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1.
Mol Cell Biol ; 44(4): 123-137, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38747374

RESUMEN

SREBP transcription factors are central regulators of lipid metabolism. Their proteolytic activation requires ER to the Golgi translocation and subsequent cleavage by site-1-protease (S1P). Produced as a proprotein, S1P undergoes autocatalytic cleavage from its precursor S1PA to mature S1PC form. Here, we report that SPRING (previously C12ORF29) and S1P interact through their ectodomains, and that this facilitates the autocatalytic cleavage of S1PA into its mature S1PC form. Reciprocally, we identified a S1P recognition-motif in SPRING and demonstrate that S1P-mediated cleavage leads to secretion of the SPRING ectodomain in cells, and in liver-specific Spring knockout (LKO) mice transduced with AAV-mSpring. By reconstituting SPRING variants into SPRINGKO cells we show that the SPRING ectodomain supports proteolytic maturation of S1P and SREBP signaling, but that S1P-mediated SPRING cleavage is not essential for these processes. Absence of SPRING modestly diminishes proteolytic maturation of S1PA→C and trafficking of S1PC to the Golgi. However, despite reaching the Golgi in SPRINGKO cells, S1PC fails to rescue SREBP signaling. Remarkably, whereas SREBP signaling was severely attenuated in SPRINGKO cells and LKO mice, that of ATF6, another S1P substrate, was unaffected in these models. Collectively, our study positions SPRING as a dedicated licensing factor for SREBP-specific activation by S1P.


Asunto(s)
Aparato de Golgi , Ratones Noqueados , Proproteína Convertasas , Animales , Ratones , Aparato de Golgi/metabolismo , Humanos , Proproteína Convertasas/metabolismo , Proproteína Convertasas/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/genética , Transducción de Señal , Células HEK293 , Hígado/metabolismo , Proteolisis , Retículo Endoplásmico/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteínas de Unión a los Elementos Reguladores de Esteroles/genética
2.
Science ; 383(6684): eadi3332, 2024 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-38359126

RESUMEN

The identification of mechanisms to store glucose carbon in the form of glycogen rather than fat in hepatocytes has important implications for the prevention of nonalcoholic fatty liver disease (NAFLD) and other chronic metabolic diseases. In this work, we show that glycogenesis uses its intermediate metabolite uridine diphosphate glucose (UDPG) to antagonize lipogenesis, thus steering both mouse and human hepatocytes toward storing glucose carbon as glycogen. The underlying mechanism involves transport of UDPG to the Golgi apparatus, where it binds to site-1 protease (S1P) and inhibits S1P-mediated cleavage of sterol regulatory element-binding proteins (SREBPs), thereby inhibiting lipogenesis in hepatocytes. Consistent with this mechanism, UDPG administration is effective at treating NAFLD in a mouse model and human organoids. These findings indicate a potential opportunity to ameliorate disordered fat metabolism in the liver.


Asunto(s)
Lipogénesis , Glucógeno Hepático , Hígado , Enfermedad del Hígado Graso no Alcohólico , Proproteína Convertasas , Serina Endopeptidasas , Uridina Difosfato Glucosa , Animales , Humanos , Ratones , Carbono/metabolismo , Glucosa/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Glucógeno Hepático/metabolismo , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Proproteína Convertasas/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Uridina Difosfato Glucosa/administración & dosificación , Uridina Difosfato Glucosa/metabolismo , Masculino , Ratones Endogámicos C57BL , Células HEK293
3.
Clin Chim Acta ; 556: 117847, 2024 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-38417778

RESUMEN

Familial hypercholesterolemia (FH) is an inherited disorder characterized by increased low-density lipoprotein LDL) cholesterol and atherosclerotic cardiovascular disease. Although initial genetic analysis linked FH to LDL receptor mutations, subsequent work demonstrated that a gain-of-function mutation in the proprotein convertase subtilisin/kexin type 9 (PCSK9), which causes LDL-R degradation, was shown to be the cause of FH. In this review, we describe the history of research on FH, its clinical phenotyping and genotyping and advances in treatment with special focus on Japan.


Asunto(s)
Hiperlipoproteinemia Tipo II , Proproteína Convertasa 9 , Humanos , Proproteína Convertasa 9/genética , Serina Endopeptidasas/metabolismo , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo , Proproteína Convertasas/uso terapéutico , Japón , Receptores de LDL/genética , Receptores de LDL/metabolismo , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/genética , Mutación
4.
Virology ; 592: 110008, 2024 04.
Artículo en Inglés | MEDLINE | ID: mdl-38335866

RESUMEN

Viral spike proteins undergo a special maturation process that enables host cell receptor recognition, membrane fusion, and viral entry, facilitating effective virus infection. Here, we investigated the protease cleavage features of ORF46, a spike-like protein in Ictalurid herpesvirus 1 (IcHV-1) sharing similarity with spikes of Nidovirales members. We noted that during cleavage, full-length ORF46 is cleaved into ∼55-kDa and ∼100-kDa subunits. Moreover, truncation or site-directed mutagenesis at the recognition sites of proprotein convertases (PCs) abolishes this spike cleavage, highlighting the crucial role of Arg506/Arg507 and Arg668/Arg671 for the cleavage modification. ORF46 cleavage was suppressed by specific N-glycosylation inhibitors or mutation of its specific N-glycosylation sites (N192, etc.), suggesting that glycoprotein ORF46 cleavage is modulated by N-glycosylation. Notably, PCs and N-glycosylation inhibitors exhibited potent antiviral effects in host cells. Our findings, therefore, suggested that PCs cleavage of ORF46, modulated by N-glycosylation, is a potent antiviral target for fish herpesviruses.


Asunto(s)
Ictalurivirus , Proproteína Convertasas , Animales , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo , Glicosilación , Proteínas Virales/genética , Proteínas Virales/metabolismo , Antivirales
5.
EMBO Rep ; 25(3): 951-970, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38287192

RESUMEN

The exquisite specificity of antibodies can be harnessed to effect targeted degradation of membrane proteins. Here, we demonstrate targeted protein removal utilising a protein degradation domain derived from the endogenous human protein Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9). Recombinant antibodies genetically fused to this domain drive the degradation of membrane proteins that undergo constitutive internalisation and recycling, including the transferrin receptor and the human cytomegalovirus latency-associated protein US28. We term this approach PACTAC (PCSK9-Antibody Clearance-Targeting Chimeras).


Asunto(s)
Proproteína Convertasa 9 , Serina Endopeptidasas , Humanos , Proproteína Convertasa 9/metabolismo , Proproteína Convertasas/metabolismo , Proteínas de la Membrana , Receptores de LDL/metabolismo
6.
FEBS J ; 291(7): 1575-1592, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38243371

RESUMEN

Ischemia/reperfusion (I/R)-induced acute kidney injury (AKI) is a common clinical syndrome with high morbidity and mortality. Ferroptosis, a newly discovered form of oxidative cell death, is involved in the pathogenesis of renal I/R injury; however, the underlying mechanism remains to be explored. Here, we reported that site 1 protease (S1P) promotes ischemic kidney injury by regulating ferroptotic cell death of tubular epithelial cells. S1P abundance was measured in hypoxia/reoxygenation (H/R)-treated Boston University mouse proximal tubular (BUMPT) cells and I/R-induced murine kidney tissue. S1P expression in BUMPT cells and kidneys was initially activated by hypoxic stimulation, accompanied by the ferroptotic response. Blocking S1P blunted H/R-induced ferroptotic cell death, which also restored sirtuin 3 (SIRT3) expression and superoxide dismutase 2 (SOD2) activity in BUMPT cells. Next, inhibition of S1P expression restored I/R-suppressed SIRT3 abundance, SOD2 activity and reduced the elevated level of mitochondria reactive oxygen species (mtROS), which attenuated tubular cell ferroptosis and renal I/R injury. In conclusion, S1P promoted renal tubular epithelial cell ferroptosis under I/R status by activating SIRT3-SOD2-mtROS signaling, thereby accelerating kidney injury. Thus, targeting S1P signaling may serve as a promising strategy for I/R kidney injury.


Asunto(s)
Lesión Renal Aguda , Ferroptosis , Daño por Reperfusión , Serina Endopeptidasas , Sirtuina 3 , Superóxido Dismutasa , Animales , Ratones , Lesión Renal Aguda/genética , Lesión Renal Aguda/patología , Células Epiteliales/metabolismo , Ferroptosis/genética , Riñón/metabolismo , Péptido Hidrolasas/metabolismo , Daño por Reperfusión/metabolismo , Sirtuina 3/genética , Sirtuina 3/metabolismo , Serina Endopeptidasas/metabolismo , Proproteína Convertasas/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo
7.
Exp Neurol ; 374: 114688, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38216110

RESUMEN

Proprotein convertase subtilisin/kexin type 6 (PCSK6) is a calcium-dependent serine proteinase that regulates the proteolytic activity of various precursor proteins and facilitates protein maturation. Dysregulation of PCSK6 expression or function has been implicated in several pathological processes including nervous system diseases. However, whether and how PCSK6 is involved in the pathogenesis of Alzheimer's disease (AD) remains unclear. In this study, we reported that the expression of PCSK6 was significantly increased in the brain tissues of postmortem AD patients and APP23/PS45 transgenic AD model mice, as well as N2AAPP cells. Genetic knockdown of PCSK6 reduced amyloidogenic processing of APP in N2AAPP cells by suppressing the activation of membrane-type 5-matrix metalloproteinase (MT5-MMP), referred to as η-secretase. We further found that PCSK6 cleaved and activated MT5-MMP by recognizing the RRRNKR sequence in its N-terminal propeptide domain in N2A cells. The mutation or knockout of this cleavage motif prevented PCSK6 from interacting with MT5-MMP and performing cleavage. Importantly, genetic knockdown of PCSK6 with adeno-associated virus (AAV) reduced Aß production and ameliorated hippocampal long-term potentiation (LTP) and long-term spatial learning and memory in APP23/PS45 transgenic mice. Taken together, these results demonstrate that genetic knockdown of PCSK6 effectively alleviate AD-related pathology and cognitive impairments by inactivating MT5-MMP, highlighting its potential as a novel therapeutic target for AD treatment.


Asunto(s)
Enfermedad de Alzheimer , Animales , Humanos , Ratones , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Modelos Animales de Enfermedad , Ratones Transgénicos , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo , Proteolisis , Serina Endopeptidasas/metabolismo , Aprendizaje Espacial
8.
Gastric Cancer ; 27(2): 292-307, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38280128

RESUMEN

BACKGROUND: Gastric cancer (GC), the fourth leading cause of cancer-related death worldwide, with most deaths caused by advanced and metastatic disease, has limited curative options. Here, we revealed the importance of proprotein convertases (PCs) in the malignant and metastatic potential of GC cells through the regulation of the YAP/TAZ/TEAD pathway and epithelial-to-mesenchymal transition (EMT) in cancer stem cells (CSC). METHODS: The general PCs inhibitor, decanoyl-RVKR-chloromethyl-ketone (CMK), was used to repress PCs activity in CSCs of various GC cell lines. Their tumorigenic properties, drug resistance, YAP/TAZ/TEAD pathway activity, and invasive properties were then investigated in vitro, and their metastatic properties were explored in a mouse xenograft model. The prognostic value of PCs in GC patients was also explored in molecular databases of GC. RESULTS: Inhibition of PCs activity in CSCs in all GC cell lines reduced tumorsphere formation and growth, drug efflux, EMT phenotype, and invasive properties that are associated with repressed YAP/TAZ/TEAD pathway activity in vitro. In vivo, PCs' inhibition in GC cells reduced their metastatic spread. Molecular analysis of tumors from GC patients has highlighted the prognostic value of PCs. CONCLUSIONS: PCs are overexpressed in GC and associated with poor prognosis. PCs are involved in the malignant and metastatic potential of CSCs via the regulation of EMT, the YAP/TAZ/TEAD oncogenic pathway, and their stemness and invasive properties. Their repression represents a new strategy to target CSCs and impair metastatic spreading in GC.


Asunto(s)
Neoplasias Gástricas , Factores de Transcripción , Humanos , Animales , Ratones , Factores de Transcripción/genética , Proteínas Señalizadoras YAP , Neoplasias Gástricas/patología , Modelos Animales de Enfermedad , Proproteína Convertasas/metabolismo , Células Madre Neoplásicas/metabolismo
9.
Arterioscler Thromb Vasc Biol ; 44(2): 328-333, 2024 02.
Artículo en Inglés | MEDLINE | ID: mdl-38059350

RESUMEN

The tremendous burden of lipid metabolism diseases, coupled with recent developments in human somatic gene editing, has motivated researchers to propose population-wide somatic gene editing of PCSK9 (proprotein convertase subtilisin/kexin type 9) within the livers of otherwise healthy humans. The best-characterized molecular function of PCSK9 is its ability to regulate plasma LDL (low-density lipoprotein) levels through promoting LDL receptor degradation. Individuals with loss-of-function PCSK9 variants have lower levels of plasma LDL and reduced cardiovascular disease. Gain-of-function variants of PCSK9 are strongly associated with familial hypercholesterolemia. A new therapeutic strategy delivers CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats; CRISPR-associated protein 9) specifically to liver cells to edit the wild-type alleles of PCSK9 with the goal of producing a loss-of-function allele. This direct somatic gene editing approach is being pursued despite the availability of US Food and Drug Administration-approved PCSK9 inhibitors that lower plasma LDL levels. Here, we discuss other characterized functions of PCSK9 including its role in infection and host immunity. We explore important factors that may have contributed to the evolutionary selection of PCSK9 in several vertebrates, including humans. Until such time that more fully understand the multiple biological roles of PCSK9, the ethics of permanently editing the gene locus in healthy, wild-type populations remains highly questionable.


Asunto(s)
Proproteína Convertasa 9 , Proproteína Convertasas , Animales , Humanos , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo , Serina Endopeptidasas/genética , Alelos , Receptores de LDL/genética
10.
AIDS ; 38(3): 317-327, 2024 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-37788081

RESUMEN

BACKGROUND: Proprotein convertase subtisilin/kexin 9 (PCSK9) raises low-density lipoprotein cholesterol (LDL-C) levels and is associated with inflammation, which is elevated in HIV and hepatitis C virus (HCV) infection. We compared PCSK9 levels in people with co-occurring HIV and HCV (HIV/HCV) vs. HIV alone, and evaluated the impact of HCV direct-acting antiviral (DAA) therapy on PCSK9. DESIGN: A prospective, observational cohort study. METHODS: Thirty-five adults with HIV/HCV and 37 with HIV alone were evaluated, all with HIV virologic suppression and without documented cardiovascular disease. Circulating PCSK9 and inflammatory biomarkers were measured at baseline and following HCV treatment or at week 52 (for HIV alone) and compared using Wilcoxon tests and Spearman correlations. RESULTS: At baseline, PCSK9 trended higher in HIV/HCV vs. HIV alone (307 vs. 284 ng/ml, P  = 0.06). Twenty-nine participants with HIV/HCV completed DAA therapy with sustained virologic response. PCSK9 declined from baseline to posttreatment 1 (median 7.3 weeks after end of therapy [EOT]) and posttreatment 2 (median 43.5 weeks after EOT), reaching levels similar to HIV alone; median within-person reduction was -60.5 ng/ml ( P  = 0.003) and -55.6 ng/ml ( P  = 0.02), respectively. Decline in PCSK9 correlated with decline in soluble (s)E-selectin and sCD163 ( r  = 0.64, P  = 0.002; r  = 0.58, P  = 0.008, respectively), but not with changes in LDL-C or other biomarkers. No significant change in PCSK9 occurred in the HIV alone group over 52 weeks. CONCLUSION: PCSK9 declined with DAA therapy in participants with HIV/HCV, correlating with declines in several inflammatory biomarkers but not LDL-C. Elevated PCSK9 with HCV may be linked to particular HCV-associated inflammatory pathways more so than cholesterol homeostasis.


Asunto(s)
Infecciones por VIH , Hepatitis C Crónica , Hepatitis C , Adulto , Humanos , Proproteína Convertasa 9 , Antivirales/uso terapéutico , Hepacivirus , LDL-Colesterol , Estudios Prospectivos , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/tratamiento farmacológico , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Proproteína Convertasas/metabolismo , Hepatitis C/complicaciones , Hepatitis C/tratamiento farmacológico , Inflamación/complicaciones , Biomarcadores
11.
Naunyn Schmiedebergs Arch Pharmacol ; 397(3): 1727-1736, 2024 03.
Artículo en Inglés | MEDLINE | ID: mdl-37721554

RESUMEN

Proprotein convertase subtilisin/kexin 9 (PCSK9) inhibitors have been shown to regulate lipid metabolism and reduce the risk of cardiovascular events. This study explores the effect and potential mechanism of PCSK9 inhibitors on lipid metabolism and coronary atherosclerosis. HepG2 cells were incubated with PCSK9 inhibitor. ApoE-/- mice were fed with a high fat to construct an atherosclerosis model, and then treated with PCSK9 inhibitor (8 mg/kg for 8 w). PCSK9 inhibitor downregulated microRNA (miRNA)-130a-3p expression in a dose-dependent manner. And, miR-130a-3p could bind directly to the 3' untranslated region (3'-UTR) region of LDLR to down-regulate LDLR expression in HepG2 cells, as confirmed by the luciferase reporter gene assay. In addition, miR-130a-3p overexpression significantly attenuated the promoting effect of PCSK9 inhibitor on LDLR and DiI-LDL uptake in HepG2 cells. More importantly, in vivo experiments confirmed that PCSK9 inhibitor could significantly inhibit miR-130a-3p levels and promote LDLR expression in liver tissues, thus regulating serum lipid profile and alleviating the progression of coronary atherosclerosis. PCSK9 inhibitor could moderately improve coronary atherosclerosis by regulating miR-130a-3p/LDLR axis, providing an exploitable strategy for the treatment of coronary atherosclerosis.


Asunto(s)
Aterosclerosis , Enfermedad de la Arteria Coronaria , MicroARNs , Ratones , Animales , Humanos , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , Proproteína Convertasa 9/farmacología , Subtilisina/metabolismo , Subtilisina/farmacología , Receptores de LDL/genética , Receptores de LDL/metabolismo , Ratones Noqueados para ApoE , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo , Proproteína Convertasas/farmacología , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/genética , Aterosclerosis/metabolismo , Hepatocitos , Células Hep G2 , MicroARNs/genética , MicroARNs/metabolismo
12.
Cancer Rep (Hoboken) ; 7(1): e1920, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-38018319

RESUMEN

BACKGROUND: Endometrial adenocarcinoma (EAC) is a malignant tumor of the endometrium. EAC is the most common female malignancy following the menopause period. About 40% of patients with EAC are linked with obesity and interrelated with hypertension, diabetes mellitus, and high circulating estrogen levels. Proprotein convertase (PC) furin was involved in the progression of EAC. RECENT FINDINGS: Furin is a protease enzyme belonging to the subtilisin PC family called PC subtilisin/kexin type 3 that converts precursor proteins to biologically active forms and products. Aberrant activation of furin promotes abnormal cell proliferation and the development of cancer. Furin promotes angiogenesis, malignant cell proliferation, and tissue invasion by malignant cells through its pro-metastatic and oncogenic activities. Furin activity is correlated with the malignant proliferation of EAC. Higher expression of furin may increase the development of EAC through overexpression of pro-renin receptors and disintegrin and metalloprotease 17 (ADAM17). As well, inflammatory signaling in EAC promotes the expression of furin with further propagation of malignant transformation. CONCLUSION: Furin is associated with the development and progression of EAC through the induction of proliferation, invasion, and metastasis of malignant cells of EAC. Furin induces ontogenesis in EAC through activation expression of ADAM17, pro-renin receptor, CD109, and TGF-ß. As well, EAC-mediated inflammation promotes the expression of furin with further propagation of neoplastic growth and invasion.


Asunto(s)
Adenocarcinoma , Furina , Humanos , Femenino , Furina/genética , Furina/metabolismo , Proproteína Convertasas/metabolismo , Subtilisinas/metabolismo , Transducción de Señal
13.
Hormones (Athens) ; 23(1): 137-140, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-37999906

RESUMEN

AIM: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a regulator of low-density-lipoprotein cholesterol (LDL-C), a major risk factor for cardiovascular (CV) disease. Since the hormone leptin has been suggested as having a role in CV risk regulation, possibly by modulating LDL receptor expression through the PCSK9 pathway, nutritional status may represent a potential regulator. Thus, evaluation of PCSK9 levels in human eating disorders appears to be of interest. In this report, we evaluate the lipoprotein profile, PCSK9, and leptin levels in subjects affected by anorexia nervosa (AN) to improve our understanding of the metabolic alterations in this disease. METHODS AND RESULTS: We designed a case-control observational study, enrolling 20 anorexic adolescent females and 20 adolescent females without AN as the control group, age- and sex-matched. Subjects affected by AN showed lower BMI, total cholesterol, and LDL-C in comparison to the control group, with lipoprotein levels in the normal range. Furthermore, adolescent girls with AN show significantly higher PCSK9 (+24%, p < 0.005) and lower leptin levels (-43%, p < 0.01), compared to the control group. CONCLUSIONS: The findings of increased levels of PCSK9 and reduced leptin levels among AN subjects warrant further research in order to unravel the role of the liver and adipose tissue in the management of PCSK9/LDL metabolism in adolescents affected by AN.


Asunto(s)
Anorexia Nerviosa , Proproteína Convertasa 9 , Femenino , Adolescente , Humanos , LDL-Colesterol , Leptina , Proproteína Convertasas/metabolismo , Serina Endopeptidasas/metabolismo
14.
J Microbiol Biotechnol ; 34(2): 425-435, 2024 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-37997262

RESUMEN

Schisandra chinensis extract (SCE) protects against hypocholesterolemia by inhibiting proprotein convertase subtilisin/kexin 9 (PCSK9) protein stabilization. We hypothesized that the hypocholesterolemic activity of SCE can be attributable to upregulation of the PCSK9 inhibition-associated low-density lipoprotein receptor (LDLR). Male mice were fed a low-fat diet or a Western diet (WD) containing SCE at 1% for 12 weeks. WD increased final body weight and blood LDL cholesterol levels as well as alanine transaminase and aspartate aminotransferase expression. However, SCE supplementation significantly attenuated the increase in blood markers caused by WD. SCE also attenuated WD-mediated increases in hepatic LDLR protein expression in the obese mice. In addition, SCE increased LDLR protein expression and attenuated cellular PCSK9 levels in HepG2 cells supplemented with delipidated serum (DLPS). Non-toxic concentrations of schisandrin A (SA), one of the active components of SCE, significantly increased LDLR expression and tended to decrease PCSK9 protein levels in DLPS-treated HepG2 cells. High levels of SA-mediated PCSK9 attenuation was not attributable to reduced PCSK9 gene expression, but was associated with free PCSK9 protein degradation in this cell model. Our findings show that PCSK9 secretion can be significantly reduced by SA treatment, contributing to reductions in free cholesterol levels.


Asunto(s)
Ciclooctanos , Hígado Graso , Lignanos , Compuestos Policíclicos , Schisandra , Masculino , Ratones , Animales , Humanos , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , Schisandra/metabolismo , Serina Endopeptidasas/genética , Subtilisina , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Células Hep G2
15.
Metabolism ; 150: 155736, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-37967646

RESUMEN

BACKGROUND: Epidemiological evidence links the proprotein convertase subtilisin/kexin 7 (PCSK7) to triglyceride (TG) metabolism. We associated the known PCSK7 gain-of-function non-coding SNP rs236918 with higher levels of plasma apolipoprotein B (apoB) and the loss-of-function coding variant p.Pro777Leu (SNP rs201598301) with lower apoB and TG. Herein, we aimed to unravel the in vivo role of liver PCSK7. METHODS: We biochemically defined the functional role of PCSK7 in lipid metabolism using hepatic cell lines and Pcsk7-/- mice. Our findings were validated following subcutaneous administration of hepatocyte-targeted N-acetylgalactosamine (GalNAc)-antisense oligonucleotides (ASOs) against Pcsk7. RESULTS: Independent of its proteolytic activity, membrane-bound PCSK7 binds apoB100 in the endoplasmic reticulum and enhances its secretion. Mechanistically, the loss of PCSK7/Pcsk7 leads to apoB100 degradation, triggering an unfolded protein response, autophagy, and ß-oxidation, eventually reducing lipid accumulation in hepatocytes. Non-alcoholic fatty liver disease (NAFLD) was induced by a 12-week high fat/fructose/cholesterol diet in wild type (WT) and Pcsk7-/- mice that were then allowed to recover on a 4-week control diet. Pcsk7-/- mice recovered more effectively than WT mice from all NAFLD-related liver phenotypes. Finally, subcutaneous administration of GalNAc-ASOs targeting hepatic Pcsk7 to WT mice validated the above results. CONCLUSIONS: Our data reveal hepatic PCSK7 as one of the major regulators of apoB, and its absence reduces apoB secretion from hepatocytes favoring its ubiquitination and degradation by the proteasome. This results in a cascade of events, eventually reducing hepatic lipid accumulation, thus supporting the notion of silencing PCSK7 mRNA in hepatocytes for targeting NAFLD.


Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Subtilisina/metabolismo , Triglicéridos/metabolismo , Hígado/metabolismo , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Proproteína Convertasas/metabolismo , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo
16.
Int J Nanomedicine ; 18: 7785-7801, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38144512

RESUMEN

Background: High-level low-density lipoprotein cholesterol (LDL-C) plays a vital role in the development of atherosclerotic cardiovascular disease. Low-density lipoprotein receptors (LDLRs) are scavengers that bind to LDL-C in the liver. LDLR proteins are regulated by proprotein convertase subtilisin/kexin type 9 (PCSK9), which mediates the degradation of LDLR and adjusts the level of the plasma LDL-C. The low expression of PCSK9 leads to the up-regulation of liver LDLRs and the reduction of plasma LDL-C. Hepatocytes are attractive targets for small interfering RNA (siRNA) delivery to silence Pcsk9 gene, due to their significant role in LDL-C regulation. Methods: Here, a type of liver-specific ionizable lipid nanoparticles is developed for efficient siRNA delivery. This type of nanoparticles shows high stability, enabling efficient cargo delivery specifically to hepatocytes, and a membrane-active polymer that reversibly masks activity until an acidic environment is reached. Results: Significantly, the siPcsk9 (siRNA targeting to Pcsk9)-loaded nanoparticles (GLP) could silence 90% of the Pcsk9 mRNA in vitro. In vivo study showed that the improved accumulation of GLP in the liver increased LDLR level by 3.35-fold and decreased plasma LDL-C by 35%. Conclusion: GLP has shown a powerful effect on reducing LDL-C, thus providing a potential therapy for atherosclerotic cardiovascular disease.


Asunto(s)
Aterosclerosis , Enfermedades Cardiovasculares , Nanopartículas , Humanos , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , LDL-Colesterol/genética , LDL-Colesterol/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo , Interferencia de ARN , Enfermedades Cardiovasculares/metabolismo , Hígado/metabolismo , Colesterol , Receptores de LDL/genética , Receptores de LDL/metabolismo , Aterosclerosis/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo
17.
Sci Rep ; 13(1): 19725, 2023 11 13.
Artículo en Inglés | MEDLINE | ID: mdl-37957262

RESUMEN

Splice modulating antisense oligomers (AOs) are increasingly used to modulate RNA processing. While most are investigated for their use as therapeutics, AOs can also be used for basic research. This study examined their use to investigate internally and terminally truncated proprotein convertase subtilisin/kexin type 9 (PCSK9) protein isoforms. Previous studies have used plasmid or viral-vector-mediated protein overexpression to study different PCSK9 protein isoforms, creating an artificial environment within the cell. Here we designed and tested AOs to remove specific exons that encode for PCSK9 protein domains and produced protein isoforms at more physiologically relevant levels. We evaluated the isoforms' expression, secretion, and subsequent impact on the low-density lipoprotein (LDL) receptor and its activity in Huh-7 cells. We found that modifying the Cis-His-rich domain by targeting exons 10 or 11 negatively affected LDL receptor activity and hence did not enhance LDL uptake although the levels of LDL receptor were increased. On the other hand, removing the hinge region encoded by exon 8, or a portion of the prodomain encoded by exon 2, have the potential as therapeutics for hypercholesterolemia. Our findings expand the understanding of PCSK9 isoforms and their impact on the LDL receptor and its activity at physiologically relevant concentrations.


Asunto(s)
Proproteína Convertasa 9 , Serina Endopeptidasas , Empalme Alternativo , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , Proproteína Convertasas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Serina Endopeptidasas/metabolismo , Humanos
18.
Development ; 150(23)2023 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-38032088

RESUMEN

Heart development is a complex process that requires asymmetric positioning of the heart, cardiac growth and valve morphogenesis. The mechanisms controlling heart morphogenesis and valve formation are not fully understood. The pro-convertase FurinA functions in heart development across vertebrates. How FurinA activity is regulated during heart development is unknown. Through computational analysis of the zebrafish transcriptome, we identified an RNA motif in a variant FurinA transcript harbouring a long 3' untranslated region (3'UTR). The alternative 3'UTR furina isoform is expressed prior to organ positioning. Somatic deletions in the furina 3'UTR lead to embryonic left-right patterning defects. Reporter localisation and RNA-binding assays show that the furina 3'UTR forms complexes with the conserved RNA-binding translational repressor, Ybx1. Conditional ybx1 mutant embryos show premature and increased Furin reporter expression, abnormal cardiac morphogenesis and looping defects. Mutant ybx1 hearts have an expanded atrioventricular canal, abnormal sino-atrial valves and retrograde blood flow from the ventricle to the atrium. This is similar to observations in humans with heart valve regurgitation. Thus, the furina 3'UTR element/Ybx1 regulon is important for translational repression of FurinA and regulation of heart development.


Asunto(s)
Regulón , Pez Cebra , Animales , Humanos , Regiones no Traducidas 3' , Regulón/genética , Morfogénesis/genética , Válvulas Cardíacas , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Proproteína Convertasas/genética , Proproteína Convertasas/metabolismo
19.
J Immunol Res ; 2023: 3291137, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37937296

RESUMEN

Acute lung injury (ALI) is a life-threatening disease that currently lacks a cure. Although stem cell-derived small extracellular vesicles (sEVs) have shown promising effects in the treatment of ALI, their underlying mechanisms and responsible components have yet to be identified. Proprotein convertase subtilisin/kexin type 6 (PCSK6) is a gene involved in inflammation and a potential target of miR-21-5p, a microRNA enriched in stem cell-derived sEVs. The current study investigated the role of PCSK6 in lipopolysaccharide (LPS)-induced ALI and its interaction with miR-21-5p. Notably, our results showed that PCSK6 expression was positively correlated with LPS stimulation. Knockdown of PCSK6 ameliorated LPS-induced inhibition of proliferation and upregulation of permeability in human BEAS-2B cells, whereas PCSK6 overexpression displayed the opposite effects. BEAS-2B cells were able to actively internalize the cocultured bone mesenchymal stem cell (MSC)-derived sEVs (BMSC-sEVs), which alleviated the cell damage caused by LPS. Overexpressing PCSK6, however, eliminated the therapeutic effects of BMSC-sEV coculture. Mechanistically, BMSC-sEVs inhibited PCSK6 expression via the delivery of miR-21-5p, which is directly bound to the PCSK6 gene. Our work provides evidence for the role of PCSK6 in LPS-induced ALI and identified miR-21-5p as a component of BMSC-derived sEVs that suppressed PCSK6 expression and ameliorated LPS-induced cell damage. These results reveal a novel molecular mechanism for ALI pathogenesis and highlight the therapeutic potential of using sEVs released by stem cells to deliver miR-21-5p for ALI treatment.


Asunto(s)
Lesión Pulmonar Aguda , Vesículas Extracelulares , Células Madre Mesenquimatosas , MicroARNs , Humanos , Lipopolisacáridos/farmacología , Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/terapia , Lesión Pulmonar Aguda/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Serina Endopeptidasas/efectos adversos , Serina Endopeptidasas/metabolismo , Proproteína Convertasas/metabolismo
20.
Nutrients ; 15(20)2023 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-37892538

RESUMEN

Platelet activation and proprotein convertase subtilisin kexin 9 (PCSK9) play pivotal roles in the progression of atherosclerosis to cardiovascular events. It has been reported that hyperlipidemia, a well-documented risk factors for cardiovascular diseases, tends increase platelet activation and PCSK9 expression. However, little is known about this specific mechanism, particularly how nutrition affects platelet activation and PCSK9 levels in hyperlipidemia conditions. This study aimed to assess how a high-fat diet influences platelet activation, its association with PCSK9, and the effects on blood pressure in an animal model. Here, male Wistar rats were divided into four groups, subjected to different high-fat diets for ten weeks with varying nutrient components. The results showed that high-fat diet-induced hypercholesterolemia and hypertriglyceridemia significantly increased the plasma levels of ß-thromboglobulin (ß-TG), p-selectin, and platelet factor 4 (PF-4). The blood pressure readings were also elevated post high-fat diet induction. Interestingly, the group with the highest percentage of saturated fatty acid and trans-fat exhibited the highest PCSK9 levels, along with the highest increase in plasma cholesterol, triglycerides, and platelet activation parameters. These findings confirm that high-fat diet-induced hypercholesterolemia and hypertriglyceridemia stimulate platelet activity and PCSK9 levels. Moreover, our results suggest that PCSK9, implicated in hypercholesterolemia and hypertriglyceridemia, may synergistically mediate platelet hyperactivity, aligning with clinical studies. Notably, our results highlight the association between a high-fat diet and PCSK9, providing insights for drug discovery targeting platelet activation in atherosclerosis-induced cardiovascular diseases.


Asunto(s)
Aterosclerosis , Enfermedades Cardiovasculares , Hipercolesterolemia , Hiperlipidemias , Hipertrigliceridemia , Animales , Ratas , Masculino , Proproteína Convertasa 9 , Hipercolesterolemia/etiología , Dieta Alta en Grasa/efectos adversos , Subtilisina , Ratas Wistar , Proproteína Convertasas/metabolismo , Aterosclerosis/etiología , Activación Plaquetaria
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