Interactive domains in the molecular chaperone human alphaB crystallin modulate microtubule assembly and disassembly.

Abstract

Small heat shock proteins regulate microtubule assembly during cell proliferation and in response to stress through interactions that are poorly understood.Novel functions for five interactive sequences in the small heat shock protein and molecular chaperone, human alphaB crystallin, were investigated in the assembly/disassembly of microtubules and aggregation of tubulin using synthetic peptides and mutants of human alphaB crystallin.The interactive sequence (113)FISREFHR(120) exposed on the surface of alphaB crystallin decreased microtubule assembly by approximately 45%. In contrast, the interactive sequences, (131)LTITSSLSSDGV(142) and (156)ERTIPITRE(164), corresponding to the beta8 strand and the C-terminal extension respectively, which are involved in complex formation, increased microtubule assembly by approximately 34-45%. The alphaB crystallin peptides, (113)FISREFHR(120) and (156)ERTIPITRE(164), inhibited microtubule disassembly by approximately 26-36%, and the peptides (113)FISREFHR(120) and (131)LTITSSLSSDGV(142) decreased the thermal aggregation of tubulin by approximately 42-44%. The (131)LTITSSLSSDGV(142) and (156)ERTIPITRE(164) peptides were more effective than the widely used anti-cancer drug, Paclitaxel, in modulating tubulinmicrotubule dynamics. Mutagenesis of these interactive sequences in wt human alphaB crystallin confirmed the effects of the alphaB crystallin peptides on microtubule assembly/disassembly and tubulin aggregation. The regulation of microtubule assembly by alphaB crystallin varied over a narrow range of concentrations. The assembly of microtubules was maximal at alphaB crystallin to tubulin molar ratios between 1:4 and 2:1, while molar ratios >2:1 inhibited microtubule assembly.Interactive sequences on the surface of human alphaB crystallin collectively modulate microtubule assembly through a dynamic subunit exchange mechanism that depends on the concentration and ratio of alphaB crystallin to tubulin. These are the first experimental results in support of the functional importance of the dynamic subunit model of small heat shock proteins.