| Popis: |
Psoriasis is a chronic inflammatory skin disease posing 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. On the other hand, Majeed syndrome, a Mendelian disorder of bone and skin inflammation is caused by homozygous mutations in LPIN2. Many observations have implicated LPIN2 in the genetic etiology of psoriasis, including its position in a psoriasis locus. We identified several non-synonymous SNPs within the LPIN2 in patients with psoriasis that are not present in healthy controls. We hypothesize that the variations in LPIN2 play a role in the susceptibility to development of psoriasis and that LPIN2 is the psoriasis susceptibility locus on 18p. We aim to examine this hypothesis by examining the properties of the wild type and mutant proteins, as well as examining any difference in function between the wild type and mutants. 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 subcloned into pYES2 vector for expression in yeast cells (Saccharomyces cerevisiae). Each construct was transformed into Saccharomyces cerevisiae for protein expression. The analysis utilizes SDS Gel Electophoresis and Western Blot. The DNA analysis indicates that each fragment has been correctly cloned into the pYES2 vector. The analyses using SDS Gel Electrophoresis and Western Blot indicate that the Wild type and p.K387E are successfully expressed in S. cerevisiae while p.S734L is expressed in S. cerevisiae but at a lower level. Expression experiments are being done on the 4 remaining mutant proteins. We were successful in artificially expressing the human Lipin2 protein in its different forms in yeast cells. We are currently optimizing the conditions to produce substantial amounts of the proteins to be studied by Circular dichroism to determine the folding patterns. Other methods will be approached to study the function. |
