View Item 
      •   Home
      • 7. Theses and Dissertations
      • Theses and Dissertations
      • View Item
      •   Home
      • 7. Theses and Dissertations
      • Theses and Dissertations
      • View Item
      JavaScript is disabled for your browser. Some features of this site may not work without it.
      Subject Lookup

      Browse

      All of DR-NTUCommunities & CollectionsTitlesAuthorsBy DateSubjectsThis CollectionTitlesAuthorsBy DateSubjects

      My Account

      Login

      Statistics

      Most Popular ItemsStatistics by CountryMost Popular Authors

      About DR-NTU

      Characterization of molecular differences between WASP and N-WASP and characterization of mutations causing Wiskott-Aldrich syndrome.

      Thumbnail
      View/Open
      Neeraj PhD Thesis.pdf (6.997Mb)
      Author
      Neeraj Jain.
      Date of Issue
      2013
      School
      School of Biological Sciences
      Abstract
      Wiskott-aldrich Syndrome is caused by mutation in gene encodes WASP. WASP and its homologue N-WASP regulate actin cytoskeleton reorganization, share more than 50% sequence identity. Knocking down of WASP expression impaired Jurkat T-cells chemotaxis towards SDF-1α and increased adhesion to fibronectin, which was rescued by exogenous expression of WASP (WASPR: shRNA resistant) but not by N-WASP. Moreover, WASP is indispensable for formation of anti-CD3/anti-CD28 stimulated membrane projections. However, N-WASP rescued the IL-2 gene transcription defect of WASPKD Jurkat T-cells suggesting both overlapping and unique functions of WASP and N-WASP in T-cells. WASP has a unique 30 amino acid (I30) region (a.a.158-187) between its WH1 domain and basic region which is absent in N-WASP. The I30 region of WASP interacts with eight (Fyn, Toca1, Nck, Grb2, FGR, Src, Lyn and Hck) out of 17 known WASP binding proteins. The I30 region of WASP is required for Jurkat T-cells chemotaxis and IL-2 gene transcription, which are the two independent activities of WASP. These defects observed in WASPΔI30 expressing WASPKD Jurkat T-cells could be due to a partially open conformation of WASP∆I30, resulting increase Hck mediated Tyr291 phosphorylation. Insertion of I30 region in N-WASP (N-WASP-I30) rescued all the impairments of WASPKD Jurkat T-cells and mediates its localization to TCR activation sites. Thus, suggesting that I30 region of WASP is one of the important determinants of functional differences between WASP and N-WASP. Two point mutations in the PRR domain of WASP (S339Y/P373S) have been reported to occur together in WAS patients. These mutations were characterized either individually (WASPS339Y, WASPP373S) or together (WASPSP/YS) for their effects in Jurkat T-cell functions. WASPPRR mutants did not rescue the defective chemotaxis of WASPKD Jurkat T-cells. Unlike WASPRS339Y, cells expressing WASPRP373S or WASPRSP/YS did not rescue the IL-2 gene transcription defect and adhesion to fibronectin suggesting that point mutation P373S is sufficient to inhibit WASP mediated Jurkat T-cell activation. Moreover, a diffused pattern of localization of WASPPRR mutants at TCR activation sites was observed suggesting that WASPPRR mutants abolish WASP function in TCR mediated signalling. Thus, both mutations individually cause some defects but together have additive effects on WASP mediated T-cell functions resulting in WAS. Most of the WAS associated missense mutations occur in the WH1 domain of WASP. Twenty five of these missense mutations were analysed for their interaction with CIB1 (WASPWH1 domain interacting protein). Six of these mutations (G40V, T45M, L46P, A47D, P58L, G70W) abolished WASP-CIB1 interaction but not WASP-WIP interaction. Four mutations (G40V, T45M, P58L, G70W) expressed poorly in WASPKD Jurkat T-cells suggesting a possible cause of disease. The remaining two mutants (L46P, A47D) expressed well but did not rescue the chemotaxis defect of WASPKD Jurkat T-cell and negatively regulate spreading of cells on anti-CD3/anti-CD28 coated coverslip. WASPKD Jurkat T-cells expressing WASPL46P expressing cells were defective in T-cell activation. Taken together, this study has led to the elucidation of the molecular difference between WASP and N-WASP and characterised the molecular defect of some of the WASP mutations causes WAS.
      Subject
      DRNTU::Science
      Type
      Thesis
      Collections
      • Theses and Dissertations

      Show full item record


      NTU Library, Nanyang Avenue, Singapore 639798 © 2011 Nanyang Technological University. All rights reserved.
      DSpace software copyright © 2002-2015  DuraSpace
      Contact Us | Send Feedback
      Share |    
      Theme by 
      Atmire NV
       

       


      NTU Library, Nanyang Avenue, Singapore 639798 © 2011 Nanyang Technological University. All rights reserved.
      DSpace software copyright © 2002-2015  DuraSpace
      Contact Us | Send Feedback
      Share |    
      Theme by 
      Atmire NV
       

       

      DCSIMG