Identification of the Region within the Neuroendocrine Polypeptide 7B2 Responsible for the Inhibition of Prohormone Convertase PC2

Horssen, A.M. van; Hurk, Wilhelmina H. van den; Bailyes, E M; Hutton, John C.; Martens, Gerard J.M.; Lindberg, Iris

doi:10.1074/jbc.270.24.14292

Cited by 71 publications

(55 citation statements)

References 30 publications

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“…Several lines of evidence suggest that 7B2 is both an inhibitor and chaperone of PC2 Martens et al, 1994;Benjannet et al, 1995;Zhu and Lindberg, 1995). Inhibition is caused by a C-terminal region of 7B2 that is slowly cleaved by PC2 (van Horssen et al, 1995;Zhu et al, 1996). Interestingly, CPE removes the C-terminal basic residues from the PC2-cleaved 7B2 and eliminates the inhibitory potency of the 7B2 fragment (Zhu et al, 1996); this could partially explain the defective endoprotease processing of prohormones seen in Cpe fat /Cpe fat mice (Naggert et al, 1995;Fricker et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

Identification and Characterization of proSAAS, a Granin-Like Neuroendocrine Peptide Precursor that Inhibits Prohormone Processing

Fricker

McKinzie²,

Sun³

et al. 2000

J. Neurosci.

246

336

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Five novel peptides were identified in the brains of mice lacking active carboxypeptidase E, a neuropeptide-processing enzyme. These peptides are produced from a single precursor, termed proSAAS, which is present in human, mouse, and rat. ProSAAS mRNA is expressed primarily in brain and other neuroendocrine tissues (pituitary, adrenal, pancreas); within brain, the mRNA is broadly distributed among neurons. When expressed in AtT-20 cells, proSAAS is secreted via the regulated pathway and is also processed at paired-basic cleavage sites into smaller peptides. Overexpression of proSAAS in the AtT-20 cells substantially reduces the rate of processing of the endogenous prohormone proopiomelanocortin. Purified proSAAS inhibits prohormone convertase 1 activity with an IC 50 of 590 nM but does not inhibit prohormone convertase 2. Taken together, proSAAS may represent an endogenous inhibitor of prohormone convertase 1.

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Section: Discussionmentioning

confidence: 99%

Identification and Characterization of proSAAS, a Granin-Like Neuroendocrine Peptide Precursor that Inhibits Prohormone Processing

Fricker

McKinzie²,

Sun³

et al. 2000

J. Neurosci.

246

336

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“…In fact, recent studies show that 7B2 represents a complex bifunctional binding protein with two domains encoding opposing functions. The N-terminal domain is responsible for the production of activatable pro-PC2 (11, 12) whereas the C-terminal domain (CT peptide) represents a potent PC2 inhibitor (13,14). The mechanism by which the N-terminal domain of 7B2 is able to effect the productive maturation of pro-PC2 to an active enzyme species is not yet clear, but it is thought to involve Golgi-mediated cellular processes rather than a direct effect on activation per se (reviewed in Ref.…”

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confidence: 99%

Functional Characterization of ProSAAS

Fortenberry

Hwang

Apletalina

et al. 2002

Journal of Biological Chemistry

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Prohormone convertases (PC) 1 and 2, enzymes found primarily in neuroendocrine tissues, are thought to mediate the proteolytic cleavage of many peptide precursors. To date, endogenous binding proteins for both PC2 (7B2) and PC1 (proSAAS) have been identified. Although 7B2 represents a potent inhibitor of PC2, the most important function of 7B2 as regards this enzyme appears to be the absolute requirement of PC2 for 7B2 in the generation of active enzyme, recently corroborated through production of a null animal that lacks PC2 activity. The purpose of the present study was to determine whether proSAAS exerts effects on PC1 other than inhibition, and to establish functional similarities and differences between 7B2 and proSAAS. We first asked whether the N-terminal domain of proSAAS (proSAAS-(1-180)) could stabilize PC1 activity, similar to the effect of the N-terminal domain of 7B2 on PC2. Recombinant His-tagged proSAAS-(1-180) had no effect on PC1 activity in vitro and was unable to protect PC1 from thermal denaturation. Transient cotransfection of proSAAS-(1-225) cDNA with PC1 cDNA into HEK 293 cells reduced the amount of PC1 activity detected in the medium. Surprisingly, cotransfection of proSAAS-(1-180) cDNA, encoding a protein that lacks the inhibitory C-terminal domain peptide, also reduced the activity of PC1 detected in the medium, but the mass of PC1 secreted into the medium was increased, suggesting a proSAAS-mediated inactivation reaction. Similar results were observed in CHO/PC1 cells stably transfected with pro-SAAS-(1-180). Stable transfection of SAAS cDNAs into AtT-20 cells was used to examine the role of proSAAS in a neuroendocrine setting. Unlike 7B2, proSAAS-(1-225) was able to slow convertase-mediated processing of proopiomelanocortin and proenkephalin; however, similarly to 7B2, proSAAS expression did not result in any accumulated differences in the content of cellular processed peptide. In summary, although both proSAAS and 7B2 potently inhibit PC enzymes via a C-terminal peptide, their intracellular interactions with PCs appear to differ significantly, with each binding protein exhibiting unique properties.The prohormone convertases (PCs) 1 are a family of serine proteinases that are believed to mediate the proteolytic cleavage of precursor proteins to mature polypeptides (for review see Ref. 1). Presently, eight members of this family have been identified based on homology to the bacterial proteolytic enzyme subtilisin. Although members of this family of proteolytic enzymes are structurally similar, their expression patterns differ. For example, furin and PACE4 are expressed ubiquitously, whereas PC4 is mainly expressed in the testes, and PC1 (also known as PC3) and PC2 are found mainly in neuroendocrine tissues (2, 3). PC1 and PC2 are involved in the processing of several neuropeptide precursors such as proinsulin (4, 5), proenkephalin (PE) (6, 7), and proopiomelanocortin (POMC) (8, 9).The first endogenous convertase binding protein identified was the neuroendocrine protein 7B2, whose fun...

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“…In addition, both have broad neuroendocrine tissue distributions [24,27,28]. One of the functions of 7B2 is to inhibit PC2 [29,30]. Similar studies showed that proSAAS is a potent and selective inhibitor of PC1 [24,31].…”

Section: Introductionmentioning

confidence: 88%

Processing of proSAAS in neuroendocrine cell lines

Mzhavia

Qian

Feng

et al. 2001

Biochemical Journal

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ProSAAS, a recently discovered granin-like protein, potently inhibits prohormone convertase (PC)1, and might also perform additional functions. In the present study, the processing of proSAAS was compared in two neuroendocrine cell lines overexpressing this protein: the AtT-20 mouse pituitary corticotrophic line and the PC12 rat adrenal phaeochromocytoma line. The processing of proSAAS was examined by pulse–chase analysis using [3H]leucine, by MS, and by chromatography and radioimmunoassay. Various smaller forms of proSAAS were detected, including peptides designated as little SAAS, PEN and big LEN. Because the PC-12 cells used in the present study do not express either PC1 or PC2, the finding that these cells efficiently cleave proSAAS indicates that these cleavages do not require either enzyme. Two of the peptides identified in AtT-20 media represent novel C-terminally truncated forms of PEN. In both cell lines, the secretion of the small proSAAS-derived peptides is stimulated by secretagogues. However, long-term treatment of wild-type AtT-20 cells with two different secretagogues (8-bromo-cAMP and a phorbol ester) does not affect levels of proSAAS mRNA; this treatment significantly increases PC1 mRNA by approx. 60–80%. The lack of co-regulation of proSAAS and PC1 mRNA implies that enzyme activity can be induced without an accompanying increase in the inhibitor. In addition, the finding that the peptides are secreted via the regulated pathway is consistent with the proposal that they may function as neuropeptides.

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Identification of the Region within the Neuroendocrine Polypeptide 7B2 Responsible for the Inhibition of Prohormone Convertase PC2

Cited by 71 publications

References 30 publications

Identification and Characterization of proSAAS, a Granin-Like Neuroendocrine Peptide Precursor that Inhibits Prohormone Processing

Identification and Characterization of proSAAS, a Granin-Like Neuroendocrine Peptide Precursor that Inhibits Prohormone Processing

Functional Characterization of ProSAAS

Processing of proSAAS in neuroendocrine cell lines

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