Hsp70 mutant proteins modulate additional apoptotic pathways and improve cell survival.
Although wild-type Hsp70 (Hsp70WT) inhibits procaspase-3 processing by preventing apoptosome formation, Hsp70WT does not block active caspase-3. Because all caspase-3 inhibitors bear canonical DXXD caspase-3 recognition motifs, we determined whether mutated Hsp70s with caspase-binding motifs act as direct caspase-3 inhibitors. Based on Hsp70 molecular modeling, the DNQP, DEVQ, and EEVD regions localized on the surface of Hsp70WT were chosen, allowing us to design mutants while trying to avoid disrupting the global fold of the molecule and losing the possibility of protein-protein interactions. We replaced DNQP with DQMD, and DEVQ and EEVD with DEVD residues that should be optimal substrates for caspase-3. The resultant Hsp70 mutants directly interacted with active caspase-3 and blocked its proteolytic activity while retaining the ability to reverse protein denaturation and disrupt the interaction between Apaf-1 and procaspase-9. The Hsp70C-terminal mutants interacted with Apaf-1 and active caspase-3 significantly longer than Hsp70WT. The Hsp70 DXXD mutants protected neuron and teratocarcinoma (NT) cells against cell death much better than Hsp70WT whether given before or after serum withdrawal. Hsp70 mutants represent a possible approach to antiapoptotic biotherapeutics. Similar rational designs could be used to engineer inhibitors of additional caspase family members.
Ran
Ruiqiong
R
Department of Neurology, Vontz Center for Molecular Studies, University of Cincinnati, Room 2327, 3125 Eden Avenue, Cincinnati, OH 45267-0536, USA. ranr@email.uc.edu
Zhou
Guoping
G
Lu
Aigang
A
Zhang
Lu
L
Tang
Yang
Y
Rigby
Alan C
AC
Sharp
Frank R
FR
D020816:Amino Acid Motifs / Q000235:genetics; D017209:Apoptosis / Q000235:genetics*; D053477:Apoptotic Protease-Activating Factor 1; D001665:Binding Sites / Q000235:genetics; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D015195:Drug Design; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D020033:Protein Isoforms / Q000235:genetics; D011506:Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured
C506592:APAF1 protein, human; D053477:Apoptotic Protease-Activating Factor 1; D016895:Culture Media, Serum-Free; D018840:HSP70 Heat-Shock Proteins; D020033:Protein Isoforms; D011506:Proteins; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases