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Abstract:Hereditary Spastic Paraplegias (HSP) are genetically diverse and clinically characterized by lower limb weakness and spasticity. The N471D and several other point mutations of human strumpellin (Str), a member of the WASH (Wiskott-Aldrich Syndrome Protein and SCAR Homologue) complex, have been shown to cause HSP type 8 (SPG8). To investigate the molecular functions of wild type and N417D Str, we generated Dictyostelium Str- cells and ectopically expressed Strwt-GFP or StrN471D-GFP in Str- and wt cells. Overexpression of both proteins apparently caused a defect in cell division as we observed a clear increase in multinucleate cells. Real time PCR analyses revealed no transcriptional changes in WASH complex subunits in Str- cells, but Western blots showed a two-fold decrease in the SWIP subunit. GFP-trap experiments in conjunction with mass-spectrometric analysis revealed many previously known as well as new Str interacting proteins and also proteins that did no longer bind to StrN471D On the cellular level, Str- cells displayed defects in cell growth, phagocytosis, macro-pinocytosis, exocytosis and lysosomal function. Expression of Strwt-GFP in Str- cells rescued all observed defects. In contrast, expression of StrN471D-GFP could not rescue lysosome morphology and exocytosis of indigestible material. Our results underscore a key role for the WASH complex and its core subunit Str in the endo-lysosomal system and highlight the fundamental importance of the Str N471 residue for maintaining lysosome morphology and dynamics. Our data indicate that the SPG8-causing N471D mutation leads to a partial loss of Strumpellin function in the endo-lysosomal system. | |||||||
Status: | aheadofprint | Type: | Journal article | Source: | PUBMED | PubMed ID: | 30061306 |
Genes addressed in this paper | ||||||||
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ccdc53 | fam21 | swip | washc5 | wshA | Topics in this paper | |||
Protein Physical Properties | X | |||||||
Disease Related | X | |||||||
Mammalian Gene Related | X | |||||||
Protein Functional Domain | X | |||||||
Strains/Constructs | X | |||||||
Non-mammalian Gene Related | X | |||||||
Cellular Location | X | |||||||
Function/Process | X | |||||||
Endocytosis | X | |||||||
Protein-Protein Interactions | X | X | X | X | X | |||
Mutants/Phenotypes | X | |||||||
Antibodies | X |