Hsp70- and Hsp90-mediated proteasomal degradation underlies TPI sugarkill 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 life span. 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. Coimmunoprecipitation 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.

FIGURE 1. Temperature enhanced turnover of TPI^sugarkill is stabilized with proteasome inhibition

A: TPI protein was assessed from TPI^sugarkill animals acutely shifted to 25° (yellow line) or 29° (red line). TPI levels were visualized by western blot. N = 5. Wildtype TPI levels do not change in response to exposure to 29° ( data not shown). B: TPI protein levels were assessed from TPI^sugarkill animals treated with 30uM MG132 (red line) or the vehicle control (0.03% DMSO, blue line) and acutely shifted to 29°. N = 3.

FIGURE 2. TPI and TPI^sugarkill have similar fractionation patterns

A: Extracts from TPI and TPI^sugarkill animals at 25° were fractionated. The pellet (P) and supernatant (S) fractions were assessed for TPI by western blot analysis. B: ATPalpha control demonstrates a pellet enriched fractionation pattern. C: Representative western blots of TPI, TPI^sugarkill, and ATPalpha. N = 3.

FIGURE 3. Hsp70 and Hsp90 levels are increased in TPI^sugarkill animals

A: Hsp90 protein was assessed from TPI^sugarkill animals acutely shifted to 25° and 29° for 24 hours. Hsp90 levels were visualized by western blot with anti-Hsp90a/b. N = 7. B: Hsp70 protein levels were assessed as in A but using anti-Hsp70. N = 7. C: TPI and TPI^sugarkill were immunoprecipitated using anti-TPI conjugated resin. Precipitated proteins were visualized by western blot. N = 3.

FIGURE 4. TPI^sugarkill is stabilized in animals with reduced Hsp90 or Hsp70

A: TPI protein levels were assessed in TPI^sugarkill animals treated with 5uM geldanamycin (GA, red line) or the vehicle control (0.03% DMSO, blue line) and acutely exposed to 29°. N = 3. B: TPI protein levels were assessed from TPI^sugarkill animals wildtype (+) or mutant (E6A or PPZ) for Hsp90 and that were exposed to 25° or 29° for 24hr. TPI levels were compared to TPI^sugarkill animals with wildtype (+) Hsp90. N = 5. C: TPI protein levels were assessed from TPI^sugarkill animals with no UAS transgene (+), UAS-Hsp70 construct, or UAS-Hsp70[K71S]. The animals were exposed to 25° or 29° for 24hr. TPI levels were compared to TPI^sugarkill animals with no transgene. N = 6.

FIGURE 5. Stabilizing TPI^sugarkill reduces the progressive mechanical stress sensitivity of TPI^sugarkill animals

The time required for recovery of mobility following mechanical stress is indicated. Animals were age matched (day 20). A & B: Vehicle only control (DMSO) and untreated (−) controls were also examined. A: MG132 administered at 10uM or 30uM improved locomotion in TPI[sgk] and did not alter wildtype animals. N = 12. B: Geldanamycin (GA) administered at 2uM or 5uM improved locomotion in TPI[sgk] and did not alter wildtype animals. N = 12. C: The Hsp90[E6A] and Hsp90[PPZ] mutations improved locomotion in TPI[sgk] and did not alter wildtype animals. N = 12. D: Wildtype Hsp70 overexpression (UAS-Hsp70) worsened locomotor function, whereas, expression of dominant-negative Hsp70[K71S] (UAS-Hsp70[K71S]) improved locomotion. Expression of these transgenes did not affect wildtype animals. N = 5.