ANGPTL3 (243-460) Human

What is ANGPTL3 (243-460) Human and why is it significant for lipid metabolism research?

ANGPTL3 (243-460) Human is a recombinant polypeptide fragment derived from the human angiopoietin-like protein 3, specifically spanning amino acids 243 to 460. This fragment represents the C-terminal fibrinogen-like domain of the full-length ANGPTL3 protein, which is critical for modulating lipoprotein metabolism. The significance of this specific fragment lies in its role in studying the protein's interactions with lipid enzymes and receptors, particularly in the context of triglyceride and HDL cholesterol regulation. While the full-length ANGPTL3 inhibits lipoprotein lipase (LPL), a key enzyme in triglyceride breakdown, research with the C-terminal fragment allows investigators to dissect domain-specific functions and interactions with potential therapeutic targets .

How does the structure of ANGPTL3 (243-460) differ from full-length ANGPTL3 in terms of functional properties?

The ANGPTL3 (243-460) fragment represents only the C-terminal fibrinogen-like domain of the full protein, which creates distinct functional differences from the complete protein. While full-length ANGPTL3 strongly inhibits LPL activity, the C-terminal fragment (243-460) retains partial binding capacity to LPL but demonstrates a role distinct from the N-terminal domain. Methodologically, when investigating lipid metabolism pathways, researchers must account for these structural differences - the C-terminal fragment is primarily used to study specific protein-protein interactions and binding activities, whereas the full-length protein provides insights into the complete physiological effects of ANGPTL3 . Studies indicate that while the N-terminal region of ANGPTL3 contains the primary LPL inhibitory activity, the C-terminal domain (243-460) contributes to other functions including HDL regulation through endothelial lipase (EL) inhibition.

What are the optimal experimental conditions for studying ANGPTL3 (243-460) interactions with lipid enzymes?

When designing experimental protocols to study ANGPTL3 (243-460) interactions with lipid enzymes, researchers should consider several methodological parameters. Based on established protocols, optimal conditions typically include physiological pH (7.4) and temperature (37°C) . For in vitro binding assays, researchers commonly use recombinant ANGPTL3 (243-460) at concentrations ranging from 1-10 μg/ml in buffer systems containing PBS with 2% BSA . When assessing interactions with lipoprotein lipase, it's crucial to use freshly prepared enzyme preparations to avoid activity loss. For cellular studies, hepatic cell lines (HepG2, Huh-7) provide appropriate models for investigating ANGPTL3's effects on LPL regulation and lipid gene expression. Experimental designs should include appropriate controls, such as using the N-terminal domain for comparison or heat-inactivated ANGPTL3 fragments to distinguish specific from non-specific interactions .

How can ANGPTL3 (243-460) be effectively used as a tool in drug development research?

ANGPTL3 (243-460) serves as a valuable tool in drug development research through multiple methodological approaches. When screening potential ANGPTL3 inhibitors, researchers can implement binding assays using this fragment to identify compounds that specifically target the C-terminal domain. In practice, this involves using labeled ANGPTL3 (243-460) in competitive binding assays to evaluate candidate molecules' affinity and specificity. For validating monoclonal antibodies like evinacumab that target ANGPTL3, the fragment can be used in epitope mapping studies to confirm binding sites . When testing antisense oligonucleotides (e.g., vupanorsen), researchers can measure the impact on ANGPTL3 (243-460) production in cell culture systems following treatment . Additionally, for CRISPR-based approaches targeting ANGPTL3, the fragment serves as a validation tool to quantify editing efficiency by measuring changes in protein expression levels in cellular models. These methodological applications provide critical insights for developing therapeutics targeting ANGPTL3-mediated lipid dysregulation.

How does ANGPTL3 (243-460) specifically affect HDL functionality compared to other lipoproteins?

ANGPTL3 (243-460) influences HDL functionality through specific molecular interactions that differ from its effects on other lipoproteins. Methodologically, researchers assess this relationship by measuring cholesterol uptake capacity (CUC), which serves as an indicator of HDL functionality . Studies have demonstrated that ANGPTL3 modulates HDL cholesterol by inhibiting endothelial lipase (EL), with the 243-460 fragment retaining this regulatory capacity . In experimental systems, ANGPTL3 (243-460) has been shown to affect reverse cholesterol transport, a critical HDL function, by altering cellular cholesterol efflux pathways . Research protocols typically involve measuring CUC using modified methods where anti-apoA1 antibodies are used to capture HDL particles, followed by assessment of BODIPY-cholesterol uptake, as demonstrated in the MASHAD cohort study . This methodology allows researchers to quantitatively evaluate how ANGPTL3 variants and fragments specifically impact HDL functionality independent of their effects on triglyceride-rich lipoproteins like VLDL .

What are the mechanisms by which ANGPTL3 governs LDL-cholesterol levels, and how can these be studied using the (243-460) fragment?

ANGPTL3 governs LDL-cholesterol levels through multiple mechanisms that can be investigated using the (243-460) fragment. Research indicates that ANGPTL3 inhibition lowers LDL-C primarily by limiting LDL particle production rather than enhancing clearance . Methodologically, investigators can study this relationship by using the ANGPTL3 (243-460) fragment in combination with antisense oligonucleotides (ASOs) targeting specific pathway components . In experimental designs, researchers commonly employ mouse models (including Angptl3−/−, Ldlr−/−, and Lipg−/− variants) to delineate the molecular pathways, with endothelial lipase (EL) identified as a key mediator of ANGPTL3's effects on LDL-C . To assess these mechanisms in vitro, protocols typically involve measuring changes in LDL receptor expression, LDL uptake rates, and VLDL-to-LDL conversion in hepatic cell models following exposure to ANGPTL3 (243-460) . Quantification of these effects requires sophisticated lipidomic analyses to track changes in lipoprotein composition and concentration .

How do ANGPTL3 polymorphisms correlate with cholesterol uptake capacity (CUC) and cardiovascular disease risk?

ANGPTL3 polymorphisms demonstrate significant correlations with cholesterol uptake capacity and cardiovascular disease risk through specific genetic variations. Research methodologies to investigate these relationships involve genotyping ANGPTL3 variants (including rs10789117, rs1748195, and rs11207997) using amplification refractory mutation system PCR with Sanger sequencing confirmation . In the MASHAD cohort study, which included 350 healthy subjects and 153 individuals who developed CVD during follow-up, a statistically significant association was found between the rs1748195 variant and CUC value (p = 0.006), while rs10899117 and rs11207997 showed no significant relationship with HDL concentration or CUC (p > 0.05) . When analyzing these correlations, researchers typically employ logistic regression models to examine relationships between variants and CUC, considering p values <0.05 as statistically significant and expressing correlations as odds ratios with 95% confidence intervals . This methodological approach allows investigators to establish how specific ANGPTL3 genetic variations might influence HDL functionality and consequently affect cardiovascular disease risk profiles.

What is the prevalence of ANGPTL3 loss-of-function mutations in different populations, and how do these impact lipid profiles?

The prevalence of ANGPTL3 loss-of-function (LOF) mutations varies across populations, with significant impacts on lipid profiles. Comprehensive sequencing studies have demonstrated that approximately 1 in 309 individuals is a heterozygous carrier for an ANGPTL3 LOF mutation . Methodologically, researchers assess these mutations using genome or exome sequencing followed by functional validation to confirm the loss of protein activity . Statistical analyses of these genetic associations typically employ linear regression models adjusted for covariates including age, sex, study cohort, CAD status, and ancestry principal components . In a study of 20,092 individuals from the Myocardial Infarction Genetics Consortium, ANGPTL3 LOF mutations were associated with significant reductions in total cholesterol (−10.9%, p=0.0008), LDL cholesterol (−11.8%, p=0.04), and triglycerides (−17.3%, p=0.01), with a smaller, non-significant effect on HDL cholesterol (−5.2%, p=0.17) . For accurate assessment, lipid measurements in participants on lipid-lowering therapy were adjusted by dividing measured total cholesterol and LDL cholesterol by 0.8 and 0.7, respectively . These methodological approaches provide robust evidence for the protective lipid effects of ANGPTL3 deficiency.

How can researchers use ANGPTL3 (243-460) to investigate the relationship between ANGPTL3 and atherosclerotic plaque formation?

Researchers can utilize ANGPTL3 (243-460) as a tool to investigate the relationship between ANGPTL3 and atherosclerotic plaque formation through several methodological approaches. In experimental designs, the fragment can be employed to study the protein's direct effects on arterial wall components, including endothelial cells, macrophages, and smooth muscle cells . One established approach involves treating these cell types with ANGPTL3 (243-460) in vitro and measuring inflammatory marker expression, lipid uptake, and foam cell formation . For in vivo studies, researchers can administer the fragment to mouse models of atherosclerosis (e.g., Apoe−/− or Ldlr−/− mice) and quantify plaque burden using techniques such as calcium scoring . Studies have demonstrated that individuals with complete ANGPTL3 deficiency show significantly lower total plaque burden (mean = 0%) compared to controls (mean = 39%) . When conducting these investigations, it's methodologically important to include appropriate controls and use imaging technologies like computed tomography angiography to assess both calcified and non-calcified plaque components .

What are the current methodologies for measuring circulating ANGPTL3 levels and correlating them with myocardial infarction risk?

Current methodologies for measuring circulating ANGPTL3 levels and correlating them with myocardial infarction (MI) risk involve several sophisticated approaches. Researchers typically quantify plasma ANGPTL3 concentrations using enzyme-linked immunosorbent assays (ELISAs) with specific antibodies that recognize both the full-length protein and relevant fragments . For robust statistical analysis, study designs often stratify populations into tertiles of ANGPTL3 concentration to assess dose-dependent relationships with MI risk . In a significant study, individuals with the lowest tertile of ANGPTL3 concentration (18-271 ng/ml) demonstrated a substantially reduced risk of MI compared to those in the highest tertile (379-1,375 ng/ml), with an adjusted odds ratio of 0.65 (95% CI: 0.55-0.77, p=2.2×10^-7) in the basic model . Methodologically, it's crucial to adjust for confounding factors such as sex, current smoking, diabetes, and hypertension (Model 1), with further refinement possible by including LDL cholesterol and log-transformed triglycerides as covariates (Model 2) . This comprehensive analytical approach allows researchers to establish independent associations between ANGPTL3 levels and cardiovascular outcomes.

How can researchers effectively design experiments to investigate ANGPTL3 (243-460) interactions with peroxisomal proteins?

When designing experiments to investigate ANGPTL3 (243-460) interactions with peroxisomal proteins such as SmarcAL1, researchers should implement a multi-faceted methodological approach. Effective experimental designs begin with co-immunoprecipitation assays using purified ANGPTL3 (243-460) and peroxisomal protein extracts to identify direct binding partners. For cellular localization studies, confocal microscopy with fluorescently-labeled ANGPTL3 (243-460) and peroxisomal markers provides spatial information about potential interactions. To assess functional consequences, researchers can introduce the fragment to peroxisome-enriched cellular fractions and measure changes in lipid metabolic enzyme activities. Recent studies have linked ANGPTL3 to SmarcAL1, a peroxisomal protein involved in lipid storage, with evidence suggesting that the 243-460 fragment may disrupt SmarcAL1 translocation, influencing triglyceride accumulation. For in vivo validation, experimental protocols can include administering labeled ANGPTL3 (243-460) to mouse models and subsequently isolating peroxisomes to quantify fragment localization and associated metabolic changes using lipidomic analyses.

What techniques can be employed to study the effect of ANGPTL3 (243-460) on hepatic fat accumulation and systemic lipid distribution?

To study the effect of ANGPTL3 (243-460) on hepatic fat accumulation and systemic lipid distribution, researchers can employ several sophisticated techniques. In mouse models, controlled injection protocols administering ANGPTL3 (243-460) fragment at defined doses (typically 1-10 mg/kg) allow researchers to observe subsequent changes in hepatic lipid content and blood triglyceride levels. Methodologically, these studies require comprehensive tissue analysis using techniques such as oil red O staining for visual assessment of lipid droplets, coupled with quantitative biochemical triglyceride extraction and measurement. For mechanistic insights, researchers can implement transcriptomic analysis of hepatic tissue to identify changes in lipid metabolism gene expression patterns following ANGPTL3 (243-460) administration. Lipidomic profiling using liquid chromatography-mass spectrometry provides detailed information about specific lipid species alterations in both liver and circulation. Additionally, stable isotope labeling approaches using 13C-labeled fatty acids or glycerol can trace lipid trafficking between tissues after ANGPTL3 (243-460) treatment, offering dynamic insights into how this fragment influences systemic lipid redistribution between circulation and hepatic storage compartments.

How do current ANGPTL3-targeting therapeutics interact with the (243-460) domain, and what methodologies are used to assess this interaction?

Current ANGPTL3-targeting therapeutics interact with the (243-460) domain in specific ways that can be assessed through various methodological approaches. Monoclonal antibodies like evinacumab (formerly REGN1500) bind to ANGPTL3 with high specificity and are developed using technologies such as VelocImmune platforms . To evaluate these interactions, researchers employ surface plasmon resonance (SPR) to determine binding kinetics and affinity constants between the therapeutic agent and ANGPTL3 (243-460) . For antisense oligonucleotides (ASOs) targeting ANGPTL3, methodological approaches include transfection of these molecules into hepatic cell lines followed by quantification of ANGPTL3 expression and secretion . Functionally, researchers assess the impact of these therapeutics on lipoprotein lipase and endothelial lipase activities in vitro using enzymatic assays with purified lipases in the presence of ANGPTL3 (243-460) with or without the inhibitory agent . In animal models, therapeutic efficacy is evaluated through subcutaneous administration of antibodies (10-25 mg/kg) or ASOs (25 mg/kg twice weekly) followed by comprehensive lipid profiling to quantify changes in triglycerides, total cholesterol, and lipoprotein fractions .

What are the emerging research directions for ANGPTL3 (243-460) in relation to non-alcoholic fatty liver disease and metabolic syndrome?

Emerging research directions for ANGPTL3 (243-460) in relation to non-alcoholic fatty liver disease (NAFLD) and metabolic syndrome are expanding our understanding of this protein fragment's role beyond basic lipid metabolism. Methodologically, researchers are investigating how ANGPTL3 (243-460) influences hepatic fat accumulation through experiments in specialized hepatic cell models and mouse models of NAFLD. These experimental designs typically involve administering ANGPTL3 (243-460) to high-fat diet-fed mice and assessing changes in hepatic steatosis, inflammation markers, and insulin sensitivity . Recent studies have established connections between ANGPTL3, peroxisomal regulation via proteins like SmarcAL1, and triglyceride storage, suggesting novel mechanistic pathways that could be targeted therapeutically. Innovative research protocols are now incorporating multi-omics approaches (transcriptomics, proteomics, and lipidomics) to comprehensively map the signaling networks affected by ANGPTL3 (243-460) in metabolic syndrome conditions . Additionally, genetic studies correlating ANGPTL3 polymorphisms with NAFLD severity provide complementary evidence for the protein's role in hepatic metabolic disorders, pointing to potential personalized therapeutic approaches based on individual genetic profiles .