Variable Autoinhibition among Deafness-Associated Variants of Diaphanous 1 (DIAPH1)

Abstract

One of the earliest mapped human deafness genes, DIAPH1, encodes the formin DIAPH1. To date, at least three distinct mutations in the C-terminal domains and two additional mutations in the N-terminal region are associated with autosomal dominant hearing loss. The underlying molecular mechanisms are not known, and the role of formins in the inner ear is not well understood. In this study we use biochemical assays to test the hypotheses that autoinhibition and/or actin assembly activities are disrupted by DFNA1 mutations. Our results indicate that C-terminal mutant forms of DIAPH1 are functional in vitro and promote actin filament assembly.Similarly, N-terminal mutants are well-folded and have quaternary structures and thermal stabilities similar to the WT protein. The strength of the autoinhibitory interactions varies widely among mutants, with the ttaa, A265S and I530S mutations having an affinity similar to WT and the 1213x and ∆ag mutations completely abolishing autoinhibition. These data indicate that, in some cases, hearing loss may be linked to reduced inhibition of actin assembly.

Formins in Human Disease

“…This mutation partially relieves the autoinhibitory DID-DAD interaction, resulting in a mildly constitutive active molecule [21]. It is likely that this also occurs with other truncation mutations mapping around this site and with the missense mutations in the DID [119,120]. The DIAPH1 gene mouse homolog, mDia1, is expressed in the organ of Corti during and after cochlear maturation, and localizes at the apical junctional complexes between the supporting cells and the hair cells [121].…”

Alterations to the broad-spectrum formin inhibitor SMIFH2 modulate potency but not specificity

“…Profilin from Schizosaccharomyces pombe was expressed in a pET plasmid in BL21-DE3* cells and was purified as described 41 . We chose this isoform of profilin since, compared to other profilins, its interaction with actin is less sensitive to actin modification at Cys734 42 . Actin was purified from rabbit skeletal muscle and labeled with pyrene-iodoacetamide as described 42 .…”

“…We chose this isoform of profilin since, compared to other profilins, its interaction with actin is less sensitive to actin modification at Cys734 42 . Actin was purified from rabbit skeletal muscle and labeled with pyrene-iodoacetamide as described 42 . For pyrene-labeled actin, the pyrene concentration was calculated using an extinction coefficient of 21,978 M −1 cm −1 at 344 nm, and the actin concentration was calculated using the correction factor of 0.127 at 290 nm ([actin] = (A 290 − 0.127 × A 344 ) × 38 μM).…”

Role of Cytoskeletal Diaphanous-Related Formins in Hearing Loss

“…Some patients who carry truncating variants (frameshift, nonsense, or in-frame deletion) in the DAD domain present with specific hematological symptoms in addition to deafness, such as thrombocytopenia, enlarged platelets, and increased bleeding tendency [ 32 , 39 , 44 , 45 , 46 , 47 , 48 , 49 ]. Interestingly, a relationship between the extent of the DAD truncation and the hematological phenotype has been suggested [ 47 ].…”