عنوان

Assembly of ASK1-MKK4-JNK3 Complex

TLpq2311052236

انگلیسی

Assembly of ASK1-MKK4-JNK3 Complex

[Thesis]

Howlader, Md Sariful Islam

Zhan, Xuanzhi

Tennessee Technological University

2019

131

M.S.

Tennessee Technological University

2019

Mitogen-activated protein kinase (MAPK) plays a pivotal role in cellular stress and immune response where external stimuli have been transduced in the cell through the phosphorylation cascades. These cascades are mainly ordered as consecutive three-tiered kinases where the upstream apoptosis signal-regulating kinase I (ASK1) can directly activate through phosphorylation of downstream MAP kinase kinase 4 (MKK4) which in turn phosphorylates and activates c-jun N-terminal kinase 3 (JNK3). However, the structure of these three kinase complexes (ASK1, MKK4, and JNK3) remains still unknown. The present study was designed to find out the assembly of ASK1-MKK4-JNK3 firstly using computational docking based on the Protein Frustratometer and the sequence identifier Evolutionary Trace forms along with rigid docking (Cluspro). To verify the proposed computational model of ASK1-MKK4-JNK3 complex, we designed a series of maltose-binding protein (MBP) fusion peptides which contain different peptide fragments derived from MKK4: MBP-P1, MBP-P2, MBP-P3, MBP-P4 and MBP-P5. After confirming appropriate cloning between pMAL-P2T vector and human MKK4 cDNA with the specific recognition site containing restriction enzyme sequence, the recombinant plasmids were then transformed into E-coli-BL21- DE3 to express by Isopropyl β-D-1-thiogalactopyranoside (IPTG), and purify using Amylose-sepharose chromatography. Direct pull-down assay was developed to evaluate the interactions between these fusion peptides with ASK1 and JNK3, respectively to find out the binding interaction among these purified proteins. We firstly selected five different forward and reverse primers from human MKK4 DNA for PCR. Amplified PCR fragments were then ligated with pMAL vector. We also got a very satisfying result after matching the sequence by Sanger's method. The purity of fusion-proteins was confirmed by the presence of Coomassie Blue staining. The result of protein-protein interaction between His-tag JNK3 and MKK4 was confirmed by SDS-PAGE analysis and Western blot. We found three peptides, P1, P2, and P5, can directly interact with JNK3, which is consistent with our computational model. We are developing ASK1-Heparin pull-down to verify the ASK1 binding with catalytic domain to test our docking result. These findings will be a great breakthrough for the novice scientist for further analysis by using sophisticated technology to get the molecular structure assembly of ASK1-MKK4-JNK3.

Biochemistry

Howlader, Md Sariful Islam

Zhan, Xuanzhi

p

[Thesis]

276903

a

Y