Biology subjects

Daw, J. A.

Publications and source records attributed to Daw, J. A..

3 recordsLinked to original sources

bioRxiv2026

Allele-selective knockdown of mutant DNAJB6 improves molecular features in a primary mouse myotube model of LGMDD1

Dominant variants in DNAJB6 cause limb-girdle muscular dystrophy D1 (LGMDD1), a progressive myopathy associated with disrupted protein homeostasis. Because complete loss of DNAJB6 is embryonic lethal in mice, we evaluated allele-selective knockdown as a strategy to reduce mutant DNAJB6 while preserving wild-type protein. We generated a knock-in mouse carrying an N-terminal 3xFLAG tag on the wild-type Dnajb6 allele, allowing endogenous wild-type and F90I DNAJB6, the murine equivalent of human F89I, to be measured within the same sample. Flag-LGMDD1 mice recapitulated the functional, myopathological, and biochemical features seen in other LGMDD1 models and in human disease. F90I DNAJB6 was more abundant and degraded more slowly than wild-type DNAJB6, and LGMDD1 muscle and primary myotubes showed altered protein handling at baseline and following proteostatic stress. Comparative proteomic analyses identified DNAJA4 as a candidate marker responsive to pathogenic DNAJB6 activity. A two-stage mismatch screen identified a lead candidate siRNA with 264-fold selectivity for F90I DNAJB6. In primary Flag-LGMDD1 myotubes, this siRNA selectively reduced F90I DNAJB6 and shifted several baseline and stress-dependent molecular abnormalities involving p62, HSP70, DNAJA4, and markers associated with unfolded protein response signaling toward control values. These findings establish cellular proof of concept for allele-selective knockdown in LGMDD1.

neuroscience↗

bioRxiv2022

DNAJB6 Isoform Specific Knockdown: Therapeutic Potential for LGMDD1

Dominant missense mutations in DNAJB6, an HSP40 co-chaperone, cause limb girdle muscular dystrophy (LGMD) D1. No treatments are currently available. Two isoforms exist, DNAJB6a and DNAJB6b, each with distinct localizations in muscle. Mutations reside in both isoforms, yet evidence suggests only DNAJB6b is responsible for disease pathogenesis. Mechanistic data supports either a toxic gain of function, a dominant negative mechanism, or a combination of both. Knockdown treatment strategies involving both isoforms carry risk as DNAJB6 knockout is embryonic lethal. We therefore developed an isoform specific knockdown approach using morpholinos. Selective reduction of each isoform was achieved in-vitro in primary mouse myotubes and human myoblasts, as well as in-vivo in mouse skeletal muscle. To assess isoform specific knockdown in LGMDD1, we created primary myotube cultures from a knock-in LGMDD1 mouse model. Using mass spectrometry, we identified an LGMDD1 protein signature related to protein homeostasis and myofibril structure. Selective reduction of DNAJB6b levels in LGMDD1 myotubes corrected much of the proteomic disease signature towards wild type levels. While additional in-vivo functional data is required, these findings suggest selective reduction of DNAJB6b may be a viable therapeutic target for LGMDD1.

neuroscience↗

bioRxiv2021

Myopathy mutations in DNAJB6 slow conformer specific substrate processing that is rescued by NEF modulation

Molecular chaperones, or heat shock proteins (HSPs), protect against the toxic misfolding and aggregation of proteins. As such, mutations or deficiencies within the chaperone network can lead to disease. In fact, dominant mutations in DNAJB6 (Hsp40/Sis1), an Hsp70 co-chaperone, leads to a protein aggregate myopathy termed Limb-Girdle Muscular Dystrophy Type D1 (LGMDD1). DNAJB6 client proteins and co-chaperone interactions in skeletal muscle are not known. Here, we used the yeast prion model client in conjunction with in vitro chaperone activity assays to gain mechanistic insights, and found that LGMDD1 mutants affect Hsp40 functions. Strikingly, the mutants changed the structure of client protein aggregates, as determined by altered distribution of prion strains. They also impair the Hsp70 ATPase cycle, dimerization, and substrate processing and consequently poison the function of wild-type protein. These results define the mechanisms by which LGMDD1 mutations alter chaperone activity and provide avenues for therapeutic intervention.

biochemistry↗