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

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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Lipid Raft Association of Carboxypeptidase E Is Necessary for Its Function as a Regulated Secretory Pathway Sorting Receptor

Journal of Biological Chemistry ◽

10.1074/jbc.m005364200 ◽

2000 ◽

Vol 275 (38) ◽

pp. 29887-29893 ◽

Cited By ~ 72

Author(s):

Savita Dhanvantari ◽

Y. Peng Loh

Keyword(s):

Lipid Raft ◽

Secretory Pathway ◽

Carboxypeptidase E ◽

Sorting Receptor ◽

Regulated Secretory Pathway

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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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Depletion of Carboxypeptidase E, a Regulated Secretory Pathway Sorting Receptor, Causes Misrouting and Constitutive Secretion of Proinsulin and Proenkephalin, But Not Chromogranin A

Endocrinology ◽

10.1210/endo.139.4.5951 ◽

1998 ◽

Vol 139 (4) ◽

pp. 2137-2145 ◽

Cited By ~ 32

Author(s):

Emmanuel Normant ◽

Y. Peng Loh

Keyword(s):

Chromogranin A ◽

Secretory Pathway ◽

Carboxypeptidase E ◽

Constitutive Secretion ◽

Sorting Receptor ◽

Regulated Secretory Pathway

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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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Systems genetics analyses in Diversity Outbred mice inform human bone mineral density GWAS and identify Qsox1 as a novel determinant of bone strength

10.1101/2020.06.24.169839 ◽

2020 ◽

Author(s):

Basel Al-Barghouthi ◽

Larry D. Mesner ◽

Gina M. Calabrese ◽

Daniel Brooks ◽

Steve M. Tommasini ◽

...

Keyword(s):

Bone Mineral Density ◽

Cortical Bone ◽

Bone Strength ◽

Bone Mineral ◽

Association Studies ◽

Systems Genetics ◽

Genome Wide Association Studies ◽

Mineral Density ◽

Diversity Outbred ◽

Wide Range

ABSTRACTGenome-wide association studies (GWASs) for osteoporotic traits have identified over 1000 associations; however, their impact has been limited by the difficulties of causal gene identification and a strict focus on bone mineral density (BMD). Here, we used Diversity Outbred (DO) mice to directly address these limitations by performing the first systems genetics analysis of over 50 complex skeletal phenotypes. We applied a network approach to cortical bone RNA-seq data to discover 46 genes likely to be causal for human BMD GWAS associations, including the novel genes SERTAD4 and GLT8D2. We also performed GWAS in the DO for a wide-range of bone traits and identified Qsox1 as a novel gene influencing cortical bone accrual and bone strength. Our results provide a new perspective on the genetics of osteoporosis and highlight the ability of the mouse to inform human genetics.

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LIVING ALONE IS ASSOCIATED WITH POORER PHYSICAL FUNCTION AND BONE MINERAL DENSITY IN ICELANDIC OLD ADULTS

Innovation in Aging ◽

10.1093/geroni/igz038.1793 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

pp. S482-S482

Author(s):

Alfons Ramel

Keyword(s):

Bone Mineral Density ◽

Older Adults ◽

Physical Function ◽

Bone Mineral ◽

Community Dwelling ◽

Living Alone ◽

Z Score ◽

Mineral Density ◽

Old Adults ◽

Wide Range

Abstract Background: Loneliness and living alone have been significant public health concerns among older adults given their association with a wide range of adverse health outcomes. Aim: The aim of this study was to examine whether living alone is associated with physical function and bone health in community-dwelling older adults. Methods: This was a secondary analysis of existing cross-sectional data of old adults (N=182, 73.7±5.7yrs, 58.2% female) from the Reykjavik capital area in Iceland. Information on socioeconomics, health, dietary intake and physical function was collected. 25-hydroxy-vitamin D (25OHD) and bone mineral density (BM were grouped retrospectively into “living alone” and into “in cohabitation”. Results: Of our subjects, 76.4% were in cohabitation and and 23.6% lived alone. Participants who lived alone were older (74.5±5.6 vs. 72.1±5.0,P=0.008) and more often female (74.4 vs. 53.2%,P=0.014), but there were no differences in education, smoking, number of medications, physical activity (PA) or body mass index (BMI). According to age and gender corrected analyses, participants in cohabitation had higher grip strength (6.2±2.4lb,P=0.011), higher 25OHD (13.1±6.3nmol/L,P=0.037) and higher BMD (z-score lumbal: 1.195±0.417,P=0.005; z-score femur: 0.421±0.219,P=0.054; z-score total: 0.846±0.290,P=0.004). Statistical correction for PA, BMI, education and fish oil intake did not change the results. Conclusion: In comparison to old adults who live in cohabitation, Icelandic old adults who live alone have poorer physical function, lower 25OHD and lower BMD, which increases their risk for wrist or hip fracture. These differences between groups were not explained by physical, dietary or social confounding variables.

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PACE4: a subtilisin-like endoprotease with unique properties

Biochemical Journal ◽

10.1042/bj3210587 ◽

1997 ◽

Vol 321 (3) ◽

pp. 587-593 ◽

Cited By ~ 41

Author(s):

Richard E. MAINS ◽

Carla A. BERARD ◽

Jean-Bernard DENAULT ◽

An ZHOU ◽

Richard C. JOHNSON ◽

...

Keyword(s):

Cell Lines ◽

Secretory Pathway ◽

Secretory Granules ◽

Fibroblast Cell ◽

Pituitary Cells ◽

Truncated Form ◽

Prohormone Convertase 1 ◽

Peptide Biosynthesis ◽

Regulated Secretory Pathway ◽

Fibroblast Cell Lines

PACE4 is one of the neuroendocrine-specific mammalian subtilisin-related endoproteases believed to function in the secretory pathway. The biosynthesis and secretion of PACE4 have been studied using transfected neuroendocrine and fibroblast cell lines, as well as primary pituitary cultures. ProPACE4 (approx. 106 kDa) is cleaved intracellularly before secretion of PACE4 (approx. 97 kDa); the N-terminal propeptide cleavage is accelerated in a truncated form of PACE4 lacking the Cys-rich C-terminal region (PACE4s). Neither PACE4 nor PACE4s is stored in regulated neuroendocrine secretory granules, whereas pro-opiomelanocortin-derived peptides and prohormone convertase 1 enter the regulated secretory pathway efficiently. The relatively slow cleavage of the proregion of proPACE4 in primary anterior pituitary cells, followed by rapid secretion of PACE4, is similar to the results for proPACE4 in transfected cell lines. The enzyme activity of PACE4 is distinct from furin and prohormone convertases, both in the marked sensitivity of PACE4 to inhibition by leupeptin and the relative insensitivity of PACE4 to inhibition by Ca2+ chelators and dithiothreitol; PACE4 is not inhibited by the α1-antitrypsin Portland variant that is very potent at inhibiting furin. The unique biosynthetic and enzymic patterns seen for PACE4 suggest a role for this neuroendocrine-specific subtilisin-like endoprotease outside the pathway for peptide biosynthesis.

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