Carboxypeptidase E, an essential element of the regulated secretory pathway, is expressed and partially co-localized with chromogranin A in chicken thymus

Chromogranin A (CgA) is widely expressed in endocrine and neuroendocrine tissues as well as in the central nervous system. We observed CgA expression (mRNA and protein) in the gastrocnemius (GAS) muscle and found that performance of CgA-deficient Chga-KO mice in treadmill exercise was impaired. Supplementation with CgA in Chga-KO mice restored exercise ability suggesting a novel role for endogenous CgA in skeletal muscle function. Chga-KO mice display (i) lack of exercise-induced stimulation of pAKT, pTBC1D1 and phospho-p38 kinase signaling, (ii) loss of GAS muscle mass, (iii) extensive formation of tubular aggregates (TA), (iv) disorganized cristae architecture in mitochondria, (v) increased expression of the inflammatory cytokines Tnfα, Il6 and Ifnγ, and fibrosis. The impaired maximum running speed and endurance in the treadmill exercise in Chga-KO mice correlated with decreased glucose uptake and glycolysis, defects in glucose oxidation and decreased mitochondrial cytochrome C oxidase activity. The lack of adaptation to endurance training correlated with the lack of stimulation of p38MAPK that is known to mediate the response to tissue damage. As CgA sorts proteins to the regulated secretory pathway, we speculate that lack of CgA could cause misfolding of membrane proteins inducing aggregation of sarcoplasmic reticulum (SR) membranes and formation of tubular aggregates that is observed in Chga-KO mice. In conclusion, CgA deficiency renders the muscle energy deficient, impairs performance in treadmill exercise and prevents regeneration after exercise-induced tissue damage.

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

Journal of Endocrinology ◽

10.1530/joe-12-0468 ◽

2013 ◽

Vol 217 (2) ◽

pp. 229-240 ◽

Cited By ~ 15

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.

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

Biochemical Journal ◽

10.1042/bj3230265 ◽

1997 ◽

Vol 323 (1) ◽

pp. 265-271 ◽

Cited By ~ 11

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.

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Chromogranin A processing and secretion: specific role of endogenous and exogenous prohormone convertases in the regulated secretory pathway.

Journal of Clinical Investigation ◽

10.1172/jci118760 ◽

1996 ◽

Vol 98 (1) ◽

pp. 148-156 ◽

Cited By ~ 79

Author(s):

N L Eskeland ◽

A Zhou ◽

T Q Dinh ◽

H Wu ◽

R J Parmer ◽

...

Keyword(s):

Chromogranin A ◽

Secretory Pathway ◽

Specific Role ◽

Prohormone Convertases ◽

Regulated Secretory Pathway

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

Molecular Endocrinology ◽

10.1210/me.2015-1166 ◽

2016 ◽

Vol 30 (1) ◽

pp. 37-47 ◽

Cited By ~ 10

Author(s):

Niamh X. Cawley ◽

Trushar Rathod ◽

Sigrid Young ◽

Hong Lou ◽

Nigel Birch ◽

...

Keyword(s):

Secretory Pathway ◽

Carboxypeptidase E ◽

Regulated Secretory Pathway

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

Endocrine Connections ◽

10.1530/ec-17-0020 ◽

2017 ◽

Vol 6 (4) ◽

pp. R18-R38 ◽

Cited By ~ 15

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.

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