Essay About Characterization Of Wiskott And Las17D Strain

Characterization of Wiskott^aldrich Syndrome(was)mutants Using Saccharomyces Cerevisiae
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Abstract
Wiskott-Aldrich syndrome (WAS) is caused by alterations in the WAS protein
(WASP), and 80% of the missense mutations are located in the WH1 domain, the
region essential for interaction with the WASP-interacting protein (WIP). It has
been suggested that loss of WASP-WIP interaction is causal to the disease. Las17p
(yeast WASP) is essential for growth at 37 1C. The growth defect of the las17D
strain can be suppressed by the expression of human WASP together with WIP.
Using the las17D strain, we have analyzed 52 missense mutations in the gene
encoding WASP and found that 13 of these mutant proteins were unable to
suppress the growth defect of the las17D strain. The majority of these 13 mutations
cause the classic WAS in humans and are located within the WH1 domain, while
none of the 12 mutations outside the WH1 domain abolished the activity ofWASP
in Saccharomyces cerevisiae cells. This suggests that some of the mutations (13 out
of 40) in the WH1 domain cause the syndrome in humans by perturbing the
WASP-WIP complex formation, while the rest of the mutations cause the
syndrome without affecting the WASP-WIP complex formation, but may affect
the activity of the complex.
Introduction
Wiskott-Aldrich syndrome (WAS) is an X-linked genetic
disease caused by recessive mutations in the gene encoding
the WAS protein (WASP) (Derry et al., 1994). WASP is a
proline-rich protein and has a modular structure organized
into several domains: a WASP homology (WH1) domain, a
basic region, a GTPase-binding domain, a proline-rich
sequence and the Verprolin homology, Cofilin homology
and the Acidic region (VCA) domain (Takenawa & Suetsugu,
2007). The VCA domain of WASP activates the Arp2/3
complex to regulate actin polymerization (Rohatgi et al.,
1999). WASP is expressed predominantly in hematopoietic
cells while its homologue N-WASP (neural), with an additional
V domain, is expressed ubiquitously (Miki et al.,
1996). Both N-WASP and WASP have been shown to
interact with Cdc42 (Symons et al., 1996) and many
cytoskeletal proteins including WASP-interacting protein
(WIP) (Ramesh et al., 1997). WASP and N-WASP adopt an
inactive conformation (Kim et al., 2000) that can be
activated by Cdc42 (Higgs & Pollard, 2000; Rohatgi et al.,
2000). Recently, it has been proposed that activation of NWASP
by Cdc42 is mediated by the transducer of Cdc42
activation (Toca-1) (Ho et al., 2004), and it was found that
Toca-1 and not Cdc42 relieves the autoinhibition of WASP
(Lim et al., 2007). Las17p is the yeast homologue of
mammalian WASP (Li, 1997; Naqvi et al., 1998), and
deletion of the gene encoding Las17p results in a Saccharomyces
cerevisiae strain with cytoskeletal and growth defects;
the las17D strains are unable to grow at elevated temperatures
and have defects in fluid-phase endocytosis and actin
patch polarization at both permissive and restrictive temperatures
(Li, 1997; Naqvi et al., 1998).
In mammalian cells, the majority of WASP molecules are
present in a complex with WIP (Sasahara et al., 2002; de la
Fuente et al., 2007).Most of the missense mutations (80%