Enzymic characterization of immunopurified prohormone convertase 2: Potent inhibition by a 7B2 peptide fragment

Lindberg, Iris; Hurk, Wilhelmina H. van den; Bui, Chau; Batie, Christopher J.

doi:10.1021/bi00016a020

Cited by 74 publications

(86 citation statements)

References 40 publications

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“…PC2 cleaves ACTH into ACTH1-17 and corticotrophin-like intermediate lobe peptide. PC2 function is modulated by 7b2, which can have differential effects on POMC processing by acting either as an inhibitor or a chaperone of PC2 (Lindberg et al 1995). Next, CPE removes C-terminal basic residues from ACTH1-17 and PAM amidates the resulting product, forming desacetyl a-MSH (DA-aMSH).…”

Section: Discussionmentioning

confidence: 99%

Cellular insulin resistance disrupts hypothalamic mHypoA-POMC/GFP neuronal signaling pathways

Nazarians-Armavil¹,

Chalmers²,

Lee³

et al. 2013

Journal of Endocrinology

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POMC neurons play a central role in the maintenance of whole-body energy homeostasis. This balance requires proper regulation of POMC neurons by metabolic hormones, such as insulin. However, the heterogeneous cellular population of the intact hypothalamus presents challenges for examining the molecular mechanisms underlying the potent anorexigenic effects of POMC neurons, and there is currently a complete lack of mature POMC neuronal cell models for study. To this end, we have generated novel, immortalized, adult-derived POMC-expressing/a-MSH-secreting cell models, mHypoA-POMC/GFP lines 1-4, representing the fluorescence-activated cell-sorted POMC population from primary POMCeGFP mouse hypothalamus. The presence of Pomc mRNA in these cell lines was confirmed, and a-MSH was detected via immunofluorescence. a-MSH secretion in the mHypoA-POMC/GFP-1 was found to increase in response to 10 ng/ml ciliary neurotrophic factor (CNTF) or 10 nM insulin as determined by enzyme immunoassay. Further experiments using the mHypoA-POMC/GFP-1 cell line revealed that 10 ng/ml CNTF increases Pomc mRNA at 1 and 2 h after treatment, whereas insulin elicited an increase in Pomc mRNA level and decreases in insulin receptor (Insr (Ir)) mRNA level at 4 h. Furthermore, the activation of IR-mediated downstream second messengers was examined by western blot analysis, following the induction of cellular insulin resistance, which resulted in a loss of insulin-mediated regulation of Pomc and Ir mRNAs. The development of these immortalized neurons will be invaluable for the elucidation of the cellular and molecular mechanisms that underlie POMC neuronal function under normal and perturbed physiological conditions.

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Section: Discussionmentioning

confidence: 99%

Cellular insulin resistance disrupts hypothalamic mHypoA-POMC/GFP neuronal signaling pathways

Nazarians-Armavil¹,

Chalmers²,

Lee³

et al. 2013

Journal of Endocrinology

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“…His-tagged proSAAS- (1-225) is, however, not nearly as potent as the LLRVKR hexapeptide (K i ϭ 5 nM), a relative order that is consistent with recent studies comparing GST-proSAAS-(1-225) and other synthetic peptides (19). The reduced potency of proSAAS-(1-225) against PC1 compared with the hexapeptide differs considerably from the situation with 7B2 and PC2, because full-length 7B2 is 10-fold more potent against PC2 than is the 7B2 CT peptide (13,10). Additionally, sequences shorter than 7B2 CT-(1-18) do not represent inhibitors of PC2 (35), indicating a smaller effective binding site for natural inhibitors for PC1 as opposed to PC2.…”

Section: His-tagged Prosaas-(1-225) But Not His-tagged Prosaas-(1-18mentioning

confidence: 93%

“…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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“…However, if proPC2 has not encountered 7B2, catalytically active PC2 cannot be generated (Zhu, and Lindberg, 1995;Muller et al, 1997). Moreover, uncleaved 7B2 as well as the CT peptide have been demonstrated to act as nanomolar inhibitors of PC2 (Martens et al, 1994;Lindberg et al, 1995).…”

Section: Abbreviationsmentioning

confidence: 99%

The proprotein convertase PC2 is involved in the maturation of prosomatostatin to somatostatin-14 but not in the somatostatin deficit in Alzheimer's disease

et al. 2003

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Abstract-A somatostatin deficit occurs in the cerebral cortex of Alzheimer's disease patients without a major loss in somatostatin-containing neurons. This deficit could be related to a reduction in the rate of proteolytic processing of peptide precursors. Since the two proprotein convertases (PC)1 and PC2 are responsible for the processing of neuropeptide precursors directed to the regulated secretory pathway, we examined whether they are involved first in the proteolytic processing of prosomatostatin in mouse and human brain and secondly in somatostatin defect associated with Alzheimer's disease. By size exclusion chromatography, the cleavage of prosomatostatin to somatostatin-14 is almost totally abolished in the cortex of PC2 null mice, while the proportions of prosomatostatin and somatostatin-28 are increased. By immunohistochemistry, PC1 and PC2 were localized in many neuronal elements in human frontal and temporal cortex. The convertases levels were quantified by Western blot, as well as the protein 7B2 which is required for the production of active PC2. No significant change in PC1 levels was observed in Alzheimer's disease. In contrast, a marked decrease in the ratio of the PC2 precursor to the total enzymatic pool was observed in the frontal cortex of Alzheimer patients. This decrease coincides with an increase in the binding protein 7B2. However, the content and enzymatic activity of the PC2 mature form were similar in Alzheimer patients and controls. Therefore, the cortical somatostatin defect is not due to convertase alteration occuring during Alzheimer's disease. Further studies will be needed to assess the mechanisms involved in somatostatin deficiency in Alzheimer's disease.

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Enzymic characterization of immunopurified prohormone convertase 2: Potent inhibition by a 7B2 peptide fragment

Cited by 74 publications

References 40 publications

Cellular insulin resistance disrupts hypothalamic mHypoA-POMC/GFP neuronal signaling pathways

Cellular insulin resistance disrupts hypothalamic mHypoA-POMC/GFP neuronal signaling pathways

Functional Characterization of ProSAAS

The proprotein convertase PC2 is involved in the maturation of prosomatostatin to somatostatin-14 but not in the somatostatin deficit in Alzheimer's disease

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