scholarly journals Glu300of Rat Carboxypeptidase E Is Essential for Enzymatic Activity but Not Substrate Binding or Routing to the Regulated Secretory Pathway

1999 ◽  
Vol 274 (17) ◽  
pp. 11582-11586 ◽  
Author(s):  
Yimei Qian ◽  
Oleg Varlamov ◽  
Lloyd D. Fricker
Keyword(s):  
Enzymatic Activity ◽  
Secretory Pathway ◽  
Substrate Binding ◽  
Carboxypeptidase E ◽  
Regulated Secretory Pathway

scholarly journals The sorting of proglucagon to secretory granules is mediated by carboxypeptidase E and intrinsic sorting signals

2013 ◽  
Vol 217 (2) ◽  
pp. 229-240 ◽  
Author(s):  
Rebecca McGirr ◽  
Leonardo Guizzetti ◽  
Savita Dhanvantari
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Alpha Helix ◽  
Intestinal Cell ◽  
Alpha Cells ◽  
Pancreatic Alpha Cells ◽  
Carboxypeptidase E ◽  
L Cells ◽  
Sorting Signals ◽  
Regulated Secretory Pathway

Proglucagon is expressed in pancreatic alpha cells, intestinal L cells and brainstem neurons. Tissue-specific processing of proglucagon yields the peptide hormones glucagon in the alpha cell and glucagon-like peptide (GLP)-1 and GLP-2 in L cells. Both glucagon and GLP-1 are secreted in response to nutritional status and are critical for regulating glycaemia. The sorting of proglucagon to the dense-core secretory granules of the regulated secretory pathway is essential for the appropriate secretion of glucagon and GLP-1. We examined the roles of carboxypeptidase E (CPE), a prohormone sorting receptor, the processing enzymes PC1/3 and PC2 and putative intrinsic sorting signals in proglucagon sorting. In Neuro 2a cells that lacked CPE, PC1/3 and PC2, proglucagon co-localised with the Golgi marker p115 as determined by quantitative immunofluorescence microscopy. Expression of CPE, but not of PC1/3 or PC2, enhanced proglucagon sorting to granules. siRNA-mediated knockdown of CPE disrupted regulated secretion of glucagon from pancreatic-derived alphaTC1–6 cells, but not of GLP-1 from intestinal cell-derived GLUTag cells. Mutation of the PC cleavage site K70R71, the dibasic R17R18 site within glucagon or the alpha-helix of glucagon, all significantly affected the sub-cellular localisation of proglucagon. Protein modelling revealed that alpha helices corresponding to glucagon, GLP-1 and GLP-2, are arranged within a disordered structure, suggesting some flexibility in the sorting mechanism. We conclude that there are multiple mechanisms for sorting proglucagon to the regulated secretory pathway, including a role for CPE in pancreatic alpha cells, initial cleavage at K70R71 and multiple sorting signals.

scholarly journals The pro region is not required for the expression or intracellular routeing of carboxypeptidase E

1997 ◽  
Vol 323 (1) ◽  
pp. 265-271 ◽  
Author(s):  
Lixin SONG ◽  
Lloyd D. FRICKER
Keyword(s):  
Confocal Microscopy ◽  
Secretory Pathway ◽  
Secretory Vesicles ◽  
Wild Type ◽  
Carboxypeptidase E ◽  
N Terminus ◽  
Regulated Secretory Pathway ◽  
Golgi Network ◽  
Pro Region ◽  
Stimulation Of

Carboxypeptidase E (CPE) is initially synthesized as a larger precursor containing an additional 14-residue propeptide that is highly conserved between human and rat. Previous studies have established that the proenzyme is enzymically active and that deletion of the pro region does not affect the expression of the active enzyme. In the present study the function of the pro region was examined both by deleting this region from CPE and by attaching this region to the N-terminus of albumin. CPE lacking the pro region is sorted into the regulated secretory pathway in AtT-20 cells, based on confocal microscopy and examination of the stimulated secretion of the protein. Stimulation of AtT-20 cells with either forskolin or phorbol 12-myristate 13-acetate induces the secretion of wild-type CPE and of CPE lacking the pro region to similar extents, indicating a similar efficiency of sorting of the mutant. When the pro region of proalbumin is replaced with the pro region of CPE followed by expression in AtT-20 cells, the protein is not sorted into the regulated pathway, based on the lack of stimulated secretion. Confocal microscopy suggests that the proCPE/albumin protein is retained in the endoplasmic reticulum to a greater extent than is proalbumin. Pulse-chase analysis indicates that the pro region of CPE is not efficiently removed from the N-terminus of albumin, and the small amount of propeptide cleavage that does occur takes place soon before secretion of the protein. In contrast, confocal microscopy indicates that the majority of the propeptide is removed from CPE, and that this cleavage occurs in the trans-Golgi network or soon after sorting into the secretory vesicles. Taken together, these results suggest that the pro region of CPE is not required for the expression or intracellular routeing of this protein.

scholarly journals Carboxypeptidase E and Secretogranin III Coordinately Facilitate Efficient Sorting of Proopiomelanocortin to the Regulated Secretory Pathway in AtT20 Cells

2016 ◽  
Vol 30 (1) ◽  
pp. 37-47 ◽  
Author(s):  
Niamh X. Cawley ◽  
Trushar Rathod ◽  
Sigrid Young ◽  
Hong Lou ◽  
Nigel Birch ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Carboxypeptidase E ◽  
Regulated Secretory Pathway

scholarly journals Dissecting carboxypeptidase E: properties, functions and pathophysiological roles in disease

2017 ◽  
Vol 6 (4) ◽  
pp. R18-R38 ◽  
Author(s):  
Lin Ji ◽  
Huan-Tong Wu ◽  
Xiao-Yan Qin ◽  
Rongfeng Lan
Keyword(s):  
Bone Mineral Density ◽  
Secretory Pathway ◽  
Bioactive Peptides ◽  
Vesicle Transport ◽  
Mineral Density ◽  
Truncated Form ◽  
Carboxypeptidase E ◽  
Wide Range ◽  
Regulated Secretory Pathway ◽  
Endocrine Tissues

Since discovery in 1982, carboxypeptidase E (CPE) has been shown to be involved in the biosynthesis of a wide range of neuropeptides and peptide hormones in endocrine tissues, and in the nervous system. This protein is produced from pro-CPE and exists in soluble and membrane forms. Membrane CPE mediates the targeting of prohormones to the regulated secretory pathway, while soluble CPE acts as an exopeptidase and cleaves C-terminal basic residues from peptide intermediates to generate bioactive peptides. CPE also participates in protein internalization, vesicle transport and regulation of signaling pathways. Therefore, in two types of CPE mutant mice, Cpefat/Cpefat and Cpe knockout, loss of normal CPE leads to a lot of disorders, including diabetes, hyperproinsulinemia, low bone mineral density and deficits in learning and memory. In addition, the potential roles of CPE and ΔN-CPE, an N-terminal truncated form, in tumorigenesis and diagnosis were also addressed. Herein, we focus on dissecting the pathophysiological roles of CPE in the endocrine and nervous systems, and related diseases.

scholarly journals Carboxypeptidase E Is a Regulated Secretory Pathway Sorting Receptor: Genetic Obliteration Leads to Endocrine Disorders in Cpefat Mice

Cell ◽  
1997 ◽  
Vol 88 (1) ◽  
pp. 73-83 ◽  
Author(s):  
David R Cool ◽  
Emmanuel Normant ◽  
Fu-sheng Shen ◽  
Hao-Chia Chen ◽  
Lewis Pannell ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Endocrine Disorders ◽  
Carboxypeptidase E ◽  
Sorting Receptor ◽  
Regulated Secretory Pathway

scholarly journals Trafficking of Mutant Carboxypeptidase E to Secretory Granules in a β-Cell Line Derived from Cpefat/Cpefat Mice

Endocrinology ◽  
2003 ◽  
Vol 144 (1) ◽  
pp. 292-298 ◽  
Author(s):  
Niamh X. Cawley ◽  
Yazmin M. Rodriguez ◽  
Alex Maldonado ◽  
Y. Peng Loh
Keyword(s):  
Cell Line ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Prohormone Convertase ◽  
Β Cell ◽  
Carboxypeptidase E ◽  
Prohormone Convertase 2 ◽  
Glucagon Like Peptide 1 ◽  
Regulated Secretory Pathway ◽  
The Stability

Abstract We have reinvestigated the stability and intracellular routing of mutant carboxypeptidase E in NIT3 cells, a pancreatic β-cell line derived from the Cpefat/Cpefat mouse. Pulse-chase experiments demonstrated that this protein has a half-life of approximately 3 h in these cells and that up to 45% of the proCPE(202) can escape degradation by the proteosome. In double-label immunofluorescence microscopy, a portion of the mutant CPE did not colocalize with calnexin, an endoplasmic reticulum marker, but was found in prohormone convertase 2-containing secretory granules, demonstrating that it had escaped degradation and arrived at a post-Golgi compartment. The mutant CPE as well as prohormone convertase 2 were secreted into the medium in a stimulated manner by treatment with the physiological secretagogue, glucagon-like peptide-1, consistent with its presence in granules of the regulated secretory pathway. The presence of mutant carboxypeptidase E in granules supports a potential role for its involvement as a sorting/retention receptor in the trafficking of proinsulin to the regulated secretory pathway.

scholarly journals Altered Expression of the Neuropeptide-Processing Enzyme Carboxypeptidase E in the Rat Brain after Global Ischemia

2001 ◽  
Vol 21 (12) ◽  
pp. 1422-1429 ◽  
Author(s):  
Kunlin Jin ◽  
Steven H. Graham ◽  
Tetsuya Nagayama ◽  
Paul C. Goldsmith ◽  
David A. Greenberg ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Rat Brain ◽  
Messenger Rna ◽  
Secretory Pathway ◽  
Global Ischemia ◽  
Altered Expression ◽  
Protein Levels ◽  
Carboxypeptidase E ◽  
Regulated Secretory Pathway

Carboxypeptidase E, an exoprotease involved in the processing of bioactive peptides released by a regulated secretory pathway, was identified in a subtractive complementary DNA library derived from an ischemic rat brain by differential screening. In situ hybridization and immunocytochemical analysis showed the presence of carboxypeptidase E messenger RNA and protein in the cerebral cortex, thalamus, striatum, and hippocampus of a healthy rat brain. After 15 minutes of transient global ischemia followed by 8 hours of reperfusion, increased levels of carboxypeptidase E messenger RNA and protein were observed in the hippocampal CA1 and CA3 regions and in the cortex, as detected by Northern and Western blot analyses and in situ hybridization. After extended reperfusion (24 to 72 hours), both carboxypeptidase E messenger RNA and protein levels were decreased. The ischemia-induced changes in carboxypeptidase E expression suggest that this enzyme may play a role in modulating the brain's response to ischemia.

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