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Progressive familial intrahepatic cholestasis (PFIC) is a rare childhood manifested disease associated with impaired bile secretion with severe pruritus yellow stool, and sometimes hepatosplenomegaly. PFIC is caused by mutations in ATP8B1, ABCB11, ABCB4, TJP2, NR1H4, SLC51A, USP53, KIF12, ZFYVE19, and MYO5B genes depending on its type. ABCB11 mutations lead to PFIC2 that encodes the bile salt export pump (BSEP). Different mutations of ABCB11 have been reported in different population groups but no data available in Pakistani population being a consanguineous one. We sequenced coding exons of the ABCB11 gene along with its flanking regions in 66 unrelated Pakistani children along with parents with PFIC2 phenotype. We identified 20 variations of ABCB11: 12 in homozygous form, one compound heterozygous, and seven heterozygous. These variants include 11 missenses, two frameshifts, two nonsense mutations, and five splicing variants. Seven variants are novel candidate variants and are not detected in any of the 120 chromosomes from normal ethnically matched individuals. Insilico analysis revealed that four homozygous missense variations have high pathogenic scores. Minigene analysis of splicing variants showed exon skipping and the addition of exon. This data is a useful addition to the disease variants genomic database and would be used in the future to build a diagnostic algorithm.
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Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP , Colestasis Intrahepática , Humanos , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/genética , Colestasis Intrahepática/genética , Pakistán , Masculino , Femenino , Niño , Preescolar , Lactante , Mutación , Exones/genética , Estudios de Cohortes , HomocigotoRESUMEN
Liver fibrosis is a severe global health problem. However, no effective antifibrotic drugs have been approved. Surf4 is primarily located in the endoplasmic reticulum (ER) and mediates the transport of secreted proteins from the ER to the Golgi apparatus. Knockout of hepatic Surf4 (Surf4 LKO) in mice impairs very-low-density lipoprotein secretion without causing overt liver damage. Here, we found that collagen levels are significantly reduced in the liver of Surf4 LKO mice compared with control Surf4 flox mice, as demonstrated by proteomics, Western blot, and quantitative reverse transcription polymerase chain reaction. Therefore, this study aims to investigate whether and how hepatic Surf4 affects liver fibrosis. We observed that CCl4-induced liver fibrosis is significantly lower in Surf4 LKO mice than in Surf4 flox mice. Mechanistically, hepatic Surf4 deficiency reduces serum amyloid A1 (SAA1) secretion and hepatic stellate cell (HSC) activation. Surf4 coimmunoprecipitates and colocalizes with SAA1. Lack of hepatic Surf4 significantly reduces SAA1 secretion from hepatocytes, and SAA1 activates cultured human HSCs (LX-2 cells). Conditioned medium (CM) from Surf4-deficient primary hepatocytes activates LX-2 cells to a much lesser extent than CM from Surf4 flox primary hepatocytes, and this reduced effect is restored by the addition of recombinant SAA1 to CM from Surf4-deficient hepatocytes. Knockdown of SAA1 in primary hepatocytes or TLR2 in LX-2 cells significantly reduces LX-2 activation induced by CM from Surf4 flox hepatocytes but not from Surf4 LKO hepatocytes. Furthermore, knockdown of SAA1 significantly ameliorates liver fibrosis in Surf4 flox mice but does not further reduce liver fibrosis in Surf4 LKO mice. We also observe substantial expression of Surf4 and SAA1 in human fibrotic livers. Therefore, hepatic Surf4 facilitates SAA1 secretion, activates HSCs, and aggravates liver fibrosis, suggesting that hepatic Surf4 and SAA1 may serve as treatment targets for liver fibrosis.
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Membrane-type I metalloproteinase (MT1-MMP/MMP14) plays a key role in various pathophysiological processes, indicating an unaddressed need for a targeted therapeutic approach. However, mice genetically deficient in Mmp14 show severe defects in development and growth. To investigate the possibility of MT1-MMP inhibition as a safe treatment in adults, we generated global Mmp14 tamoxifen-induced conditional knockout (Mmp14kd) mice and found that MT1-MMP deficiency in adult mice resulted in severe inflammatory arthritis. Mmp14kd mice started to show noticeably swollen joints two weeks after tamoxifen administration, which progressed rapidly. Mmp14kd mice reached a humane endpoint 6 to 8 weeks after tamoxifen administration due to severe arthritis. Plasma TNF-α levels were also significantly increased in Mmp14kd mice. Detailed analysis revealed chondrocyte hypertrophy, synovial fibrosis, and subchondral bone remodeling in the joints of Mmp14kd mice. However, global conditional knockout of MT1-MMP in adult mice did not affect body weight, blood glucose, or plasma cholesterol and triglyceride levels. Furthermore, we observed substantial expression of MT1-MMP in the articular cartilage of patients with osteoarthritis. We then developed chondrocyte-specific Mmp14 tamoxifen-induced conditional knockout (Mmp14chkd) mice. Chondrocyte MT1-MMP deficiency in adult mice also caused apparent chondrocyte hypertrophy. However, Mmp14chkd mice did not exhibit synovial hyperplasia or noticeable arthritis, suggesting that chondrocyte MT1-MMP is not solely responsible for the onset of severe arthritis observed in Mmp14kd mice. Our findings also suggest that highly cell-type specific inhibition of MT1-MMP is required for its potential therapeutic use.
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Cartílago Articular , Osteoartritis , Animales , Ratones , Glucemia , Peso Corporal , Metaloproteinasa 14 de la Matriz/genética , Osteoartritis/inducido químicamente , Osteoartritis/genéticaRESUMEN
Extracellular matrix (ECM) resolution by matrix metalloproteinases (MMPs) is a well-documented mechanism. MMPs play a dual and complex role in modulating ECM degradation at different stages of liver fibrosis, depending on the timing and levels of their expression. Increased MMP-1 combats disease progression by cleaving the fibrillar ECM. Activated hepatic stellate cells (HSCs) increase expression of MMP-2, -9, and -13 in different chemicals-induced animal models, which may alleviate or worsen disease progression based on animal models and the stage of liver fibrosis. In the early stage, elevated expression of certain MMPs may damage surrounding tissue and activate HSCs, promoting fibrosis progression. At the later stage, downregulation of MMPs can facilitate ECM accumulation and disease progression. A number of phytochemicals modulate MMP activity and ECM turnover, alleviating disease progression. However, the effects of phytochemicals on the expression of different MMPs are variable and may depend on the disease models and stage, and the dosage, timing and duration of phytochemicals used in each study. Here, we review the most recent advances in the role of MMPs in the effects of phytochemicals on liver fibrogenesis, which indicates that further studies are warranted to confirm and define the potential clinical efficacy of these phytochemicals.
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Matriz Extracelular , Cirrosis Hepática , Animales , Cirrosis Hepática/tratamiento farmacológico , Matriz Extracelular/metabolismo , Fitoquímicos/farmacología , Fitoquímicos/uso terapéutico , Progresión de la EnfermedadRESUMEN
[This corrects the article DOI: 10.3389/fcvm.2021.764024.].
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BACKGROUND: Postprandial dyslipidemia is a causative risk factor for cardiovascular disease. The majority of absorbed dietary lipids are packaged into chylomicron and then delivered to circulation. Previous studies showed that Surf4 (surfeit locus protein 4) mediates very low-density lipoprotein secretion from hepatocytes. Silencing hepatic Surf4 markedly reduces the development of atherosclerosis in different mouse models of atherosclerosis without causing hepatic steatosis. However, the role of Surf4 in chylomicron secretion is unknown. METHODS: We developed inducible intestinal-specific Surf4 knockdown mice (Surf4IKO) using Vil1Cre-ERT2 and Surf4flox mice. Metabolic cages were used to monitor mouse metabolism. Enzymatic kits were employed to measure serum and tissue lipid levels. The expression of target genes was detected by qRT-PCR and Western Blot. Transmission electron microscopy and radiolabeled oleic acid were used to assess the structure of enterocytes and intestinal lipid absorption and secretion, respectively. Proteomics was performed to determine changes in protein expression in serum and jejunum. RESULTS: Surf4IKO mice, especially male Surf4IKO mice, displayed significant body weight loss, increased mortality, and reduced metabolism. Surf4IKO mice exhibited lipid accumulation in enterocytes and impaired fat absorption and secretion. Lipid droplets and small lipid vacuoles were accumulated in the cytosol and the endoplasmic reticulum lumen of the enterocytes of Surf4IKO mice, respectively. Surf4 colocalized with apoB and co-immunoprecipitated with apoB48 in differentiated Caco-2 cells. Intestinal Surf4 deficiency also significantly reduced serum triglyceride, cholesterol, and free fatty acid levels in mice. Proteomics data revealed that diverse pathways were altered in Surf4IKO mice. In addition, Surf4IKO mice had mild liver damage, decreased liver size and weight, and reduced hepatic triglyceride levels. CONCLUSIONS: Our findings demonstrate that intestinal Surf4 plays an essential role in lipid absorption and chylomicron secretion and suggest that the therapeutic use of Surf4 inhibition requires highly cell/tissue-specific targeting.
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Aterosclerosis , Mucosa Intestinal , Humanos , Masculino , Animales , Ratones , Mucosa Intestinal/metabolismo , Células CACO-2 , Absorción Intestinal/fisiología , Grasas de la Dieta , Quilomicrones/metabolismo , Metabolismo de los Lípidos/genética , Triglicéridos/metabolismo , Aterosclerosis/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismoRESUMEN
Surfeit 4 is a polytopic transmembrane protein that primarily resides in the endoplasmic reticulum (ER) membrane. It is ubiquitously expressed and functions as a cargo receptor, mediating cargo transport from the ER to the Golgi apparatus via the canonical coat protein complex II (COPII)-coated vesicles or specific vesicles. It also participates in ER-Golgi protein trafficking through a tubular network. Meanwhile, it facilitates retrograde transportation of cargos from the Golgi apparatus to the ER through COPI-coated vesicles. Surf4 can selectively mediate export of diverse cargos, such as PCSK9 very low-density lipoprotein (VLDL), progranulin, α1-antitrypsin, STING, proinsulin, and erythropoietin. It has been implicated in facilitating VLDL secretion, promoting cell proliferation and migration, and increasing replication of positive-strand RNA viruses. Therefore, Surf4 plays a crucial role in various physiological and pathophysiological processes and emerges as a promising therapeutic target. However, the molecular mechanisms by which Surf4 selectively sorts diverse cargos for ER-Golgi protein trafficking remain elusive. Here, we summarize the most recent advances in Surf4, focusing on its role in lipid metabolism.
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Metabolismo de los Lípidos , Proteínas de la Membrana , Humanos , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Proproteína Convertasa 9/metabolismo , Transporte de Proteínas/genética , Transporte de Proteínas/fisiologíaRESUMEN
Low-density lipoprotein receptor (LDLR) mediates clearance of plasma LDL cholesterol, preventing the development of atherosclerosis. We previously demonstrated that membrane type 1-matrix metalloproteinase (MT1-MMP) cleaves LDLR and exacerbates the development of atherosclerosis. Here, we investigated determinants in LDLR and MT1-MMP that were critical for MT1-MMP-induced LDLR cleavage. We observed that deletion of various functional domains in LDLR or removal of each of the five predicted cleavage sites of MT1-MMP on LDLR did not affect MT1-MMP-induced cleavage of the receptor. Removal of the hemopexin domain or the C-terminal cytoplasmic tail of MT1-MMP also did not impair its ability to cleave LDLR. On the other hand, mutant MT1-MMP, in which the catalytic domain or the MT-loop was deleted, could not cleave LDLR. Further Ala-scanning analysis revealed an important role for Ile at position 167 of the MT-loop in MT1-MMP's action on LDLR. Replacement of Ile167 with Ala, Thr, Glu, or Lys resulted in a marked loss of the ability to cleave LDLR, whereas mutation of Ile167 to a non-polar amino acid residue, including Leu, Val, Met, and Phe, had no effect. Therefore, our studies indicate that MT1-MMP does not require a specific cleavage site on LDLR. In contrast, an amino acid residue with a hydrophobic side chain at position 167 in the MT-loop is critical for MT1-MMP-induced LDLR cleavage.
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Elevated plasma levels of low-density lipoprotein-C (LDL-C) increase the risk of atherosclerotic cardiovascular disease. Circulating LDL is derived from very low-density lipoprotein (VLDL) metabolism and cleared by LDL receptor (LDLR). We have previously demonstrated that cargo receptor Surfeit 4 (Surf4) mediates VLDL secretion. Inhibition of hepatic Surf4 impairs VLDL secretion, significantly reduces plasma LDL-C levels, and markedly mitigates the development of atherosclerosis in LDLR knockout (Ldlr-/-) mice. Here, we investigated the role of Surf4 in lipoprotein metabolism and the development of atherosclerosis in another commonly used mouse model of atherosclerosis, apolipoprotein E knockout (apoE-/-) mice. Adeno-associated viral shRNA was used to silence Surf4 expression mainly in the liver of apoE-/- mice. In apoE-/- mice fed a regular chow diet, knockdown of Surf4 expression significantly reduced triglyceride secretion and plasma levels of non-HDL cholesterol and triglycerides without causing hepatic lipid accumulation or liver damage. When Surf4 was knocked down in apoE-/- mice fed the Western-type diet, we observed a significant reduction in plasma levels of non-HDL cholesterol, but not triglycerides. Knockdown of Surf4 did not increase hepatic cholesterol and triglyceride levels or cause liver damage, but significantly diminished atherosclerosis lesions. Therefore, our findings indicate the potential of hepatic Surf4 inhibition as a novel therapeutic strategy to reduce the risk of atherosclerotic cardiovascular disease.
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Aterosclerosis , Enfermedades Cardiovasculares , Animales , Apolipoproteínas E/metabolismo , Aterosclerosis/metabolismo , Enfermedades Cardiovasculares/metabolismo , Colesterol/metabolismo , LDL-Colesterol/metabolismo , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Triglicéridos/metabolismoRESUMEN
BACKGROUND: Traumatic brain edema occurs commonly brain injury, and most manifests as pericontusional edema of brain contusions. On the basis of evidence-based medicine, apart from recommending craniotomy and mannitol, there are few particularly effective measures to prevent and treat traumatic brain edema. It is uncertain whether an early complementary acupuncture treatment would improve long-term outcomes of patients with traumatic brain edema. The aim of this study is to assess the efficacy and the safety of early complementary acupuncture for patients with traumatic brain edema. METHODS: This study is an actively accruing, single-center, single-blinded, 2-arm, randomized controlled trial. Patients with traumatic brain injury, a Glasgow Coma Scale score of 6â¼12, and brain edema on computed tomography scan will be divided into 2 groups on the basis of stratified block randomization. All patients will receive conventional treatment, and the study group will undergo additional acupuncture therapy (start within 72âhours after the injury) once a day for 28âdays. The primary outcome is the dichotomized Glasgow Outcome Score at 6âmonths and 12âmonths after injury, and the secondary outcomes are the Glasgow Coma Scale, the volume of traumatic brain edema, the serum levels of C-reactive protein and interleukin-6, and the Modified Barthel Index. DISCUSSION: This study will provide data regarding the efficacy of early complementary acupuncture for traumatic brain edema. If the study yields positive results, its findings may offer insights into a valuable complementary option of acupuncture for traumatic brain edema that could provide pilot evidence for large, randomized, controlled trials.Trial registration: This trial has been published in the Chinese Clinical Trial Register, http://www.chictr.org.cn/edit.aspx?pid=141208&htm=4 (Identifier: ChiCTR2100053794, registered on December 3, 2021).
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Acupuntura , Edema Encefálico/terapia , Lesiones Traumáticas del Encéfalo/complicaciones , Terapia por Acupuntura/métodos , Adolescente , Adulto , Anciano , Edema Encefálico/etiología , Lesiones Traumáticas del Encéfalo/terapia , Humanos , Persona de Mediana Edad , Pronóstico , Resultado del Tratamiento , Adulto JovenRESUMEN
The adrenal gland produces steroid hormones to play essential roles in regulating various physiological processes. Our previous studies showed that knockout of hepatic Surf4 (Surf4LKO) markedly reduced fasting plasma total cholesterol levels in adult mice, including low-density lipoprotein and high-density lipoprotein cholesterol. Here, we found that plasma cholesterol levels were also dramatically reduced in 4-week-old young mice and non-fasted adult mice. Circulating lipoprotein cholesterol is an important source of the substrate for the production of adrenal steroid hormones. Therefore, we investigated whether adrenal steroid hormone production was affected in Surf4LKO mice. We observed that lacking hepatic Surf4 essentially eliminated lipid droplets and significantly reduced cholesterol levels in the adrenal gland; however, plasma levels of aldosterone and corticosterone were comparable in Surf4LKO and the control mice under basal and stress conditions. Further analysis revealed that mRNA levels of genes encoding enzymes important for hormone synthesis were not altered, whereas the expression of scavenger receptor class B type I (SR-BI), low-density lipoprotein receptor (LDLR) and 3-hydroxy-3-methyl-glutaryl-CoA reductase was significantly increased in the adrenal gland of Surf4LKO mice, indicating increased de novo cholesterol biosynthesis and enhanced LDLR and SR-BI-mediated lipoprotein cholesterol uptake. We also observed that the nuclear form of SREBP2 was increased in the adrenal gland of Surf4 LKO mice. Taken together, these findings indicate that the very low levels of circulating lipoprotein cholesterol in Surf4LKO mice cause a significant reduction in adrenal cholesterol levels but do not significantly affect adrenal steroid hormone production. Reduced adrenal cholesterol levels activate SREBP2 and thus increase the expression of genes involved in cholesterol biosynthesis, which increases de novo cholesterol synthesis to compensate for the loss of circulating lipoprotein-derived cholesterol in the adrenal gland of Surf4LKO mice.
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Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes degradation of low-density lipoprotein receptor (LDLR) and plays a central role in regulating plasma levels of LDL cholesterol levels, lipoprotein(a) and triglyceride-rich lipoproteins, increasing the risk of cardiovascular disease. Additionally, PCSK9 promotes degradation of major histocompatibility protein class I and reduces intratumoral infiltration of cytotoxic T cells. Inhibition of PCSK9 increases expression of LDLR, thereby reducing plasma levels of lipoproteins and the risk of cardiovascular disease. PCSK9 inhibition also increases cell surface levels of major histocompatibility protein class I in cancer cells and suppresses tumor growth. Therefore, PCSK9 plays a vital role in the pathogenesis of cardiovascular disease and cancer, the top two causes of morbidity and mortality worldwide. Monoclonal anti-PCSK9 antibody-based therapy is currently the only available treatment that can effectively reduce plasma LDL-C levels and suppress tumor growth. However, high expenses limit their widespread use. PCSK9 promotes lysosomal degradation of its substrates, but the detailed molecular mechanism by which PCSK9 promotes degradation of its substrates is not completely understood, impeding the development of more cost-effective alternative strategies to inhibit PCSK9. Here, we review our current understanding of PCSK9 and focus on the regulation of its expression and functions.
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Lipids exert many essential physiological functions, such as serving as a structural component of biological membranes, storing energy, and regulating cell signal transduction. Dysregulation of lipid metabolism can lead to dyslipidemia related to various human diseases, such as obesity, diabetes, and cardiovascular disease. Therefore, lipid metabolism is strictly regulated through multiple mechanisms at different levels, including the extracellular matrix. Membrane-type I matrix metalloproteinase (MT1-MMP), a zinc-dependent endopeptidase, proteolytically cleaves extracellular matrix components, and non-matrix proteins, thereby regulating many physiological and pathophysiological processes. Emerging evidence supports the vital role of MT1-MMP in lipid metabolism. For example, MT1-MMP mediates ectodomain shedding of low-density lipoprotein receptor and increases plasma low-density lipoprotein cholesterol levels and the development of atherosclerosis. It also increases the vulnerability of atherosclerotic plaque by promoting collagen cleavage. Furthermore, it can cleave the extracellular matrix of adipocytes, affecting adipogenesis and the development of obesity. Therefore, the activity of MT1-MMP is strictly regulated by multiple mechanisms, such as autocatalytic cleavage, endocytosis and exocytosis, and post-translational modifications. Here, we summarize the latest advances in MT1-MMP, mainly focusing on its role in lipid metabolism, the molecular mechanisms regulating the function and expression of MT1-MMP, and their pharmacotherapeutic implications.
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Aterosclerosis/metabolismo , Metabolismo de los Lípidos , Metaloproteinasa 14 de la Matriz/metabolismo , Obesidad/metabolismo , Transducción de Señal , Adipocitos/metabolismo , Animales , Membrana Celular/metabolismo , Matriz Extracelular/metabolismo , Humanos , Macrófagos/metabolismo , Músculo Liso Vascular/metabolismo , Receptores de LDL/metabolismoRESUMEN
Plasma LDL is produced from catabolism of VLDL and cleared from circulation mainly via the hepatic LDL receptor (LDLR). Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes LDLR degradation, increasing plasma LDL-C levels. Circulating PCSK9 is mainly secreted by the liver, whereas VLDL is exclusively secreted by hepatocytes. However, the mechanism regulating their secretion is not completely understood. Surfeit 4 (Surf4) is a cargo receptor localized in the ER membrane. It recruits cargos into coat protein complex II vesicles to facilitate their secretion. Here, we investigated the role of Surf4 in VLDL and PCSK9 secretion. We generated Surf4 liver-specific knockout mice and found that knockout of Surf4 did not affect PCSK9 secretion, whereas it significantly reduced plasma levels of cholesterol, triglyceride, and lipid-binding protein apolipoprotein B (apoB). In cultured human hepatocytes, Surf4 coimmunoprecipitated and colocalized with apolipoprotein B100, and Surf4 silencing reduced secretion of apolipoprotein B100. Furthermore, knockdown of Surf4 in LDLR knockout (Ldlr-/-) mice significantly reduced triglyceride secretion, plasma levels of apoB and non-HDL-C, and the development of atherosclerosis. However, Surf4 liver-specific knockout mice and Surf4 knockdown in Ldlr-/- mice displayed similar levels of liver lipids and plasma alanine aminotransferase activity as control mice, indicating that inhibition of Surf4 does not cause notable liver damage. Expression of stearoyl-CoA desaturase-1 was also reduced in the liver of these mice, suggesting a reduction in de novo lipogenesis. In summary, hepatic deficiency of Surf4 reduced VLDL secretion and the development of atherosclerosis but did not cause significant hepatic lipid accumulation or liver damage.
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Aterosclerosis/metabolismo , Lipoproteínas VLDL/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Células Cultivadas , Proteínas de la Membrana/deficiencia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proproteína Convertasa 9/deficiencia , Proproteína Convertasa 9/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/metabolismoRESUMEN
Plasma low-density lipoprotein (LDL) is primarily cleared by LDL receptor (LDLR). LDLR can be proteolytically cleaved to release its soluble ectodomain (sLDLR) into extracellular milieu. However, the proteinase responsible for LDLR cleavage is unknown. Here we report that membrane type 1-matrix metalloproteinase (MT1-MMP) co-immunoprecipitates and co-localizes with LDLR and promotes LDLR cleavage. Plasma sLDLR and cholesterol levels are reduced while hepatic LDLR is increased in mice lacking hepatic MT1-MMP. Opposite effects are observed when MT1-MMP is overexpressed. MT1-MMP overexpression significantly increases atherosclerotic lesions, while MT1-MMP knockdown significantly reduces cholesteryl ester accumulation in the aortas of apolipoprotein E (apoE) knockout mice. Furthermore, sLDLR is associated with apoB and apoE-containing lipoproteins in mouse and human plasma. Plasma levels of sLDLR are significantly increased in subjects with high plasma LDL cholesterol levels. Thus, we demonstrate that MT1-MMP promotes ectodomain shedding of hepatic LDLR, thereby regulating plasma cholesterol levels and the development of atherosclerosis.
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Apolipoproteína B-100/sangre , Apolipoproteínas E/sangre , Aterosclerosis/patología , Lipoproteínas LDL/sangre , Metaloproteinasa 14 de la Matriz/metabolismo , Receptores de LDL/metabolismo , Animales , Apolipoproteínas E/genética , Línea Celular Tumoral , Ésteres del Colesterol/metabolismo , Dependovirus/genética , Femenino , Células HEK293 , Células Hep G2 , Humanos , Masculino , Metaloproteinasa 14 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Plasma levels of cholesterol, especially low-density lipoprotein cholesterol (LDL-C), are positively correlated with the risk of cardiovascular disease. Buildup of LDL in the intima promotes the formation of foam cells and consequently initiates atherosclerosis, one of the main underlying causes of cardiovascular disease. Hepatic LDL receptor (LDLR) is mainly responsible for the clearance of plasma LDL. Mutations in LDLR cause familiar hypercholesterolemia and increase the risk of premature coronary heart disease. Proprotein convertase subtilisin/kexin-type 9 (PCSK9) promotes LDLR degradation and thereby plays a critical role in the regulation of plasma cholesterol metabolism. PCSK9 can bind to LDLR and reroute the receptor to lysosomes for degradation, increasing both circulating LDL-C levels and the risk of cardiovascular disease. PCSK9 is mainly regulated by sterol response element binding protein 2 (SREBP2) at the transcriptional level. Furthermore, many proteins have been identified as interacting with PCSK9, regulating plasma cholesterol levels. Pharmacotherapeutic inhibition of PCSK9 dramatically reduces plasma levels of LDL cholesterol and significantly reduces cardiovascular events. In this article, we summarize the latest advances in PCSK9, mainly focusing on the structure, function, and regulation of the protein, the underlying molecular mechanisms, and its pharmacotherapeutic applications.
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Metabolismo de los Lípidos , Proproteína Convertasa 9/metabolismo , Subtilisina/metabolismo , Humanos , Proproteína Convertasa 9/química , Receptores de LDL/metabolismo , Subtilisina/químicaRESUMEN
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory protein that promotes low-density lipoprotein receptor (LDLR) degradation and thereby regulating plasma levels of LDL cholesterol. Previous studies have revealed the role of the C-terminal domain (CTD) of PCSK9 in its secretion, however, how CTD regulates PCSK9 secretion is not completely understood. Additionally, SEC24A, the cargo adaptor protein of the coat protein complex II, has been implicated in the secretion of mouse PCSK9. Here, we investigated how CTD and SEC24 regulated PCSK9 secretion in humans. We found that mutant PCSK91-528, in which amino acids from 529 to the end (amino acid 692) were deleted, was maturated and secreted from cells as effectively as the wild-type protein. On the other hand, lacking amino acids 454 to 692 in mutant PCSK91-453 significantly reduced its maturation and secretion, but to a lesser extent when compared to mutants PCSK91-446, PCSK91-445 and PCSK91-444, that all markedly impaired PCSK9 maturation. However, mutant PCSK91-444 virtually eliminated PCSK9 secretion while PCSK91-446 and PCSK91-445 could still be adequately detected in culture medium. Interestingly, mutation of Pro445 to other amino acid residues considerably impaired the secretion of mutant PCSK91-445 but not the full-length protein. We also found that natural variants in CTD including S462P, S465L, E482G, R495Q and A522T impaired PCSK9 secretion. Further, the knockdown of SEC24A, SEC24B, SEC24C but not SEC24D reduced secretion of the full-length PCSK9 but not mutant PCSK91-446. Therefore, SEC24A, SEC24B, and SEC24C facilitate endogenous PCSK9 secretion from cultured human hepatocytes, that are most likely mediated by the CTD of PCSK9. Our studies also indicate that the CTD of PCSK9 may allosterically and independently modulate the stability of the hinge region. Collectively, these data revealed that the CTD of PCSK9 and the hinge region play a critical role in PCSK9 maturation and secretion.