Hsp70- and Hsp90-mediated proteasomal degradation underlies TPIsugarkill pathogenesis in Drosophila

Abstract

Triosephosphate isomerase (TPI) deficiency is a severe glycolytic enzymopathy that causes progressive locomotor impairment and neurodegeneration, susceptibility to infection, and premature death. The recessive missense TPI sugarkill mutation in Drosophila melanogaster exhibits phenotypes analogous to human TPI deficiency such as progressive locomotor impairment, neurodegeneration, and reduced lifespan. We have shown that the TPI sugarkill protein is an active stable dimer, however, the mutant protein is turned over by the proteasome reducing cellular levels of this glycolytic enzyme. As proteasome function is often coupled with molecular chaperone activity, we hypothesized that TPI sugarkill is recognized by molecular chaperones that mediate the proteasomal degradation of the mutant protein. Co-immunoprecipitation data and analyses of TPI sugarkill turnover in animals with reduced or enhanced molecular chaperone activity indicate that both Hsp90 and Hsp70 are important for targeting TPI sugarkill for degradation. Furthermore, molecular chaperone and proteasome activity modified by pharmacological or genetic manipulations resulted in improved TPI sugarkill protein levels and rescue some but not all of the disease phenotypes suggesting that TPI deficiency pathology is complex. Overall, these data demonstrate a surprising role for Hsp70 and Hsp90 in the progression of neural dysfunction associated with TPI deficiency.

Triosephosphate isomerase I170V alters catalytic site, enhances stability and induces pathology in a Drosophila model of TPI deficiency

“…Ten fly heads were obtained in triplicate from each genotype and processed as outlined previously [21]. Proteins were resolved by SDS–PAGE, transferred onto 0.45 µm PVDF membrane.…”

Evidence of a triosephosphate isomerase non-catalytic function critical to behavior and longevity

“…S4). TPI sgk animals display reduced TPI protein levels in vivo (Seigle et al, 2008;Hrizo and Palladino, 2010), and TPI levels were further reduced when TPI sgk was paired with the TPI JS10 null allele (Fig. 4A,C) …”

“…After 24 hours, ten fly heads were collected in triplicate from each genotype and processed as outlined previously (Hrizo and Palladino, 2010). Briefly, the fly heads were ground by pestle in 80 ml 26 SDS-PAGE sample buffer (4% SDS, 4% b-mercaptoethanol, 130 mM Tris-HCl pH 6.8, 20% glycerol) and centrifuged for 5 minutes at 5000 g to pellet the exoskeleton.…”

“…The affected methionine is present near the dimer interface yet does not seem to result in a shift in monomer-dimer populations in vivo (Seigle et al, 2008). However, the TPI sgk mutation has been shown to induce abnormal proteasomal degradation of TPI resulting in reduced total TPI protein (Seigle et al, 2008;Hrizo and Palladino, 2010). Interestingly, we have previously shown that this loss-of-function mutation can be attenuated by overexpressing mutant TPI sgk (Celotto et al, 2006).…”

Genetically encoded redox sensor identifies the role of ROS in degenerative and mitochondrial disease pathogenesis

“…Locomotor function stress-sensitivity (a.k.a. bang-sensitivity) was assayed as previously(Celotto et al, 2006a; Celotto et al, 2006b; Hrizo and Palladino, 2010). The elav-Gal4 strain is from the Bloomington Drosophila stock center.…”