Inhibition of DNAJ-HSP70 interaction improves strength in muscular dystrophy

Abstract

Introduction: 41Limb-girdle muscular dystrophies (LGMD) are a family of hereditary muscle diseases 42 that are inherited in an autosomal recessive or dominant manner [1]. Most recessively 43 inherited LGMDs are postulated to be caused by a loss-of-function mechanism, 44 because the protein is absent in patient muscle tissue and many mutations lead to 45 premature stops or frameshifts. However, the mechanisms of dominantly inherited 46LGMDs are less clear: they may be due to haploinsufficiency, a gain of new toxic 47 function or a dominant interaction leading to protein dysfunction. 48 49One common pathologic feature seen in some dominantly inherited myopathies, 50including LGMD1D, myofibrillar myopathy and inclusion body myopathy, are protein 51 aggregates and myofiber vacuolation [2]. The inclusions in LGMD1D muscle are 52 heterogeneous and contain myofibrillar proteins, such as desmin and myotilin [3, 4]. 53Additionally, they contain proteins that aggregate in neurodegenerative disorders, such 54 as TDP-43 and SQSTM1 [3, 4]. Together, these features suggest that a subset of 55 inherited myopathies might involve an impairment in protein quality control. Indeed, one 56 family of muscle diseases with myofibrillar disorganization (e.g. myofibrillar myopathies) 57 are caused by missense mutations in Z-disc associated structural proteins, such as 58 desmin, myotilin or filamin-C that lead to their aggregation [2]. Likewise, myofibrillar 59 myopathies are also associated with mutations in molecular chaperones, such as 60 BAG3, HSPB5 and DNAJB6 [2, 5, 6]. These "chaperonopathies" are autosomal 61 dominantly inherited and lead to the accumulation and aggregation of Z-disc proteins, 62 such as desmin and filamin-C. Moreover, several chaperones are known to be 63 4 necessary for sarcomere development and maintenance [7]. Our aim was to better 64 understand the mechanisms associated with dominantly inherited LGMD1D caused by 65 mutations in DNAJB6 in order to gain insight into the relationship between protein 66 quality control and myopathy. 67 68 DNAJB6 is a ubiquitously expressed HSP40 co-chaperone that facilitates HSP70 69 functionality via its J domain [8]. DNAJB6, like other DNAJB family members, has a 70 canonical J domain, a C-terminal dimerization motif and a glycine-and phenylalanine-71 rich region (termed a G/F domain) [8]. Interestingly, all reported LGMD1D mutations 72 reside within the G/F domain [3, 4,[9][10][11]. DNAJB6 has two isoforms, DNAJB6a and 73DNAJB6b, which differ in the length of their C-terminal region and in their cellular 74 localization. DNAJB6a is longer and localizes to the nucleus; whereas DNAJB6b is 75 shorter and cytoplasmic [12]. Although both isoforms share the G/F domain containing 76LGMD1D disease mutations, it is thought that DNAJB6b is the pathogenic isoform [3, 77 12]. Patients with LGMD1D have insidious onset of weakness in the second to sixth 78 decade of life that affects their ability to ambulate [3, 4, 9, 10, 13]. The muscle tissue of 79LGMD1D patients is characteristically...

Co-Chaperones in Targeting and Delivery of Misfolded Proteins to the 26S Proteasome

“…Another study on chaperone regulation found that the underlying mechanism of the inherited muscular dystrophy disease LGMD1D is due to increased affinity between HSP70 and a mutated variant of the JDP DNAJB6, thereby locking HSP70 to this complex, preventing it from completing its cellular tasks. By inhibiting the interaction between HSP70 and DNAJB6, HSP70 molecules were released, relieving disease models from symptoms of muscular dystrophy [ 166 ].…”

Distinctive chaperonopathy in skeletal muscle associated with the dominant variant inDNAJB4

“…The variant site, phenylalanine at 90, is within the conserved G/F domain in DNAJB4. Similar to the DNAJB6-F93L mutant, the DNAJB4-F90L mutant enhanced the formation of TDP-43-positive nuclear stress granules when expressed in cells, and the abrogation of its association with HSP70 suppressed TDP-43 stress granule formation 22 . These results suggested that the F90L variant in DNAJB4 has a gain-of- toxicity with HSP70 on TDP-43.…”

“…Because LGMDD1-mutant DNAJB6b showed a toxic gain-of-function effect through the interaction with HSP70 in cellulo models 22 , we hypothesized that DNAJB4 mutant also have a similar toxic gain-of-function effect. To test this, we first analyzed the subcellular localization of wild-type and mutant DNAJB4 in HeLa cells and found that DNAJB4-F90L did not change its localization (Fig.…”

“…Expanded poly-Q (polyQ23 and polyQ74) in human huntingtin were digested with HindIII/EcoRI and ligated into a vector pEGFP-C1 containing a GFP tag. For heat shock experiments, human DNAJB4 (Addgene) and DNAJB6 (Addgene) constructs were made as previously described 55 . The DNAJB4 and DNAJB6 cDNAs were digested with HindIII/XhoI and ligated into the vector pcDNA3.1 containing a GFP tag or a V5 tag.…”

“…We performed heat shock experiments as previously described 55 . HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum, penicillin/streptomycin.…”