Abstract
Mutations in one allele of the human LIS1 gene cause a severe brain malformation, lissencephaly. Although most LIS1 mutations involve deletions, several point mutations with a single amino acid alteration were described. Patients carrying these mutations reveal variable phenotypic manifestations. We have analyzed the functional importance of these point mutations by examining protein stability, folding, intracellular localization, and protein-protein interactions. Our data suggest that the mutated proteins were affected at different levels, and no single assay could be used to predict the lissencephaly phenotype. Most interesting are those mutant proteins that retain partial folding and interactions. In the case of LIS1 mutated in F31S, the cellular phenotype may be modified by overexpression of specific interacting proteins. Overexpression of the PAF-AH alpha1 subunit dissolved aggregates induced by this mutant protein and increased its half-life. Overexpression of NudE or NudEL localized this mutant protein to spindle poles and kinetochores but had no effect on protein stability. Our results implicate that there are probably different biochemical and cellular mechanisms obstructed in each patient yielding the varied lissencephaly phenotypes.
Highlights
Mutations in one allele of the human LIS1 gene cause a severe brain malformation, lissencephaly
In the case of LIS1 mutated in F31S, the cellular phenotype may be modified by overexpression of specific interacting proteins
Our analysis included several assays to determine whether there were global effects of amino acid substitutions; protein folding was tested by limited trypsin cleavage, LIS1 stability was monitored by examining protein degradation, and its intracellular localization was inspected by immunostaining
Summary
Mutations in one allele of the human LIS1 gene cause a severe brain malformation, lissencephaly. Our data suggest that the mutated proteins were affected at different levels, and no single assay could be used to predict the lissencephaly phenotype Most interesting are those mutant proteins that retain partial folding and interactions. Abnormalities in the migration of neurons into the embryonic cortex lead to loss of normal convolutions of the human neocortex, known as lissencephaly One cause for such an acute brain malformation is mutation(s) in the LIS1 gene [1]. Our previous analysis indicated that truncated or internally deleted LIS1 protein is unlikely to fold correctly [11] This finding was supported by a study done in cells derived from lissencephaly patients, where no protein was detected from mutated alleles that were expected to result in a truncated protein [12]. H149R [13], G162S [10], and
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