Carboxypeptidase E Activity Is Deficient in Mice with the fat Mutation

Fricker, Lloyd D.; Berman, Yemiliya; Leiter, Edward H.; Devi, Lakshmi A.

doi:10.1074/jbc.271.48.30619

Cited by 160 publications

(121 citation statements)

References 33 publications

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“…CPE is a key enzyme in the insulin secretory pathway, and disruptions in this pathway are known to alter the function and survival of pancreatic ␤-cells, and to cause diabetes in humans and animals (37,38). In the CPE fat/fat mouse strain, a single point mutation in CPE is sufficient to produce an animal with multiple disorders including obesity and diabetes (28,39). Importantly, one study found CPE polymorphisms associated with type 2 diabetes in humans (40).…”

Section: Discussionmentioning

confidence: 99%

Carboxypeptidase E mediates palmitate-induced β-cell ER stress and apoptosis

Jeffrey

Alejandro

Luciani

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

142

153

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Obesity is a principal risk factor for type 2 diabetes, and elevated fatty acids reduce ␤-cell function and survival. An unbiased proteomic screen was used to identify targets of palmitate in ␤-cell death. The most significantly altered protein in both human islets and MIN6 ␤-cells treated with palmitate was carboxypeptidase E (CPE). Palmitate reduced CPE protein levels within 2 h, preceding endoplasmic reticulum (ER) stress and cell death, by a mechanism involving CPE translocation to Golgi and lysosomal degradation. Palmitate metabolism and Ca 2؉ flux were also required for CPE proteolysis and ␤-cell death. Chronic palmitate exposure increased the ratio of proinsulin to insulin. CPE null islets had increased apoptosis in vivo and in vitro. Reducing CPE by Ϸ30% using shRNA also increased ER stress and apoptosis. Conversely, overexpression of CPE partially rescued ␤-cells from palmitate-induced ER stress and apoptosis. Thus, carboxypeptidase E degradation contributes to palmitate-induced ␤-cell ER stress and apoptosis. CPE is a major link between hyperlipidemia and ␤-cell death pathways in diabetes.2D difference gel electrophoresis proteomics ͉ free fatty acids ͉ hyperproinsulinemia ͉ mechanisms of ␤-cell lipotoxicity ͉ type 2 diabetes T here is a strong association between type 2 diabetes and obesity.High levels of circulating lipids, including free fatty acids, are a prominent clinical feature of type 2 diabetes and represent an important risk factor for this disease (1, 2). But exactly how elevated lipids might lead to diabetes remains unresolved. Fatty acids increase basal insulin secretion (3) and the relative levels of circulating proinsulin (4). Chronic exposure to the free fatty acid palmitate has been shown to impair glucose-stimulated insulin release (i.e., lipotoxicity) (5-10). ␤-Cell apoptosis can be initiated by high levels of palmitate (6,7,(11)(12)(13)(14), which may account in part for alterations in insulin secretory function (13). A number of studies have established palmitate targets in the ␤-cell, including lipid metabolism (15, 16), mitochondrial function (17-23), and prosurvival transcription factors such as Pdx1 (24,25). Recently, a role for endoplasmic reticulum (ER) stress in lipotoxicity has been demonstrated in multiple cell types, including ␤-cells (11,26,27). The effects of palmitate on ␤-cell survival are likely mediated by a number of mechanisms.In the present study, we conducted unbiased proteomic screens using human islets and MIN6 ␤-cells to elucidate targets of palmitate. Carboxypeptidase E (CPE) was the most significantly changed protein in both screens. Mice lacking CPE develop hyperproinsulinemia and hyperglycemia (28), but the involvement of this protein in ␤-cell apoptosis has not been reported. Palmitate caused the rapid intracellular redistribution and degradation of CPE via mechanisms that required palmitate metabolism, K ATP channel closure, Ca 2ϩ influx, and protease activity. We further showed that CPE levels control ␤-cell ER stress and apoptosis. Thus, CPE is a cri...

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Section: Discussionmentioning

confidence: 99%

Carboxypeptidase E mediates palmitate-induced β-cell ER stress and apoptosis

Jeffrey

Alejandro

Luciani

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

142

153

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“…The mutant CPE is inactive and is rapidly degraded prior to transport into the Golgi (29). Without active CPE, the Cpe fat /Cpe fat mice have greatly elevated levels of peptide processing intermediates with C-terminal basic residues, and reduced levels of the fully processed peptides (28,30,31). However, low levels of mature peptides are detected, indicating that another enzyme is able to partially compensate for the absence of CPE activity.…”

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confidence: 89%

Characterization of the Enzymatic Properties of the First and Second Domains of Metallocarboxypeptidase D

Novikova

Eng

Lin

et al. 1999

Journal of Biological Chemistry

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Carboxypeptidase D (CPD) contains three domains with homology to other metallocarboxypeptidases. To further characterize the various domains, we constructed a series of point mutants with a critical active site Glu of duck CPD converted to Gln. The proteins were expressed in the baculovirus system, purified to homogeneity, and characterized. Point mutations within both the first and second domains eliminated enzyme activity, indicating that the third domain is inactive toward dansyl-Phe-Ala-Arg. CPD removed only the C-terminal Lys or Arg from peptides, with the first domain more efficient toward Arg and the second domain more efficient toward Lys. Peptides containing Pro in the penultimate position were poorly cleaved by either domain. Cleavage of a peptide with Ala in the penultimate position was most efficient, with the relative order Ala > Met > Ser, Phe > Tyr > Trp > Thr > Gln, Asp, Leu, Gly Ͼ Ͼ Ͼ Ͼ Pro for CPD with both domains active. There were only minor differences between the first and the second domains regarding the influence of the penultimate amino acid. The first domain was optimally active at pH 6.3-7.5, whereas the second domain was optimally active at pH 5.0 -6.5. Thus, the first and second carboxypeptidase domains have complementary enzyme activities. Furthermore, the finding that CPD with both domains active shows a broad activity to a wide range of substrates is consistent with a role for this enzyme in the processing of many proteins that transit the secretory pathway.

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“…Obese Cpe fat /Cpe fat mice suffer from hyperproinsulinemia associated with expression of a mutated CPE, which results in destruction of the CPE protein in the rough endoplasmic reticulum (20,21). The pancreatic ␤-cells of these mice are clearly granulated but the content of the granules appears to be less dense (20), suggesting storage of proinsulin instead of mature insulin.…”

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confidence: 99%

Proinsulin Targeting to the Regulated Pathway Is Not Impaired in Carboxypeptidase E-deficientCpe /Cpe Mice

Irminger

Verchere

Meyer

et al. 1997

Journal of Biological Chemistry

103

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Sorting of proinsulin from the trans-Golgi network to secretory granules is critical for its conversion to insulin as well as for regulated insulin secretion. The proinsulin sorting mechanism is unknown. Recently, carboxypeptidase E (CPE) was proposed as a sorting receptor for prohormones. To know whether CPE is implicated in proinsulin sorting, pancreatic islets were isolated from CPE-deficient Cpe fat /Cpe fat mice and Cpe fat /؉ controls, pulse-labeled ([ 3 H]leucine), and then chased in basal medium (90 min) to examine constitutive secretion followed by medium with secretagogues (60 min) to stimulate regulated secretion. Secretion of labeled proinsulin via the constitutive pathway was <2% even in Cpe fat /Cpe fat islets. After a 150-min chase, only 13% of radioactivity remained as proinsulin in Cpe fat /؉ islets compared with 46% in Cpe fat /Cpe fat islets, reflecting slower conversion. Regulated secretion was stimulated to an equal extent from Cpe fat /؉ and Cpe fat /Cpe fat mice with 20% of the total content of labeled (pro)insulin released during the 60-min stimulatory period. It is concluded that in CPE-deficient Cpe fat / Cpe fat mice, proinsulin is efficiently routed to the regulated pathway and its release can be effectively stimulated by secretagogues. CPE is thus not essential for sorting proinsulin to granules.Proinsulin is directed to the regulated secretory pathway of the pancreatic ␤-cell and is converted to bioactive insulin in immature secretory granules (1). At least three enzymes are needed for generating native insulin, namely the conversion endoproteases PC1 (also known as PC3) and PC2 (2, 3) and carboxypeptidase E (also called carboxypeptidase H or CPE 1 ) for trimming residual basic C-terminal residues (4 -6). Correct targeting to secretory granules is essential for the cell to produce and store bioactive insulin, which can then be released upon stimulation of the ␤-cell by glucose or other secretagogues. One of the crucial steps in targeting takes place in the TGN (trans-Golgi network) (7). The proprotein must somehow be recognized selectively in the TGN and then be transferred to the immature secretory granules, instead of being released through the constitutive or default pathway (3).One hypothesis (reviewed in Ref.3) accounting for targeting within the TGN implicates a sorting receptor with a broad specificity in the TGN membrane, which recognizes a sorting domain and thereby binds proteins to be routed to the regulated pathway. Sorting domains have been reported for several prohormones including prosomatostatin (8, 9), chromogranin (10), and the basic proline-rich protein (11). An N-terminal sorting domain has been reported for proopiomelanocortin (POMC) (12, 13), although this is controversial (14). A putative C-terminal sorting domain has also recently been proposed for the prohormone convertase PC2 (15). There is not as yet any direct experimental evidence for a sorting domain for proinsulin, although the comparison of structural features of many prohormones suggests that a regio...

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Carboxypeptidase E Activity Is Deficient in Mice with the fat Mutation

Abstract: Carboxypeptidase E (CPE) is involved in the biosyn

Cited by 160 publications

References 33 publications

Carboxypeptidase E mediates palmitate-induced β-cell ER stress and apoptosis

Carboxypeptidase E mediates palmitate-induced β-cell ER stress and apoptosis

Characterization of the Enzymatic Properties of the First and Second Domains of Metallocarboxypeptidase D

Proinsulin Targeting to the Regulated Pathway Is Not Impaired in Carboxypeptidase E-deficientCpe /Cpe Mice

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