Anne Houdusse — Page 2

PubMed2002

Isolation and characterization of an aggresome determinant in the NF2 tumor suppressor.

Schwannomin (Sch) is the product of the NF2 tumor suppressor gene. The NF2 gene is mutated in patients affected by neurofibromatosis type 2, a syndrome associated with multiple tumors of the nervous system. Here we found that Sch, when its N-terminal FERM domain was misfolded by the pathogenetic mutation Delta F118, formed aggresomes, i.e. aggregates that cluster at the centrosome as a result of microtubule-dependent transport. Strikingly the related protein ezrin affected by the same mutation did not form aggresomes even though its FERM domain was similarly misfolded. By studying ezrin/Sch chimeras, we delineated a sequence of 61 amino acids in the C terminus of Sch that determined the formation of aggresomes. Aggresome formation by these chimeras was independent from their rate of degradation. Sch(535-595) was sufficient to induce aggresomes of a green fluorescent fusion protein in vivo and aggregates of a glutathione S-transferase fusion protein in vitro. Taken together, these results suggest that aggresome formation is controlled primarily by aggresome determinants, which are distinct from degradation determinants, or from misfolding, through which aggresome determinants might be exposed.

Animals↗

PubMed2002

Biochemical and functional characterization of Rab27a mutations occurring in Griscelli syndrome patients.

Rab27a is a member of the Rab family of small GTPase proteins, and thus far is the first member to be associated with a human disease (ie, the Griscelli syndrome type 2). Mutations in the Rab27a gene cause pigment as well as cytotoxic granule transport defects, accounting for the partial albinism and severe immune disorder characteristics of this syndrome. So far, 3 Rab27a missense mutations have been identified. They open a unique opportunity to designate critical structural and functional residues of Rab proteins. We show here that the introduction of a proline residue in the alpha 4 (Ala152Pro) or beta 5 (Leu130Pro) loop, observed in 2 of these spontaneous mutants, dramatically affects both guanosine triphosphate (GTP) and guanosine diphosphate (GDP) nucleotide-binding activity of Rab27a, probably by disrupting protein folding. The third mutant, Trp73Gly, is located within an invariant hydrophobic triad at the switch interface, and was previously shown in active Rab3A to mediate rabphilin3A effector interaction. Trp73Gly is shown to display the same nucleotide-binding and GTPase characteristics as the constitutively active mutant Gln78Leu. However, in contrast to Gln78Leu, Trp73Gly mutant construct neither interacts with the Rab27a effector melanophilin nor modifies melanosome distribution and cytotoxic granule exocytosis. Substitutions introduced at the 73 position, including the leucine residue present in Ras, did not restore Rab27a protein functions. Taken together, our results characterize new critical residues of Rab proteins, and identify the Trp73 residue of Rab27a as a key position for interaction with the specific effectors of Rab27a, both in melanocytes and cytotoxic cells.

Albinism↗