Seven secretory mammalian kexin-like subtilases have been identified that cleave a variety of precursor proteins at monobasic and dibasic residues. The recently characterized pyrolysin-like subtilase SKI-1 cleaves proproteins at nonbasic residues. In this work we describe the properties of a proteinase K-like subtilase, neural apoptosis-regulated convertase 1 (NARC-1), representing the ninth member of the secretory subtilase family. Biosynthetic and microsequencing analyses of WT and mutant enzyme revealed that human and mouse pro-NARC-1 are autocatalytically and intramolecularly processed into NARC-1 at the (Y,I)VV(V,L)(L,M)2 motif, a site that is representative of its enzymic specificity. In vitro peptide processing studies and͞or Ala substitutions of the P1-P5 sites suggested that hydrophobic͞aliphatic residues are more critical at P1, P3, and P5 than at P2 or P4. NARC-1 expression is highest in neuroepithelioma SK-N-MCIXC, hepatic BRL-3A, and in colon carcinoma LoVo-C5 cell lines. In situ hybridization and Northern blot analyses of NARC-1 expression during development in the adult and after partial hepatectomy revealed that it is expressed in cells that have the capacity to proliferate and differentiate. These include hepatocytes, kidney mesenchymal cells, intestinal ileum, and colon epithelia as well as embryonic brain telencephalon neurons. Accordingly, transfection of NARC-1 in primary cultures of embryonic day 13.5 telencephalon cells led to enhanced recruitment of undifferentiated neural progenitor cells into the neuronal lineage, suggesting that NARC-1 is implicated in the differentiation of cortical neurons.cleavage specificity ͉ hypercholesterolemia ͉ neurogenesis ͉ hepatogenesis
The gene encoding the proprotein convertase subtilisin/kexin type 9 (PCSK9) is linked to familial hypercholesterolemia, as are those of the low-density lipoprotein receptor (LDLR) and apolipoprotein B. PCSK9 enhances LDLR degradation, resulting in low-density lipoprotein accumulation in plasma. To analyze the role of hepatic PCSK9, total and hepatocyte-specific knockout mice were generated. They exhibit 42% and 27% less circulating cholesterol, respectively, showing that liver PCSK9 was responsible for two thirds of the phenotype. We also demonstrated that, in liver, PCSK9 is exclusively expressed in hepatocytes, representing the main source of circulating PCSK9. The data suggest that local but not circulating PCSK9 regulates cholesterol levels. Although transgenic mice overexpressing high levels of liver and circulating PCSK9 led to the almost complete disappearance of the hepatic LDLR, they did not recapitulate the plasma cholesterol levels observed in LDLRdeficient mice. Single LDLR or double LDLR/PCSK9 knockout mice exhibited similar cholesterol profiles, indicating that PCSK9 regulates cholesterol homeostasis exclusively through the LDLR. Finally, the regenerating liver of PCSK9-deficient mice exhibited necrotic lesions, which were prevented by a high-cholesterol diet. However, lipid accumulation in hepatocytes of these mice was markedly reduced under both chow and high-cholesterol diets, revealing that PCSK9 deficiency confers resistance to liver steatosis. Conclusion: Although PCSK9 is a target for controlling hypercholesterolemia, our data indicate that upon hepatic damage, patients lacking PCSK9 could be at risk. (HEPATOLOGY 2008;48:646-654.) P roprotein convertase subtilisin/kexin type 9 (PCSK9) 1 is the ninth member of the proprotein convertase family. 2 The first seven members, including furin, cleave protein precursors of hormones, growth factors, receptors, or surface glycoproteins at basic sites (after Arg or Lys residues). The eighth member, SKI-1 3 or S1P, 4 is known to cleave membrane-bound transcription factors such as the SREBPs 5 in their luminal domains, resulting in the release of their DNA-binding domain. Proprotein convertases can also inactivate secreted substrates, such as endothelial lipase 6 and PCSK9. 7 PCSK9 is synthesized as a precursor that undergoes autocatalytic cleavage of its N-terminal prosegment in the ER, 1 a step required for its exit from this compartment and its efficient secretion. Secreted PCSK9 remains associated with its prosegment. 1 Different from the other proprotein convertases, this serine protease has no known substrate other than itself. In addition, the tight association of the prosegment with the active site 8 raises the question of the existence of an in trans PCSK9 protease
The proprotein convertase PCSK9 gene is the third locus implicated in familial hypercholesterolemia, emphasizing its role in cardiovascular diseases. Loss of function mutations and gene disruption of PCSK9 resulted in a higher clearance of plasma low density lipoprotein cholesterol, likely due to a reduced degradation of the liver low density lipoprotein receptor (LDLR). In this study, we show that two of the closest family members to LDLR are also PCSK9 targets. These include the very low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) implicated in neuronal development and lipid metabolism. Our results show that wild type PCSK9 and more so its natural gain of function mutant D374Y can efficiently degrade the LDLR, VLDLR, and ApoER2 either following cellular co-expression or re-internalization of secreted human PCSK9. Such PCSK9-induced degradation does not require its catalytic activity. Membrane-bound PCSK9 chimeras enhanced the intracellular targeting of PCSK9 to late endosomes/lysosomes and resulted in a much more efficient degradation of the three receptors. We also demonstrate that the activity of PCSK9 and its binding affinity on VLDLR and ApoER2 does not depend on the presence of LDLR. Finally, in situ hybridization show close localization of PCSK9 mRNA expression to that of VLDLR in mouse postnatal day 1 cerebellum. Thus, this study demonstrates a more general effect of PCSK9 on the degradation of the LDLR family that emphasizes its major role in cholesterol and lipid homeostasis as well as brain development.Familial hypercholesterolemia is mainly characterized by elevated plasma LDL 2 cholesterol that is highly correlated with cardiovascular diseases (1). The main player in regulating the circulating cholesterol is the low density lipoprotein receptor (LDLR), which is expressed mostly in the liver. Recently, natural mutations in the proprotein convertase PCSK9 (2, 3) have been identified and associated with the third locus implicated in familial hypercholesterolemia (4 -6). The major function of PCSK9 seems to be an enhancement of the degradation of the LDLR (7, 8) in acidic subcellular compartments (3), likely endosomes/lysosomes (9, 10). This can occur either via an extracellular endocytotic route (11), or possibly by a direct cellular circuit not involving cell surface endocytosis of the LDLR (12). The gain of function PCSK9 mutations D374Y (13, 14) or D374H (15) have the highest impact on the development of hypercholesterolemia (16), likely through enhanced binding (17) and degradation of the LDLR (18, 19). The major binding site of LDLR to PCSK9 seems to reside within its first epidermal growth factor-like repeat namely EGF-A (20). Finally, it was recently suggested that the PCSK9-induced degradation of the cell surface LDLR does not require its proteolytic activity (21). One of the unanswered questions is the target specificity of PCSK9, and it is not known, nor obvious, whether other members of the LDLR family are also affected by PCSK9. This family consists of str...
Using reverse transcriptase-PCR and degenerate oligonucleotides derived from the active-site residues of subtilisin͞kexin-like serine proteinases, we have identified a highly conserved and phylogenetically ancestral human, rat, and mouse type I membrane-bound proteinase called subtilisin͞kexin-isozyme-1 (SKI-1). Computer databank searches reveal that human SKI-1 was cloned previously but with no identified function. In situ hybridization demonstrates that SKI-1 mRNA is present in most tissues and cells. Cleavage specificity studies show that SKI-1 generates a 28-kDa product from the 32-kDa brain-derived neurotrophic factor precursor, cleaving at an RGLT2SL bond. In the endoplasmic reticulum of either LoVo or HK293 cells, proSKI-1 is processed into two membrane-bound forms of SKI-1 (120 and 106 kDa) differing by the nature of their N-glycosylation. Late along the secretory pathway some of the membrane-bound enzyme is shed into the medium as a 98-kDa form. Immunocytochemical analysis of stably transfected HK293 cells shows that SKI-1 is present in the Golgi apparatus and within small punctate structures reminiscent of endosomes. In vitro studies suggest that SKI-1 is a Ca 2؉ -dependent serine proteinase exhibiting a wide pH optimum for cleavage of pro-brainderived neurotrophic factor.
Objective-Proprotein convertase subtilisin/kexin 9 (PCSK9) promotes the degradation of the low-density lipoprotein receptor (LDLR), and its gene is the third locus implicated in familial hypercholesterolemia. Herein, we investigated the role of PCSK9 in adipose tissue metabolism. Methods and Results-At 6 months of age, Pcsk9Ϫ/Ϫ mice accumulated Ϸ80% more visceral adipose tissue than wild-type mice. This was associated with adipocyte hypertrophy and increased in vivo fatty acid uptake and ex vivo triglyceride synthesis. Moreover, adipocyte hypertrophy was also observed in Pcsk9 Ϫ/Ϫ Ldlr Ϫ/Ϫ mice, indicating that the LDLR is not implicated. Rather, we show here by immunohistochemistry that Pcsk9 Ϫ/Ϫ males and females exhibit 4-and Ϸ40-fold higher cell surface levels of very-low-density lipoprotein receptor (VLDLR) in perigonadal depots, respectively. Expression of PCSK9 in the liver of Pcsk9 Ϫ/Ϫ females reestablished both circulating PCSK9 and normal VLDLR levels. In contrast, specific inactivation of PCSK9 in the liver of wild-type females led to Ϸ50-fold higher levels of perigonadal VLDLR. Key Words: PCSK9 Ⅲ VLDL receptor Ⅲ adipose tissue metabolism Ⅲ fatty acid uptake Ⅲ proprotein convertase P roprotein convertase subtilisin/kexin 9 (PCSK9) is the ninth member of the family of proprotein convertases (encoded by the genes PCSK1 to PCSK9) that share identity with subtilisin and kexin. 1 The first 8 members of the family cleave protein precursors of hormones, growth factors, receptors, and transmembrane transcription factors that transit through or reside in the secretory pathway. 2 In contrast, PCSK9 has no known substrates but itself. It undergoes an autocatalytic cleavage of its N-terminal prosegment, 1,3 which remains trapped in the catalytic pocket. 4 PCSK9 shortens the half-life of the low-density lipoprotein receptor (LDLR), 5 a process independent of its catalytic activity. 6 Gain-of-function mutations in the PCSK9 gene lead to autosomal dominant hypercholesterolemia, 7 as do mutations in the LDLR and APOB genes. Other PCSK9 mutations or polymorphisms responsible for loss of function result in hypocholesterolemia. 8 PCSK9 is highly expressed in the liver, 1 where it binds the LDLR and promotes its internalization and degradation in endosomal/lysosomal compartments. 5 Thus, mice lacking PCSK9 (Pcsk9 Ϫ/Ϫ ) exhibit a 2-to 3-fold increase of the LDLR protein in liver homogenates, and a substantial accumulation of the receptor at the hepatocyte cell surface. 9,10 This leads to hypocholesterolemia (Ϫ40%), with a Ϸ5-fold drop in low-density lipoprotein (LDL) cholesterol levels. In humans, where 70% of cholesterol is associated with LDLs, versus only 30% in mice, the hypocholesterolemia due to PCSK9 deficiency is even more dramatic. Two women lacking functional PCSK9 exhibited LDL cholesterol levels of Ϸ0.4 mmol/L (15 mg/dL). 11,12 PCSK9 is now considered a promising target to treat hypercholesterolemia and prevent coronary heart disease. Conclusion-InHuman PCSK9 is abundant in plasma, 13 and analysis of mice tha...
Background-The proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes independently of its enzymatic activity the degradation of the low-density lipoprotein (LDL) receptor. PCSK9 gain of function in humans leads to autosomal dominant hypercholesterolemia, whereas the absence of functional PCSK9 results in Ϸ7-fold lower levels of LDL cholesterol. This suggests that lowering PCSK9 may protect against atherosclerosis. Methods and Results-We investigated the role of PCSK9 in atherosclerosis in C57BL/6 wild-type (WT), apolipoprotein E-deficient, and LDL receptor-deficient mouse models. Circulating cholesterol levels, fast protein liquid chromatography profiles, aortic cholesteryl esters (CE), and plaque sizes were determined. Intima-media thicknesses were measured by ultrasound biomicroscopy. First, mice expressing null (knockout [KO]), normal (WT), or high (transgenic [Tg]) levels of PCSK9 were fed a 12-month Western diet. KO mice accumulated 4-fold less aortic CE than WT mice, whereas Tg mice exhibited high CE and severe aortic lesions. Next we generated apolipoprotein E-deficient mice, known to spontaneously develop lesions, that expressed null (KO/e), normal (WT/e), or high (Tg/e) levels of PCSK9. After a 6-month regular diet, KO/e mice showed a 39% reduction compared with WT/e mice in aortic CE accumulation, whereas Tg/e mice showed a 137% increase. Finally, LDL receptor-deficient mice expressing no (KO/L), normal (WT/L), or high (Tg/L) levels of PCSK9 were fed a Western diet for 3 months. KO/L and Tg/L mice exhibited levels of plasma cholesterol and CE accumulation similar to those of WT/L mice, suggesting that PCSK9 modulates atherosclerosis mainly via the LDL receptor. Conclusions-Altogether, our results show a direct relationship between PCSK9 and atherosclerosis. PCSK9 overexpression is proatherogenic, whereas its absence is protective. (Circulation. 2012;125:894-901.)Key Words: atherosclerosis Ⅲ cardiovascular diseases Ⅲ cholesterol Ⅲ lipoproteins Ⅲ proprotein convertases A therosclerosis is a progressive degenerative pathology of large arteries that leads to ischemic heart diseases, which are expected to remain one of the leading causes of mortality until at least 2030. 1 The different stages of atherosclerotic plaque development have been reviewed extensively. [2][3][4][5] In brief, high levels of circulating atherogenic lipoproteins favor the accumulation of oxidized low-density lipoproteins (LDLs) in the subendothelial space. The latter are taken up by invading macrophages that, if they cannot efflux excess cholesterol to high-density lipoprotein, become foam cells. Foamy cells then release cytokines that recruit more macrophages into the building plaque. After formation of a necrotic core, plaques may ultimately become unstable; their fibrous cap can rupture and cause the formation of thrombus that can lead to death. Although inflammation is an important contributor to atherosclerosis, accumulation of oxidized LDLs in the subendothelial compartment is a key triggering event in atherosclerosis...
The secretory factor VEGF-C has been directly implicated in various physiological processes during embryogenesis and human cancers. However, the importance of the conversion of its precursor proVEGF-C to mature VEGF-C in tumorigenesis, and vessel formation and the identity of the protease(s) that regulate these processes is/are not known. The intracellular processing of proVEGF-C that occurs within the dibasic motif HSIIRR(227)SL suggests the involvement of the proprotein convertases (PCs) in this process. In addition, furin and VEGF-C were found to be coordinately expressed in adult mouse tissues. Cotransfection of the furin-deficient colon carcinoma cell line LoVo with proVEGF-C and different PC members revealed that furin, PC5, and PC7 are candidate VEGF-C convertases. This finding is consistent with the in vitro digestions of an internally quenched synthetic fluorogenic peptide mimicking the cleavage site of proVEGF-C ((220)Q-VHSIIRR downward arrow SLP(230)). The processing of proVEGF-C is blocked by the inhibitory prosegments of furin, PC5, and PACE4, as well as by furin-motif variants of alpha2-macroglobulin and alpha1-antitrypsin. Subcutaneous injection of CHO cells stably expressing VEGF-C into nude mice enhanced angiogenesis and lymphangiogenesis, but not tumor growth. In contrast, expression of proVEGF-C obtained following mutation of the cleavage site (HSIIRR(227)SL to HSIISS(227)SL) inhibits angiogenesis and lymphangiogenesis as well as tumor growth. Our findings demonstrate the processing of proVEGF-C by PCs and highlight the potential use of PC inhibitors as agents for inhibiting malignancies induced by VEGF-C.
The proprotein convertase PC5/6 cleaves protein precursors after basic amino acids and is essential for implantation in CD1/129/Sv/ C57BL/6 mixed-background mice. Conditional inactivation of Pcsk5 in the epiblast but not in the extraembryonic tissue bypassed early embryonic lethality but resulted in death at birth. PC5/6-deficient embryos exhibited Gdf11-related phenotypes such as altered anteroposterior patterning with extra vertebrae and lack of tail and kidney agenesis. They also exhibited Gdf11-independent phenotypes, such as a smaller size, multiple hemorrhages, collapsed alveoli, and retarded ossification. In situ hybridization revealed overlapping PC5/6 and Gdf11 mRNA expression patterns. In vitro and ex vivo analyses showed that the selectivity of PC5/6 for Gdf11 essentially resides in the presence of a P1 Asn in the RSRR2N cleavage motif. This work identifies Gdf11 as a likely in vivo specific substrate of PC5/6 and opens the way to the identification of other key substrates of this convertase.Furin-like ͉ Meox2-cre ͉ Pcsk5
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