Distinct Molecular Events during Secretory Granule Biogenesis Revealed by Sensitivities to Brefeldin A

Fernández, Carlos; Haugwitz, Michael; Eaton, Benjamin A.; Moore, Hsiao-Ping

doi:10.1091/mbc.8.11.2171

Cited by 59 publications

(73 citation statements)

References 55 publications

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“…In parallel studies, we did not observe precursor release when similar methods were used to monitor processing and release of the precursors of NGF (8) or . Indeed, previous work by others (18,27) has shown that large amounts of the precursors of proteins released by the regulated secretory pathway, such as pro-opiomelanocorticotrophin and pro-renin, are also constitutively released from AtT-20 cells. Although these differences could simply reflect overexpression of pro-BDNF saturating the sorting machinery in the transGolgi network (8), constitutive release of the precursor could also be of biological significance.…”

Section: Discussionmentioning

confidence: 99%

“…Generation of 28-kDa BDNF Occurs in the ER-To determine where in the cell the 28-kDa form of BDNF is generated, we metabolically labeled vv:pro-BDNF infected cells with [ 35 S]Cys-Met for 3 h in the presence or absence of brefeldin A (BFA, 5 g/ml), a molecule that inhibits anterograde vesicular transport from the ER (18). The cells were analyzed immediately or after a further 2-h chase period without BFA.…”

Section: Pro-bdnf Processing In Vv:bdnf-infected Att-20mentioning

confidence: 99%

See 1 more Smart Citation

Biosynthesis and Post-translational Processing of the Precursor to Brain-derived Neurotrophic Factor

Mowla

Farhadi

Pareek

et al. 2001

Journal of Biological Chemistry

508

372

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We examined the biosynthesis and post-translational processing of the brain-derived neurotrophic factor precursor (pro-BDNF) in cells infected with a pro-BDNFencoding vaccinia virus. Metabolic labeling, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis reveal that pro-BDNF is generated as a 32-kDa precursor that is N-glycosylated and glycosulfated on a site, within the pro-domain. Some pro-BDNF is released extracellularly and is biologically active as demonstrated by its ability to mediate TrkB phosphorylation. The precursor undergoes N-terminal cleavage within the trans-Golgi network and/or immature secretory vesicles to generate mature BDNF (14 kDa). Small amounts of a 28-kDa protein that is immunoprecipitated with BDNF antibodies is also evident. This protein is generated in the endoplasmic reticulum through N-terminal cleavage of pro-BDNF at the Arg-Gly-Leu-Thr 57 -2-SerLeu site. Cleavage is abolished when Arg 54 is changed to Ala (R54A) by in vitro mutagenesis. Blocking generation of 28-kDa BDNF has no effect on the level of mature BDNF and blocking generation of mature BDNF with ␣ 1 -PDX, an inhibitor of furin-like enzymes, does not lead to accumulation of the 28-kDa form. These data suggest that 28-kDa pro-BDNF is not an obligatory intermediate in the formation of the 14-kDa form in the constitutive secretory pathway. Brain-derived neurotrophic factor (BDNF)1 along with nerve growth factor (NGF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) are members of the neurotrophin family of trophic factors (1). The neurotrophins play essential roles in the development, survival, and function of a wide range of neurons in both the peripheral and central nervous systems.The neurotrophins have a number of shared characteristics, including similar molecular weights (13.2-15.9 kDa), isoelectric points (in the range of 9 -10), and ϳ50% identity in primary structure. They exist in solution as noncovalently bound dimers. Six cysteine residues conserved in the same relative positions give rise to three intra-chain disulfide bonds (2, 3). The neurotrophins interact with two cell surface receptors, the low affinity P75 receptor (4) and the Trk family of high affinity tyrosine kinase receptors (5). NGF preferentially binds TrkA, BDNF and NT4/5 bind TrkB, and NT-3 binds TrkC (and TrkA to a lesser extent).Sequence data predict that mature neurotrophins are generated through the proteolytic processing of higher molecular weight precursors (31-35 kDa), a process that has been extensively studied with respect to the production of NGF (6, 7). Almost nothing is known, however, about the biosynthesis and post-translational processing of the other members of the neurotrophin family. Recent data from our laboratory show that cells with a regulated secretory pathway, including central nervous system neurons, release mature (i.e. fully processed) NGF (8) and NT-3 (9) via the constitutive secretory pathway, whereas mature BDNF is packaged in vesicles and released through the regulated pathway (8). Furthermore, BDNF i...

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Section: Discussionmentioning

confidence: 99%

Section: Pro-bdnf Processing In Vv:bdnf-infected Att-20mentioning

confidence: 99%

Biosynthesis and Post-translational Processing of the Precursor to Brain-derived Neurotrophic Factor

Mowla

Farhadi

Pareek

et al. 2001

Journal of Biological Chemistry

508

372

View full text Add to dashboard Cite

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“…For example, the processing of proinsulin begins in immature secretory granules in ␤-cells (26). Also, the generation of ACTH and ␤-endorphin from the larger precursor proopiomelanocortin (POMC) in AtT-20 cells is predominantly a post-TGN event (27,28), although POMC processing may be initiated in the TGN (29 -31).…”

mentioning

confidence: 99%

Localization of Metallocarboxypeptidase D in AtT-20 Cells

Varlamov

Eng

Novikova

et al. 1999

Journal of Biological Chemistry

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Carboxypeptidase D (CPD) is a recently discovered metallocarboxypeptidase that is predominantly located in the trans-Golgi network (TGN), and also cycles between the cell surface and the TGN. In the present study, the intracellular distribution of CPD was examined in AtT-20 cells, a mouse anterior pituitary-derived corticotroph. CPD-containing compartments were isolated using antibodies to the CPD cytosolic tail. The immunopurified vesicles contained TGN proteins (TGN38, furin, syntaxin 6) but not lysosomal or plasma membrane proteins. The CPD-containing vesicles also contained neuropeptide-processing enzymes and adrenocorticotropic hormone, a product of proopiomelanocortin proteolysis. Electron microscopic analysis revealed that CPD is present within the TGN and immature secretory granules but is virtually absent from mature granules, suggesting that CPD is actively removed from the regulated pathway during the process of granule maturation. A second major finding of the present study is that a soluble truncated form of CPD is secreted mainly via the constitutive pathway in AtT-20 cells, indicating that the lumenal domain does not contain signals for the sorting of CPD to mature secretory granules. Taken together, these data are consistent with the proposal that CPD participates in the processing of proteins within the TGN and immature secretory vesicles.

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“…3A); we observed increased secretion of intact POMC (Fig. 3B), possibly via increased constitutive secretion (Fernandez et al 1997).…”

Section: In Trans Expression Of Pc1 Propeptide Inhibits Pc1-mediated mentioning

confidence: 51%

Prohormone convertase 1 (PC1) processing and sorting: effect of PC1 propeptide and proSAAS

Lee¹,

Prodhomme²,

Lindberg³

2004

Journal of Endocrinology

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Prohormone convertase 1 (PC1) is a serine proteinase responsible for the proteolytic processing of many precursor proteins within the regulated secretory pathway. The activity of PC1 is potentially regulated by two endogenous inhibitors, the PC1 propeptide and proSAAS. Here we have investigated the effect of proSAAS and propeptidecontaining constructs on PC1 carboxy-terminal processing and activity. In AtT-20 cells, proSAAS expression inhibited both C-terminal PC1 processing and proopiomelanocortin (POMC) processing under pulse/chase conditions. SAAS CT peptide-propeptide chimeric constructs had no effect on the cleavage of PC1 and POMC under pulse/chase conditions. However, a construct containing the propeptide alone reduced C-terminal PC1 processing under pulse/chase conditions and also inhibited POMC processing. In contrast, experiments using HEK293 cells transiently expressing PC1 plus the respective constructs demonstrated significant inhibition of zymogen processing and decreased C-terminal processing of PC1 by the SAAS CT peptide portion of the chimera. Our results suggest that the PC1 propeptide expressed in trans is able to act as an endogenous inhibitor of PC1, but that SAAS CT peptide-containing/propeptide constructs cannot function as effective inhibitors of precursor maturation in the regulated pathway.

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Distinct Molecular Events during Secretory Granule Biogenesis Revealed by Sensitivities to Brefeldin A

Cited by 59 publications

References 55 publications

Biosynthesis and Post-translational Processing of the Precursor to Brain-derived Neurotrophic Factor

Biosynthesis and Post-translational Processing of the Precursor to Brain-derived Neurotrophic Factor

Localization of Metallocarboxypeptidase D in AtT-20 Cells

Prohormone convertase 1 (PC1) processing and sorting: effect of PC1 propeptide and proSAAS

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