Identification of a novel LDLR p.Glu179Met variant in Thai families with familial hypercholesterolemia and response to treatment with PCSK9 inhibitor.

Familial hypercholesterolemia (FH) is a genetic disease characterized by elevated LDL-C levels. In this study, two FH probands and 9 family members from two families from northeastern Thailand were tested for LDLR, APOB, and PCSK9 variants by whole-exome sequencing, PCR-HRM, and Sanger sequencing. In silico analysis of LDLR was performed to analyse its structure‒function relationship. A novel variant of LDLR (c.535_536delinsAT, p.Glu179Met) was detected in proband 1 and proband 2 in homozygous and heterozygous forms, respectively. A total of 6 of 9 family members were heterozygous for LDLR p.Glu179Met variant. Compared with proband 2, proband 1 had higher baseline TC and LDL-C levels and a poorer response to lipid-lowering therapy combined with a PCSK9 inhibitor. Multiple sequence alignment showed that LDLR p.Glu179Met was located in a fully conserved region. Homology modelling demonstrated that LDLR p.Glu179Met variant lost one H-bond and a negative charge. In conclusion, a novel LDLR p.Glu179Met variant was identified for the first time in Thai FH patients. This was also the first report of homozygous FH patient in Thailand. Our findings may expand the knowledge of FH-causing variants in Thai population, which is beneficial for cascade screening, genetic counselling, and FH management to prevent coronary artery disease.

The prevalence and treatment patterns of familial hypercholesterolemia among Thai patients with premature coronary artery disease.

OBJECTIVES: Familial hypercholesterolemia (FH) is an autosomal dominant genetic disorder that is characterized by severe hypercholesterolemia. The prevalence of FH in Thailand has not been reported. Therefore, this study aimed to investigate the prevalence of FH and treatment patterns among Thai patients with premature coronary artery disease (pCAD). METHODS: A total of 1,180 pCAD patients at two heart centers from northeastern and southern Thailand between October 2018 and September 2020 were enrolled. FH was diagnosed using the Dutch Lipid Clinic Network (DLCN) criteria. pCAD was diagnosed in men aged < 55 years and women aged < 60 years. RESULTS: The prevalence of definite/probable FH, possible FH, and unlikely FH in pCAD patients was 1.36% (n = 16), 24.83% (n = 293), and 73.81% (n = 871), respectively. Definite/probable FH in pCAD patients had a significantly higher frequency of STEMI but a lower frequency of hypertension than those with unlikely FH. After discharge, most pCAD patients (95.51%) received statin therapy. Definite/probable FH patients had a higher frequency of high-intensity statin therapy than those with possible FH and unlikely FH. After follow-up for 3-6 months, approximately 54.72% of pCAD patients with DLCN scores ≥ 5 had a reduction in LDL-C > 50% from baseline. CONCLUSIONS: The prevalence of definite/probable FH, particularly possible FH, was high among pCAD patients in this study. The early diagnosis of FH among Thai pCAD patients should be performed for the early treatment and prevention of CAD.

Silver-enhanced colloidal gold dip strip immunoassay integrated with smartphone-based colorimetry for sensitive detection of cardiac marker troponin I.

Cardiac troponin I (cTnI) is a specific cardiac biomarker for diagnosis of acute myocardial infarction (AMI). A sensitive and simple point-of-care test (POCT) is still required for early detection of AMI. To address this need, we developed a dip strip assay based on sandwich immunoassay coupled with a silver enhancement system. Pre-incubation and silver enhancement were introduced to the dip strip to increase sensitivity. Due to the catalytic reaction of the silver enhancement solution, the red color of AuNPs changed to dark brown as silver ions precipitated and enlarged the AuNPs. The obtained results were easily seen by the naked eye. For quantitative analysis, the color intensity of the results was analyzed using a smartphone with RGB color picker application. The effects of operating parameters (volume of AuNP-Ab conjugate, volume of sample, incubation time, and analysis time) were investigated and optimized. Under optimal conditions, the limit of detection (LOD) by the naked eye was 0.5 ng/mL. The LOD with silver enhancement was 50-fold lower than without. For quantitative analysis using the smartphone, linearity of detection was observed through the range of 0.5-50 ng/mL (R2 = 0.9952) and the LOD was 0.12 ng/mL. The developed method was successfully applied to detection of cTnI in serum samples, achieving analytical recoveries and %RSD in the ranges of 96.10-119.17% and 2.91-5.13%, respectively. Additionally, this developed assay was not cross reactive with the potentially interfering serum proteins. These results showed the great potential of this dip strip assay as an alternative POCT for detection of serum cTnI.

Extracellular Vesicle-Mediated IL-1 Signaling in Response to Doxorubicin Activates PD-L1 Expression in Osteosarcoma Models.

The expression of programmed cell death ligand 1 (PD-L1) in tumors is associated with tumor cell escape from T-cell cytotoxicity, and is considered a crucial effector in chemoresistance and tumor relapse. Although PD-L1 induction has been observed in patients after chemotherapy treatment, the mechanism by which the drug activates PD-L1 expression remains elusive. Here, we identified the extracellular vesicles (EVs) as a molecular mediator that determines the effect of doxorubicin on PD-L1 expression in osteosarcoma models. Mechanistically, doxorubicin dependently stimulates the release of extracellular vesicles, which mediate autocrine/paracrine signals in osteosarcoma cells. The recipient cells were stimulated by these EVs and acquired the ability to promote the expression of inflammatory cytokines interleukin (IL)-1ß and IL-6. In response to doxorubicin, IL-1ß, but not IL-6, allowed- osteosarcoma cells to promote the expression of PD-L1, and the elimination of IL-1ß/IL-1 receptor signaling with IL-1 receptor antagonist reduced PD-L1 expression. Together, these findings provided insights into the role of EV release in response to chemotherapy that mediates PD-L1 expression via the IL-1 signaling pathway, and suggested that the combination of a drug targeting IL-1 or PD-L1 with chemotherapy could be an effective treatment option for osteosarcoma patients.

Arylesterase activity but not PCSK9 levels is associated with chronic kidney disease in type 2 diabetes.

PURPOSE: Oxidative stress and dyslipidemia have been found to be associated with the progression of chronic kidney disease (CKD) in type 2 diabetes mellitus (T2DM) patients. Paraoxonase 1 (PON-1) activity, and proprotein convertase subtilisin kexin type 9 (PCSK9) levels play an important role regarding anti-oxidants, and lipid metabolism, respectively. The aim of this study was to investigate the association of PON-1 activity, and PCSK9 levels with CKD in T2DM. METHODS: A total of 180 T2DM (87 CKD, and 93 non-CKD) with age-, and gender-matched subjects were recruited in this study. PON-1 activity was measured with two kinds of substrate: paraoxon for paraoxonase (PONase) activity and phenylacetate for arylesterase (AREase) activity. PCSK9 levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: AREase activity was significantly lower in CKD compared with non-CKD (225.53 ± 108.73 vs. 257.45 ± 106.12 kU/L, p = 0.044) in T2DM, whereas there was no significant difference in PONase activity and PCSK9 levels between CKD and non-CKD groups. In addition, multivariate logistic regression analysis showed that the lowest tertile of AREase increased the risk for CKD in T2DM (OR 3.251; 95% CI 1.333-7.926, p = 0.010), whereas PONase activity and PCSK9 levels were not associated with CKD in T2DM. CONCLUSION: Reduced AREase activity can increase the risk for CKD in T2DM patients. AREase activity, but not PONase activity and PCSK9 levels, may be used as the biomarker for predicting the progression of CKD in T2DM.

Structure-Function Relationships of LDL Receptor Missense Mutations Using Homology Modeling.

Mutations in the low-density lipoprotein receptor (LDLR), which cause familial hypercholesterolemia (FH), present a variable clinical FH phenotype. To date, over 1600 FH-causing mutations have been found worldwide. The aim of this study was to investigate the structure-function relationships of LDLR mutations by using homology modeling. Structural analysis of 36 missense mutations of known receptor activity (33 severe, 1 mild, and 2 non-pathogenic phenotypes) using sequence comparison and homology modeling was performed. Severe phenotypes had less than 2% to 32% of residual LDLR activity. Mild phenotypes had 76-92% of residual LDLR activity. Finally, non-pathogenic phenotypes had normal residual LDLR activity. Sequence comparisons showed that most of the severe phenotypes were located within the fully conserved residues of LDLR, while most of the mild and non-pathogenic phenotypes were located within the poorly conserved residues. Homology modeling demonstrated several phenomena for severe phenotypes: disruption of disulfide bond formation, disturbance of the calcium binding sites, and perturbation of LDLR hydrophobic conserved packing. In contrast, mild and non-pathogenic phenotypes did not disturb the critical region of LDLR. In addition, the root mean square deviation (RMSD) values of severe phenotype tended to be higher than the mild and non-pathogenic phenotypes, and the mean of solvent accessible surface area (ASA) of the residues in wild type structure for the severe phenotype was lower than mild and non-pathogenic phenotypes. These findings provide a better understanding in the structure-function relationships of LDLR mutations and may be useful in predicting FH severity based on future genotyping.

The effect of APOE, CETP, and PCSK9 polymorphisms on simvastatin response in Thai hypercholesterolemic patients.

AIM: To investigate the effect of apolipoprotein E (APOE), cholesteryl ester transfer protein (CETP) and proprotein convertase subtilisin kexin type 9 (PCSK9) polymorphisms on the lipid-lowering response to simvastatin therapy in Thai hypercholesterolemic patients. METHOD: Two hundred and twenty-five hypercholesterolemic patients in southern Thailand were enrolled and treated with simvastatin 20 or 40 mg per day for 3 months. Serum lipids were measured before and after the therapy. APOE, CETP TaqIB, and PCSK9 (R46L, I474V, and E670G) polymorphisms were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: After 3 months of simvastatin therapy, subjects with APOE2 (Total cholesterol [TC]: -30.89% vs-13.56%, P < .05, LDL-C: -45.00% vs -17.73%, P < .05) and APOE3 carriers (TC: -26.22% vs -13.56%, P < .05, LDL-C: -37.14% vs -17.73%, P < .05) had greater TC and LDL-C reduction compared to APOE4 carriers, whereas CETP TaqIB B2B2 genotype showed lower TC (-16.37% vs -24.92%, P = .016) and LDL-C (-22.54% vs -35.19%, P = .028) reduction compared to CETP TaqIB B1 carriers. In addition, PCSK9 474IV carriers showed greater LDL-C (-50.57% vs -32.99%) reduction compared to PCSK9 474II carriers. Combined effect analyses showed that individuals carrying more risk alleles tended to have lower TC and LDL-C (P for trend = .000 and .000, respectively) reduction in response to simvastatin therapy. CONCLUSION: APOE4 carriers and the CETP TaqIB B2B2 genotype were associated with a decreased response, but PCSK9 474IV carriers tended to be associated with an increased response to simvastatin therapy in Thai hypercholesterolemic patients.

Studies of the CETP TaqIB and ApoE Polymorphisms in Southern Thai Subjects with the Metabolic Syndrome.

Several genetic factors have been investigated responsible for metabolic syndrome (MetS). The aim of this study was to investigate the association between cholesteryl ester transfer protein (CETP) TaqIB and apolipoprotein E (ApoE) polymorphisms and MetS in 378 subjects from Southern Thailand. Subjects were divided into MetS+ (n = 121) and MetS- (n = 257) groups according to the criteria of National Cholesterol Education Program Adult Treatment Panel III (NCEP ATPIII). The CETP TaqIB and ApoE polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Logistic regression analysis revealed no association of CETP TaqIB and ApoE variants with MetS, after adjustment for age and sex. However, ε4 allele had a significantly increased odds ratio (OR) of reduced high-density lipoprotein-cholesterol (HDL-C) levels when compared with ε3 allele (OR 1.91; 95% CI 1.11-3.29, p = 0.020). This suggests that CETP TaqIB and ApoE polymorphisms may not be considered as genetic risk factors for MetS in a Southern Thai population. However, ε4 allele which is associated with one metabolic component, low HDL-C levels, might predispose the subjects to develop metabolic disturbances.

Combined PCSK9 and APOE polymorphisms are genetic risk factors associated with elevated plasma lipid levels in a Thai population.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) and apolipoprotein E (ApoE) play a key role in the regulation of lipid metabolism. We aimed to investigate the effects of PCSK9 (R46L, I474V, and E670G) and APOE polymorphisms on lipid levels in a Southern Thai population. A total of 495 participants (307 urban, 188 rural) were recruited for the study. Anthropometric and biochemical variables were evaluated. PCSK9 and APOE polymorphisms were analyzed using PCR-RFLP. The 46L urban male carriers had significantly higher diastolic blood pressure (DBP) and fasting blood sugar compared with non-carriers. In contrast, the 46L urban female carriers had significantly lower total cholesterol (TC) and LDL-C levels compared with non-carriers. The 474V rural female carriers had significantly lower HDL-C levels than non-carriers. The 670G urban female carriers showed significantly higher TC and LDL-C levels compared with non-carriers. APOE4 carriers had increased TC and LDL-C levels relative to APOE3 carriers in the urban males. APOE2 carriers had decreased TC and/or LDL-C levels compared with APOE3 carriers in urban males and females. A significant trend of increased TC and LDL-C levels was observed in non-APOE4-PCSK9 670EE carriers to APOE4-PCSK9 670EG carriers in urban subjects. In summary, R46L, I474V, and E670G may be genetic risk factors for cardiovascular disease (CVD) in urban males, rural females, and urban females, respectively. In contrast, R46L had a favorable lipid profiles that may protect against CVD in urban females. The combination of PCSK9 E670G and APOE polymorphisms may represent an independent factor for the determination of lipid levels.

Two-dye based arrayed primer extension for simultaneous multigene detection in lipid metabolism.

BACKGROUND: Cardiovascular disease (CVD) is one of the major causes of death worldwide. Numerous genetic risk factors in lipid metabolism, including mutations of LDLR, APOB, and PCSK9, as well as polymorphisms of CETP and APOE, have been found to associate with CVD. METHODS: In this study, a two-dye based arrayed primer extension (APEX) microarray assay for simultaneous multigene (LDLR, APOB, PCSK9, CETP, and APOE) detection was developed. The DNA templates, originating from 1 DNA sample of known genotype and 7 blind DNA samples, were amplified by uniplex PCR. RESULTS: Optimized conditions for the APEX reaction were determined to include a hybridization temperature of 55°C and a DNA template size of 50-150bp. The total assay including PCR, purification, fragmentation, APEX reaction, and image analysis could be performed in 6h. In total, 48 genotypes were identified among 8 individual DNA samples by APEX analysis. CONCLUSIONS: The data suggest that this APEX microarray offers a robust, fast, and versatile option for screening these genotypes in hypercholesterolemia patients.

Lead inhibits paraoxonase 2 but not paraoxonase 1 activity in human hepatoma HepG2 cells.

Lead is an environmental toxicant of great concern for humans and animals. Lead-induced liver damage and malfunction are partly due to a disturbance of the cellular antioxidant balance. Paraoxonase 1 (PON1) and PON2 are highly expressed in the liver and have been proposed as antioxidative enzymes. In this study, the effects of lead on PON1 and PON2 activities were investigated in human hepatoma HepG2 cells by exposing the cells to various concentrations of lead acetate for 24, 48, or 72 h. The results show that a significant increase in reactive oxygen species was observed even at the lowest concentration of lead treatment. However, only the highest concentration of lead significantly influenced cell viability. Lead had no influence on cell-associated PON1 activity, but it significantly decreased cytoplasmic PON2 activity in a concentration- and time-dependent manner. This reduction was rescued by the addition of calcium. A significant increase of PON2 transcript was observed by real-time polymerase chain reaction, while PON2 protein expression did not change in the western blot analysis. Taken together, these results indicate that lead reduces PON2, but not PON1, activity and that this reduction is reversed by calcium. Lead-induced oxidative stress and decreased PON2 activity lead to the upregulation of PON2 transcript.

Decreased paraoxonase 1 activity and increased oxidative stress in low lead-exposed workers.

Paraoxonase 1 (PON1) has been proposed as an antioxidant enzyme. Although lead-inhibited PON1 activity has been demonstrated mostly based on in vitro experiments, it is uncertain whether this phenomenon is relevant in pathogenesis of lead-induced oxidative stress in the lead exposure. We examined associations of blood lead levels (BLL) and PON1 activity along with oxidative stress parameters in lead exposure workers. We determined malondialdehyde (MDA), conjugated diene (CD), total peroxides (TP), total antioxidant status (TAS), the oxidative stress index (OSI), and PON1 activity in earthenware factory workers (n = 60) and control subjects (n = 65). The lead-exposed group significantly increased lipid peroxidation parameters and OSI compared to the control group (p < 0.001). The lead-exposed group had significantly decreased PON1 activity and TAS levels compared to the control group (p < 0.001). Multiple linear regression analysis revealed that BLL were significantly correlated with decreased TAS (r = -0.496) and PON1 activity (r = -0.434), but with increased CD (r = 0.694), TP (r = 0.614), MDA (r = 0.788), and OSI (r = 0.722). Interestingly, BLL at 10 µg/dL significantly decreased PON1 activity and increased oxidative stress parameters with insignificant changes in other biochemical and hematological parameters. Altogether, the reduction of PON1 activity may associate in an imbalance in pro-oxidants and antioxidants, leading to oxidative damage in lead-exposed workers even at low BLL.

Effects of Curcuma comosa on the expression of atherosclerosis-related cytokine genes in rabbits fed a high-cholesterol diet.

AIM OF THE STUDY: Curcuma comosa has been known to have potential use in cardiovascular diseases, but its immunoregulatory role in atherosclerosis development and liver toxicity has not been well studied. We therefore investigated the effects of Curcuma comosa on the expression of atherosclerosis-related cytokine genes in rabbits fed a high-cholesterol diet. MATERIALS AND METHODS: Twelve male New Zealand White rabbits were treated with 1.0% cholesterol for one month and were subsequently treated with 0.5% cholesterol either alone, or in combination with 5mg/kg/day of simvastatin or with 400mg/kg/day of Curcuma comosa powder for three months. The expression of IL-1, MCP-1, TNF-α, IL-10, and TGF-ß in the isolated abdominal aorta and liver were determined by real-time RT-PCR. Liver toxicity was determined by hepatic enzyme activity. RESULTS: Curcuma comosa significantly decreased the expression of pro-inflammatory cytokines, leading to a stronger reduction in IL-1, MCP-1, and TNF-α expression compared to that was suppressed by simvastatin treatment. However, neither Curcuma comosa nor simvastatin affected the expression of anti-inflammation cytokines. In the liver, Curcuma comosa insignificantly decreased the expression of pro-inflammatory cytokines and significantly increased the expression of the anti-inflammatory cytokine IL-10 without altering the activity of hepatic enzymes. In contrast, simvastatin significantly increased the MCP-1 and TNF-α expressions and serum ALT level, without affecting the expression of anti-inflammatory cytokines. CONCLUSIONS: In this study, we demonstrated that Curcuma comosa exerts anti-inflammatory activity in the aorta and liver without causing liver toxicity, indicating that Curcuma comosa is a potential candidate as an alternative agent in cardiovascular disease therapy.

Two novel D151Y and M391T LDLR mutations causing LDLR transport defects in Thai patients with familial hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in the low density lipoprotein receptor (LDLR) gene. Two novel LDLR mutations, D151Y and M391T, had been previously identified in unrelated Thai patients with heterozygous FH. To confirm that these mutations cause FH, the functional characteristics of D151Y and M391T, which are located in the fourth cysteine repeat of the ligand-binding domain and in the sixth YWTD repeat of the epidermal growth factor precursor homology domain, respectively, were studied. METHODS: CHO-ldlA7 cells were transfected with wild type and mutant LDLR cDNAs. Thereafter, the localization, expression, and ability of LDL uptake of LDLR were evaluated by confocal laser scanning microscope (CLSM), and flow cytometry. RESULTS: CLSM revealed both D151Y and M391T LDLR were partially retained in the endoplasmic reticulum, with the remaining residual activity observed by LDL uptake. Similarly, flow cytometric analysis showed a significant reduction of LDLR expression to 18% and 38% and of LDL uptake to 15% and 71% in D151Y and M391T LDLR, respectively. CONCLUSIONS: The transport defect of LDLR contributes to the pathology of FH. These data are useful for an insight inspires the development of novel lipid-lowering drugs with beneficial therapeutic value.

Human paraoxonase 2.

Human paraoxonase 2 (PON2), which is a member of the paraoxonase family, possesses unique properties that distinguish it from PON1 and PON3. PON2 is ubiquitously expressed in many different tissue types and is highly expressed in the vital organs, such as the heart and lungs. Early research revealed that PON2 is exclusively intracellularly found, wherein it functions as an anti-oxidative protein by reducing intracellular and local oxidative stress. Studies in the last five years have demonstrated that PON2 protects against atherosclerosis by preventing low-density lipoprotein (LDL) oxidation, reversing the oxidation of mildly oxidised LDL, inhibiting monocyte chemotaxis, and increasing cholesterol efflux. Recently, emerging evidence has proposed that PON2 is an anti-atherosclerotic and may be associated with cardiovascular disease (CVD). The number of investigations concerning the relationship between two common PON2 polymorphisms and CVD among different ethnic groups and regions is rapidly growing. Here, we briefly review the developments in PON2 research by focusing on past and recent findings.

Effect of atorvastatin on paraoxonase1 (PON1) and oxidative status.

It has been proposed that paraoxonase1 (PON1), a high density lipoprotein (HDL)-associated esterase/lactonase, has anti-atherosclerotic properties. The activity of PON1 is influenced by PON1 polymorphisms. However, the influence of PON1 polymorphisms on PON1 activity and oxidative stress in response to lipid-lowering drugs remains poorly understood. The objective of the present study was to investigate the effects of atorvastatin on PON1 activity and oxidative status. The influence of PON1 polymorphisms on PON1 activity and oxidative status in response to atorvastatin treatment was also evaluated. In total, 22 hypercholesterolemic patients were treated with atorvastatin at a dose of 10 mg/day for 3 months. Lipid profile, lipid oxidation markers (malondialdehyde (MDA), conjugated diene (CD), total peroxides (TP)), total antioxidant substance (TAS), oxidative stress index (OSI), and paraoxonase1 activity were determined before and after treatment. L55M, Q192R, and T(-107)C PON1 polymorphisms were also determined. Atorvastatin treatment significantly reduced the levels of total cholesterol (24.5%), low density lipoprotein (LDL) cholesterol (25.4%), triglycerides (24.4%), CD (4.4%), MDA (15.2%), TP (13.0%) and OSI (24.0%), and significantly increased the levels of TAS (27.3%), and PON1 activity (14.0%). Interestingly, the increase in PON1 activity and the reduction in oxidative stress in response to atorvastatin were influenced only by the PON1 T-107C polymorphism. Atorvastatin treatment improved the lipid profile, lipid oxidation, and oxidative/antioxidative status markers including the activity of PON1 towards paraoxon. These beneficial effects may be attributed to the antioxidant properties of statins and the increase in PON1 activity. The increase in PON1 activity was enhanced by the PON1 T-107C polymorphism.

Paraoxonasel phenotype distribution in Thais.

OBJECTIVE: To determine the PON1 activity and phenotype distribution in a Thai population. STUDY DESIGN: Prospective Descriptive study. MATERIAL AND METHOD: Between October 2001 and April 2002, 160 healthy Thai individuals aged 20-74 years were assessed for PON1 activity and the phenotype distribution by using dual substrate method. RESULTS: The means +/- SD of basal, salt-stimulated paraoxonase and arylesterase activities were 239.7 +/- 83.9 nmol/min/mL 555.2 +/- 222.2 nmol/min/mL and 147.6 +/- 33.8 micromol/min/mL respectively. The authors observed a wide interindividual variability up to 6.9-fold for paraoxonase activity and 4.6-fold for arylesterase activity. The authors found a range of ssPON/ARE ratio from 1.04 to 7.05 and three distinctive phenotype modals of AA (1.04-2.25), AB (2.44-4.29), and BB (4.53-7.05) with frequencies of 14.4% (AA), 51.9% (AB), and 33.7% (BB). The authors also observed the association of sex with lipid parameters and PON1 activity. CONCLUSION: The distribution of PON1 phenotype in Thais is clearly trimodal with high frequency in BB phenotype.

Molecular modeling of D151Y and M391T mutations in the LDL receptor.

The low-density lipoprotein receptor (LDLR) is a key regulator of cholesterol homeostasis, and defects in the function of LDLR result in familial hypercholesterolemia (FH). In the present study, we performed structural analyses of two novel LDLR mutations, D151Y and M391T. Both mutations occurred in conserved residues of LDLR. The D151Y mutation, in the ligand binding domain, caused an elimination of a hydrogen bond in the calcium binding site, higher solvent accessibility and a loss of negative charge in the Y151 residue. On the other hand, the M391T mutation, in the beta-propeller of the epidermal growth factor (EGF) precursor homology domain, caused an additional hydrogen bond to form, higher solvent accessibility and a distortion of the beta-strand. These data suggest that the irregular structures of the mutated LDLRs are likely to cause the functional defect that contributes to the pathology of FH.

Different roles of Sp family members in HIV-1 Tat-mediated manganese superoxide dismutase suppression in hepatocellular carcinoma cells.

The expression of manganese superoxide dismutase (MnSOD) is regulated by agents associated with cancer development. It has been shown that infection with the human immunodeficiency virus type 1 (HIV-1) is associated with the development of liver cancer and that the transactivating transcriptional factor (Tat) of human HIV-1 reduces the expression of MnSOD in several cell types. However, the role of Tat in the expression of MnSOD in hepatocellular carcinoma is unknown. Furthermore, the precise mechanisms whereby Tat suppresses MnSOD expression in hepatocellular carcinoma cells remain unclear. In this report, we build on our original observations that Tat changes the distribution of Sp family members on the MnSOD promoter, which accounts for Tat-dependent changes in basal expression. In hepatic cells, Tat expression upregulates Sp1/Sp3, which play different roles in regulating MnSOD transcription. While overexpression of Sp1 stimulates, overexpression of Sp3 represses transcriptional activity. The transcription repression effect of Sp3 is not due to Sp3 competing for the binding site with Sp1 because only the full-length Sp3 but not the truncated Sp3 suppresses MnSOD promoter activity. These findings suggest a novel mechanism by which Tat modulates the repression of the MnSOD gene and establish a link between HIV infection and liver cancer.

HIV-1 Tat regulates the SOD2 basal promoter by altering Sp1/Sp3 binding activity.

Regulation of the basal manganese superoxide dismutase (SOD2) promoter depends on the transcriptional activity of the Sp family of transcription factors. Here we report that reduced expression in the presence of Tat is independent of induction with Tumor necrosis factor alpha and that Tat affects the interaction of Sp1 and Sp3 with the basal promoter. Footprinting and electrophoretic mobility shift assay (EMSA) analyses with extracts from HeLa cells showed that Sp1/Sp3 complexes populate the proximal SOD2 promoter, and that Tat leads to an increase in the binding activity of Sp3. In Drosophila S2 cells, both Sp1 and Sp3 activated the basal SOD2 promoter (88.1 +/- 39.4 fold vs. 10.3 +/- 3.5 fold, respectively), demonstrating a positive, yet lower transcriptional regulatory function for Sp3. Additionally, the inability of Sp3 to synergistically affect promoter activity indicates an efficient competition of Sp3 with Sp1 for the multiple Sp binding sites in the SOD2 basal promoter. Tat potentiated both Sp1 and Sp3 activation of the promoter in S2 cells, though the activity of Sp3 was still lower than that of Sp1. Thus, the consequence of a shift by Tat to increased Sp3-containing complexes on the basal SOD2 promoter is decreased SOD2 expression. Together, our studies demonstrate the functional importance of the interaction of Sp1, Sp3, and Tat, revealing a possible mechanism for the attenuation of basal manganese superoxide dismutase expression.