Publication: Matriks metalloproteinaz enzimlerine özgü tanımlayıcı ve enzim aktivitesini durdurucu peptit yapılarının geliştirilmesi
Abstract
MATRİKS METALLOPROTEİNAZ ENZİMLERİNE ÖZGÜ TANIMLAYICI VE ENZİM AKTİVİTESİNİ DURDURUCU PEPTİT YAPILARININ GELİŞTİRİLMESİ Bu çalışmada, matriks metalloproteinaz-2 (MMP-2) ve matriks metalloproteinaz-9 (MMP-9) enzimlerine özgü, tanımlayıcı ya da aktivitesini bloke edici peptit yapılarının, Faj Gösterim Teknolojisi kullanılarak tanımlanması ve enzim aktivitesi üzerine etkilerinin incelenmesi amaçlandı. MMP-2’ye ve MMP-9’a bağlanan peptitler, 12 amino asitlik (a.a.) peptit kütüphanesinden, sırası ile ikili ve üçlü biyopanning döngüsü ile seçildi. Seçilen 30’ar adet faj peptit klonunun MMP-2’ye ve MMP-9’a olan özgünlükleri faj ELISA (Enzime Bağlı İmmün Testi) yöntemi ile test edildi. MMP-2’ye bağlanan 19 adet ve MMP-9’a bağlanan 13 adet fajın yüzeyinde sunulan peptit yapılara ait nükleotit dizisi, dizi analizi yöntemi ile tanımlandı. Peptitlerin MMP-2 enzim aktivitesine olan etkisinin jelatinaz zimogram yöntemi ile test edilebilmesi için, faj yüzeyinde sunulan peptit yapıları ile ve bu yapıların nükleotit dizilerine göre sentezlenen, sentetik peptitler kullanılarak zimogram analizleri yapıldı. Sentetik peptitler ile yapılan zimogram analizlerinde MMP-2 ve MMP-9’a karşı seçilen 8 adet peptidin MMP-2 enziminin aktivitesine olan etkisi incelendi. Analizler sonucunda, MMP2-28 peptidinin MMP-2 aktivitesini 1000 μM konsantrasyonda % 55 oranında inhibe ettiği tespit edildi. MMP-2 aktivitesini etkilemediği belirlenen MMP9-27 ve MMP9-3 numaralı peptitlerin MMP-9’a özgü bloklayıcı olmaya aday peptitler olduğu belirlendi. Tez kapsamında ilk kez MMP-2 ve MMP-9 aktivitesine özgün olarak bloklayabilecek 12 a.a.’lik peptit yapıları elde edildi. Belirlenen peptitler jelatinazların rol oynadığı hastalıkların tanı veya tedavisi için kullanılabilecek aday peptitlerdir.
IDENTIFICATION OF MATRIX METALLOPROTEINASE ENZYME-SPECIFIC DESCRIPTIVE OR ACTIVITY BLOCKING PEPTIDE STRUCTURES In this study, it was aimed to identify matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) enzymes-specific descriptive or activity blocking peptide structures by using “Phage Display Technology” and to study the effects on enzyme activity. Peptides which bind to MMP-2 and MMP-9 were selected from 12 amino acid (a.a.) peptide library with two and three rounds of biopanning, respectively. Specificity of 30 selected phage clones to MMP-2 and MMP-9 was tested by phage ELISA (Enzyme Linked Immunosorbent Assay). Sequences of 19 MMP-2 binding and 13 MMP-9 binding phage-displayed peptides were deduced by DNA sequencing. To test the effect of peptides on MMP-2 enzyme activity using gelatinase zymography method, zymograph assays were performed with phage-diplayed peptides and with synthetic peptides which were synthesized based on deduced sequences of phage-displayed peptides. In the zymograph assays performed with synthetic peptides, the effects of eight peptides, which were selected against MMP-2 and MMP-9, on MMP-2 enzyme activity, were investigated. It has been detected that MMP2-28 peptide at concentrations of 1000 µM inhibited 55 % of MMP-2 activity. MMP9-27 and MMP9-3 peptides that have no effect on MMP-2 activity, are candidate MMP-9 specific blocking peptides. In the context of the thesis, MMP-2 and MMP-9 activity blocking 12 a.a. peptide structures have been obtained for the first time. Identified peptides are candidates for the diagnosis or treatment of diseases which gelatinases take role in.
IDENTIFICATION OF MATRIX METALLOPROTEINASE ENZYME-SPECIFIC DESCRIPTIVE OR ACTIVITY BLOCKING PEPTIDE STRUCTURES In this study, it was aimed to identify matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) enzymes-specific descriptive or activity blocking peptide structures by using “Phage Display Technology” and to study the effects on enzyme activity. Peptides which bind to MMP-2 and MMP-9 were selected from 12 amino acid (a.a.) peptide library with two and three rounds of biopanning, respectively. Specificity of 30 selected phage clones to MMP-2 and MMP-9 was tested by phage ELISA (Enzyme Linked Immunosorbent Assay). Sequences of 19 MMP-2 binding and 13 MMP-9 binding phage-displayed peptides were deduced by DNA sequencing. To test the effect of peptides on MMP-2 enzyme activity using gelatinase zymography method, zymograph assays were performed with phage-diplayed peptides and with synthetic peptides which were synthesized based on deduced sequences of phage-displayed peptides. In the zymograph assays performed with synthetic peptides, the effects of eight peptides, which were selected against MMP-2 and MMP-9, on MMP-2 enzyme activity, were investigated. It has been detected that MMP2-28 peptide at concentrations of 1000 µM inhibited 55 % of MMP-2 activity. MMP9-27 and MMP9-3 peptides that have no effect on MMP-2 activity, are candidate MMP-9 specific blocking peptides. In the context of the thesis, MMP-2 and MMP-9 activity blocking 12 a.a. peptide structures have been obtained for the first time. Identified peptides are candidates for the diagnosis or treatment of diseases which gelatinases take role in.