Lipid Metabolism In The Activity Of Protein Structure And Function,

Partâ… Structural and Functional Properties of Apolipoprotein A-â…¤and Its Deletion MutantsBackground:Hypertriglyceridemia is known to be an independent risk factor for coronaryartery disease (CAD). In addition to non-genetic factors such as overweight, diet,heavy smoking, excessive alcohol consumption, genetic factors are important indetermining serum triglyceride levels, accounting for 21-40ï¼….APOA-â…¤is located proximal to the well-characterized APOA-â… /C-â…¢/A-â…£genecluster on human 11q23. Mice expressing a human APOA-â…¤transgene showed adecrease in plasma triglyceride concentrations to one-third of those in control mice;conversely, knockout mice lacking apoa-â…´had four times as much plasmatriglycerides as controls. The levels of very low-density lipoprotein (VLDL) particleswere increased in the homozygous knockout mice and decreased in the transgenicmice compared with controls. VLDL levels in a heterozygous knockout mouse wereintermediate between homozygous knockout and control mice. In humans, singlenucleotide polymorphisms (SNPs) across the APOA-â…¤locus were found to besignificantly associated with plasma triglyceride levels in several independent studies.These findings indicate that APOA-â…¤is an important determinant of plasmatriglyceride levels, a major risk factor for coronary artery disease.The APOA-â…¤gene consists of 4 exons and encodes a 366-amino acid protein.Far-UV circular dichroism analysis reveals that apoA-â…¤possesses 32％α-helixcontent. Protein structure analyses predicted several amphipathic helical domains andan N-terminal signal peptide, characteristic features of lipid-binding apolipoproteins, in both human and mouse apoA-â…¤. The apoA-â…¤protein is secreted by the liver and istransport primarily on large high-density lipoprotein (HDL).Objectives:In this study, we compared the structural and functional properties of wild-typeapoA-â…¤(wtapoA-â…¤) and its 6 deletion mutants, each occurring on a separate segment.Our aim is to explore how each segment influences the structure and function ofapoA-â…¤, and whether there is a relationship between the segments and functions ofapoA-â…¤. We hope that our findings could shed some lights on the potentialmechanisms of the prominent triglyceride-lowering effects of apoA-â…¤.Methods:Six deletion mutants of apoA-â…¤were designed, according to structure predictionand hydrophobility analysis, and generated by DNA recombition. These mutants,named as A-â…¤(â–³(1-51)), A-â…¤(â–³(51-128)), A-â…¤(â–³(132-188)), A-â…¤(â–³,(192-238)),A-â…¤(â–³(246-299)), A-â…¤(â–³(301-343)), delete corresponding fragments between thetwo number from amino-terminal to carboxyl-terminal in turn. Both of wtapoA-â…¤andthe deletion mutants were expressed with pET30b (+) as the expression vector andBL21 (DE3) as the host bacterial, respectively. After purified by Ni²⁺ affinitychromatography, all of the recombinant proteins were examined about the propertiesof their structures and functions. Circular dichrosim (CD) was employed to determinethe secondary structure and conformation stability of apoA-â…¤; Turbility clearanceassay was used to assess their abilities to bind DMPC liposome; LPL activation assaywas used to observe their capacities to promote TG hydrolysis by LPL.Results:CD Assays:Theα-helix content of wild-type apoA-â…¤(wtapoA-â…¤) in detergent-binding statewas 46.26±5.08ï¼…. Guanidine was used as a chemical denaturant to assess theconformation stability of apoA-â…¤. CD results showed that free energy of denaturation(â–³G_D⁰) of wtapoA-â…¤was 1.94±0.14 kcal/mol apoA-â…¤and concentration ofguanidine at the midpoint of denaturation was 2.02±0.19. Turbility Clearance Assay:The mutations with deletion from 192 to 238 and 301 to 343 showedsubstantially reduced activities of binding lipids, for their binding rate constants (K_1/2)were low enough, compared with wtapoA-â…¤(p＜0.05). Specially, the former mutantfell more obviously. And mutant A-â…¤(â–³(56-227)) showed a similar decrease in lipidaffinity. While on the contrary mutants A-â…¤(â–³(51-128)) and A-â…¤(â–³(132-188))displayed increase in lipid binding rates. And other mutants exhibited the samecapacities of disrupting DMPC liposomes as wtapoA-â…¤(p＞0.05).LPL Activation Assay:All mutants showed reduced activation ability to LPL. The extent of activation ofmutants A-â…¤(â–³(1-51)),A-â…¤(â–³(51-128)),A-â…¤(â–³(246-299)) and A-â…¤(â–³(301-343))were about 2/3 of wtapoA-â…¤. And mutant A-â…¤(â–³(132-188)) reserved approximately1/3 activation ability. Although most mutants retained some activating ability, deletionof fragment between 192 and 238 caused almost complete loss of activity.Conclusion:1. ApoA-â…¤exhibits a highα-helix content of 46ï¼…and a low free energy of stabilityof its alpha-helical segments (â–³G_D⁰) of 1.94 kcal/mol. ApoA-â…¤adopts a looselyfolded conformation in solution, compared to apoA-â… .2. ApoA-â…¤interacts with bilayer vesicles of dimyristoylphosphatidylcoline to formdiscoidal complexes. Each fragment of apoA-â…¤has very different influence onthe lipid association property of this protein. The central and C-terminal region ofpolypeptide chain seems to play important roles in mediating the interactionbetween apoA-â…¤and lipid. And the domain proximal to N-terminus of apoA-â…¤may give rise to low structure plasticity.3. ApoA-â…¤could activate lipoprotein lipase (LPL) effectively. Our data demonstratethat the central region of polypeptide chain is of special importance for the LPLactivation function of apoA-â…¤. And the structure integrality of apoA-â…¤also playsan essential role. Partâ…¡Lipodystrophy and obesity represent extreme and opposite ends of the adiposityspectrum and have typically been attributed to alterations in the expression or functionof distinct sets of genes. Previous sdudies have demonstrated that lipin1 deficiencyimpairs adipocyte differentiation and causes lipodystrophy in the mouse. Using twodifferent tissue-specific lipin transgenic mouse strains, it has been demonstrated thatenhanced lipin1 expression in either adipose tissue or skeletal muscle promotesobesity. Thus, variations in lipin1 levels alone are sufficient to induce extreme statesof adiposity and may represent a mechanism by which adipose tissue and skeletalmuscle modulate fat mass and energy balance.Our primary experimental results show: (A). The mouse lipin1 protein was notsuccessfully produced in E.coli as a whole, probably dueing to the existence of severalrare codons for E.coli. And the N-terminal fragment was overproduced as inclusionbodies in E.coli BL21(DE3). We successfully produced rabbit polyclonal antibodiesagainst this fragment. (B). Fluorescence localiztion analysis revealed that mouselipin1 protein is predominantly located in the nucleus of 3T3-L1 cell. Point mutationanalysis revealed that Cys(30) and Gly(84) in the N-terminal region are essential tolocalization of lipin1.(C).We eatablished a primary relation between lipin1 and thestructure of nuclear envelope and endoplasmic reticulum by RNAi test withfluorescence.(D). The construction of adipocyte differentiation model would offer achance to study the function of lipin1 deeply.In conclusion, we got the complete coding sequence of mouse lipin1 gene andproduced rabbit polyclonal antibodies. We studied the localization of mouse lipin1protein. And we also analyzed the relation between lipin1 and cell structure.