oa Molecular Characterization, Activity Assay Of Lipin2 Protein And Its Role In Psoriasis

Background: Psoriasis is a common and recurrent proliferative inflammatory skin disease that causes areas of thickened, inflamed, red skin, often with silvery scales. It poses a considerable worldwide health problem due to its high prevalence, associated morbidity and high health-care costs. It is a multifactorial "complex" disorder, with compelling evidence for a genetic predisposition. Many observations implicate LPIN2 in the genetic etiology of psoriasis, including its position in a minor psoriasis locus; and Majeed syndrome (a Mendelian disorder of bone and skin inflammation) which is caused by homozygous mutations in LPIN2 is often associated with psoriasis in affected individuals and in carriers. We identified several non-synonymous SNPs within LPIN2 in patients with psoriasis that are not present in healthy controls. Objectives: We hypothesize that the identified LPIN2 variations play a role in the genetic etiology of psoriasis and that LPIN2 is the psoriasis susceptibility locus on 18p. We aim to examine this hypothesis through studying the molecular characterization and stability of wild type and variant proteins of LIPIN2, as well as examining the effect of mutation in the phosphatase function of these proteins by colorimetric assay using phosphate containing substrate. Methods: We have obtained custom synthesized cDNA clones encoding the full Lipin2 wild type protein and the six identified mutant proteins (p.K387E, p.S734L, p.A331S, p.L504F, p.P348L, p.E601K). The cDNA clones were sub-cloned and expressed in two expression hosts E. coli and yeast (S. cerevisiae). The recombinant proteins were purified by Ni-Affinity Chromatography, analyzed by SDS Gel Electrophoresis and Western Blot analysis. The effect of mutation in protein stability was studied using Circular Dichroism (CD) spectroscopy, chemically by monitoring spectral changes with unfolding induced by denaturant and thermally by studying CD spectra at different temperatures. In addition the effect of mutation on phosphatase activity of the recombinant proteins was studied colorimetrically by monitoring the hydrolysis of inorganic phosphate from organic phosphate source. Results: DNA analysis, SDS-PAGE and Western Blot analyses indicate that the Wild type and the six mutants are successfully sub-cloned and expressed in the expression hosts. Large scale expressions for all clones are carried out. The pure proteins were studied for their protein stability by CD spectroscopy and showed that there are no significant differences in stability. The activity assay showed that one of the mutants (p.P348L) has higher phosphatase activity which a proximately two times more than the wild type. Conclusion: We have successfully expressed the human Lipin2 protein and its different forms in E. coli and yeast cells. We optimized the conditions to produce substantial amounts of the proteins to be studied by CD spectroscopy to determine the folding patterns, protein stability as well as to study their phosphatase activity by colorimetric method. Other methods will be approached to study the subcellular localization of the proteins and x-ray crystallography.