Functional characterization of the Wiskott Aldrich Syndrome Protein (WASP)

• Main Author: Lim, Rina Pei Zhi
• Other Authors: Thirumaran s/o Thanabalu
• Format: Theses and Dissertations
• Language: English
• Published: 2011
• Subjects: DRNTU::Science::Biological sciences::Genetics
• Online Access: https://hdl.handle.net/10356/46285
• Institution: Nanyang Technological University

Wiskott Aldrich Syndrome (WAS) is an X-linked recessive disease with clinical symptoms such as thrombocytopenia, eczema and recurrent infections due to immune deficiency. Identification of the WASP gene led to the discovery of other WAS-related diseases, X-linked thrombocytopenia (XLT) and X-linked congenital neutropenia (XLN) that present milder symptoms of WAS. WASP is a 502 –residue multidomain protein, comprising of, from the N to the C terminus, WH1 domain, basic region (BR), GTPase binding domain (GBD), proline rich region (PRR) and the VCA domain. The two fragments of YFP (Yellow Fluorescent Protein) molecule (YFP1-154 and YFP155-238) were fused to the N- and C-terminus of WASP respectively as a probe (WASP Reporter) for Bi-molecular fluorescence complementation (BiFC) to elucidate the conformation of WASP inside the cell. When WASP adopts a closed conformation, the two fragments of YFP are close to each other resulting in fluorescence and reduction in fluorescence indicates that WASP is in an open conformation. WASP was found to be in a closed conformation in yeast and this conformation was stabilized by WIP (WASP Interacting Protein) or WIRE (WIP related) as seen by the enhanced the fluorescence from the WASP reporter. On the other hand, Toca1 (Transducer of cdc42-dependent actin assembly) or Nck1 (Nonbinding catalytic region of tyrosine kinase) caused a reduction in YFP fluorescence, suggesting that SH3 domain-containing proteins altered the conformation to an open one. Cells expressing WASP reporter with mutations in WH1, GBD and VCA domains showed reduced fluorescence even in the presence of WIP suggesting that the conformation was affected by these mutations.