Inhibition of DNAJ-HSP70 interaction improves strength in muscular dystrophy
Rocio Bengoechea, Andrew R. Findlay, Ankan K. Bhadra, Hao Shao, Kevin C. Stein, Sara K. Pittman, Jil A.W. Daw, Jason E. Gestwicki, Heather L. True, Conrad C. Weihl
Rocio Bengoechea, Andrew R. Findlay, Ankan K. Bhadra, Hao Shao, Kevin C. Stein, Sara K. Pittman, Jil A.W. Daw, Jason E. Gestwicki, Heather L. True, Conrad C. Weihl
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Research Article Cell biology Muscle biology

Inhibition of DNAJ-HSP70 interaction improves strength in muscular dystrophy

Abstract

Dominant mutations in the HSP70 cochaperone DNAJB6 cause a late-onset muscle disease termed limb-girdle muscular dystrophy type D1 (LGMDD1), which is characterized by protein aggregation and vacuolar myopathology. Disease mutations reside within the G/F domain of DNAJB6, but the molecular mechanisms underlying dysfunction are not well understood. Using yeast, cell culture, and mouse models of LGMDD1, we found that the toxicity associated with disease-associated DNAJB6 required its interaction with HSP70 and that abrogating this interaction genetically or with small molecules was protective. In skeletal muscle, DNAJB6 localizes to the Z-disc with HSP70. Whereas HSP70 normally diffused rapidly between the Z-disc and sarcoplasm, the rate of diffusion of HSP70 in LGMDD1 mouse muscle was diminished, probably because it had an unusual affinity for the Z-disc and mutant DNAJB6. Treating LGMDD1 mice with a small-molecule inhibitor of the DNAJ-HSP70 complex remobilized HSP70, improved strength, and corrected myopathology. These data support a model in which LGMDD1 mutations in DNAJB6 are a gain-of-function disease that is, counterintuitively, mediated via HSP70 binding. Thus, therapeutic approaches targeting HSP70-DNAJB6 may be effective in treating this inherited muscular dystrophy.

Authors

Rocio Bengoechea, Andrew R. Findlay, Ankan K. Bhadra, Hao Shao, Kevin C. Stein, Sara K. Pittman, Jil A.W. Daw, Jason E. Gestwicki, Heather L. True, Conrad C. Weihl

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Figure 3

LGMDD1-mutant dysfunction is corrected by abrogating HSP70 interaction.

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LGMDD1-mutant dysfunction is corrected by abrogating HSP70 interaction. ...
(A) HeLa cells were cotransfected with plasmids expressing Flag-HSP70 and WT or LGMDD1-mutant (F89I, F93L, P96R, or F100I) GFP-DNAJB6b. In some cases, a second mutation in the J domain (H31Q) was also present. Twenty-four hours later, cells were lysed, and GFP-DNAJB6 was immunoprecipitated and subsequently immunoblotted for GFP or Flag. Quantitation of 3 replicates is shown in Supplemental Figure 2A. (B) Tetracycline-inducible isogenic 293 cell lines expressing (WT, H31Q, F93L, F93L-H31Q, P96R, or P96R-H31Q) V5-DNAJB6b were induced with tetracycline for 24 hours and then removed for the indicated durations. Lysates were immunoblotted for V5 and GAPDH. (C) Quantitation of V5-DNAJB6b levels from 3 independent experiments. *P ≤ 0.05, **P < 0.005, and ***P < 0.0005, by paired Student’s t test for comparisons between groups; Bonferroni-corrected P < 0.001 for multiple comparisons. (D) HeLa cells were transfected with the indicated constructs and subjected to heat shock at 42°C for 1 hour, and the percentage of ethidium homodimer-1–positive cells was quantitated. Data are presented as the percentage of cells found positive/dead. **P < 0.005 and ***P < 0.0005, by paired Student’s t test for comparisons between groups. (E) HeLa cells were cotransfected with mCherry-TDP43 and WT or LGMDD1-mutant (F89I, F93L, P96R, or F100I) GFP-DNAJB6b. In some cases, a second mutation in the J domain (H31Q) was also present. After 12 hours of transfection, cells were treated for 16 hours with either YM-01, JG231, JG98 (HSP70 inhibitors), or SW02 (HSP70 activator). The percentage of cells with TDP43-positive nuclear aggregate granules was quantified following 1 hour of heat shock. *P < 0.05, **P < 0.005, and ***P < 0.0005, by paired Student’s t test for comparisons between groups; Bonferroni-adjusted P < 0.0025 for multiple comparisons. n = 300 cells analyzed per condition. The blots and graphs are representative of 3 independent experiments.