabs involved in secretory vesicle exocytosis in mammalian cells. Clearly, the protein machinery for secretory vesicle formation, docking, and fusion, in the secretory process, is exquisitely organized to allow regulated secretion to occur. Organelle proteins of the secretory vesicle are critical for the biosynthesis of multiple peptide hormones and neurotransmitters, catecholamines, and neuroeffectors that are secreted to regulate target organ systems. Proteins in both the soluble and membrane fractions of chromaffin secretory vesicles were represented by the main categories of prohormones, protease and enzyme systems for the production of active neuropeptides and catecholamine transmitters, as well as proteins involved in maintaining the unique intravesicular environment for secretory vesicle functions. The prohormone category of proteins comprised a major portion of proteins in both the soluble and membrane fractions. The major prohormones, or proneuropeptide precursors, were identified in this study as chromogranins A, B, and C. Related granins were also identified. The precursors for enkephalin and neuropeptide Y (NPY) were also present. Newly identified peptide components were represented by cathelicidins that inhibit microbial growth,25,26 and growth factor related proteins.27-29 The presence of these proteins in gel slices of different apparent molecular weights suggested proteolysis of these proteins. In addition, identification of tryptic peptides Journal of Proteome Research • Vol. 6, No. 5, 2007 1661

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Figure 6. Functional protein systems for secretory vesicle production, storage, and release of peptide hormones, neurotransmitters, and neurohumoral agents. The main function of the secretory vesicle organelle is to produce, store, and release active peptide hormones, neurotransmitters, and neurohumoral factors. The primary steps for these secretory vesicle functions are represented by processes required in the formation of secretory vesicles and exocytotic mechanisms to allow secretion of secretory vesicle contents. On the basis of the proteomic data obtained in this study of proteins in chromaffin secretory vesicles, functional protein systems involved in secretory vesicle function utilizes proteins for (1) production of hormones, neurotransmitters, and neuromodulatory factors; (2) generating selected internal vesicular conditions for reducing condition, acidic pH conditions maintained by ATPases, and chaperones for protein folding; and (3) vesicular trafficking mechanisms to allow mobilization of secretory vesicles for exocytosis, which utilizes proteins for nucleotide-binding, calcium regulation, and vesicle exocytosis. Among these protein systems within secretory vesicles, a significant number of GTP nucleotide-binding proteins were identified that are predicted to be involved in vesicular trafficking. In addition, a small number of proteins were identified that are known to be involved in cell adhesion. Overall, these protein systems are coordinated to allow the secretory vesicle to achieve release of active biomolecules for cell-cell communication in the control of neuroendocrine functions.

representative of the C- and N-terminal ends of the identified protein provided further validation of proteolysis. These prohormones and proproteins undergo limited proteolysis in these secretory vesicles by several protease and protease inhibitor systems. New and existing components involved in proteolysis were identified in this category. Newly identified components included cathepsin B and cathepsin D, cystatin C, ubiquitin, and TIMP (tissue inhibitor of metalloproteinase 1). While cathepsins B and D have been known to function in lysosomes, results from this study support novel secretory vesicle functions for these cathepsins. Indeed, recent immunoelectron microscopic studies indicate the localization of cathepsin B,42 as well as cathepsin L,86 in these chromaffin secretory vesicles. The presence of polyubiquitin represents a newly identified protease system within these secretory vesicles. Cystatin C and TIMP are also newly identified protease inhibitor components of chromaffin secretory vesicles. Previously known prohormone convertases 1 and 2 (PC1 and PC2) and 1662


carboxypeptidase E proteases,1,2 combined with 7B238 and proSAAS37 protease regulators, were also identified. The newly identified proteases and inhibitors contribute to the complexity of existing secretory vesicle protease systems. This study also identified enzymes responsible for the metabolism of catecholamines in these secretory vesicles. Results identified tyrosine 3-hydroxylase, dopamine betamonooxygenase, phenylethanolamine N-methyltransferase, and the synaptic vesicle monoamine transporter that participate in catecholamine production and storage.3-6 The special internal environment within secretory vesicles allows proteases and catecholamine synthesizing enzymes to function. In vivo conditions for reducing equivalents, acidic internal pH (approximately pH 5.0-6.0), and protein folding are required for enzymatic functions.18,87 Indeed, these secretory vesicles contain cytochrome and glutathione regulatory proteins that modulate the internal redox environment, membrane ATPases as proton pumps for maintenance of the acidic pH


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within these vesicles, and chaperonins that modulate protein folding.

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Release of bioactive neurohumoral molecules stored in secretory vesicles involves calcium regulation. Numerous annexin protein isoforms were identified in chromaffin secretory vesicles that are known to function as calcium-dependent phospholipid binding proteins.67-69 Calcium release from chromaffin granules during exocytosis is regulated by annexin70 in the control of exocytosis for calcium-dependent vesicularmediated secretion.71 In addition, the Ca2+-binding protein calnuc72 was also identified as a membrane component of chromaffin vesicles. Prior studies demonstrated calnuc as a Golgi protein. Results of this study showing the presence of calnuc in regulated secretory vesicles suggests that it may undergo trafficking into secretory vesicles of the regulated secretory pathway Regulation of exocytotic release of secretory vesicle contents utilizes cell signaling mechanisms to initiate and stimulate vesicular secretion of neuropeptides, catecholamines, and neurohumoral agents. This study identified a multitude of Rab and Rho signal transduction components present in the membranes of regulated secretory vesicles from neuroendocrine chromaffin cells. These GTP-binding signal transduction proteins, synaptobrevin, and synaptotagmin work with associated structural and Ca2+-regulating protein components of the cell, including cell adhesion molecules, to mediate the Ca2+dependent process of exocytosis. These identified protein components in chromaffin secretory vesicles represent multiple functional categories that participate in the biosynthesis, storage, and secretion of potent peptide hormones and neurotransmitters, catecholamines, and neurohumoral agents in response to specific cellular stimuli that induce regulated exocytosis. Numerous protein components were identified which were not previously known to reside in these regulated secretory vesicles.88,89 Proteins for prohormone processing in the biosynthesis of molecules for cell-cell communication combined with proteins for signal transduction mechanisms in exocytosis represented the largest portions of the identified secretory vesicle proteins. Proteins that regulate the internal vesicular conditions for oxidation-reduction, proton transport, and protein folding are present in these secretory vesicles. Furthermore, proteins for calcium regulation and structural proteins for vesicle exocytosis are present in the secretory vesicle. Overall, this proteomic study demonstrates the complexity of proteins whose numerous functions are coordinated and organized for secretory vesicle production and exocytosis of active molecules that participate in regulating target physiological processes in normal and disease conditions. Abbreviations: HPLC, high pressure liquid chromatography; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; GeLC-MS/MS, systematically sectioned one-dimensional SDS-PAGE gel followed by enzymatic digestion and LC-MS/MS analysis; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Acknowledgment. Grant support from the National Institutes of Health to V.H. is appreciated. References (1) Hook, V. Y. H.; Azaryan, A. V.; Hwang, S.-R.; Tezapsidis, N. Proteases and the emerging role of protease inhibitors in prohormone processing. FASEB J. 1994, 8, 1269-1278.


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PR060503P


Proteomics of Neuroendocrine Secretory Vesicles Reveal Distinct

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