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.

Alterations to the broad-spectrum formin inhibitor SMIFH2 improve potency

“…41 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). Extinction coefficients for formins at 280 nm were calculated using Expasy ProtParam 43 : DIAPH1 (22,920 M −1 cm −1 ), DIAPH2 (25,900 M −1 cm −1 ), FMNL3 (28,420 M −1 cm −1 ), FMN2 (36,900 M −1 cm −1 ), Delphilin (25,440 M −1 cm −1 ), INF2 (47,440 M −1 cm −1 ), and DAAM2 (29,910 M −1 cm −1 ).…”

Metallothionein-3 attenuates the effect of Cu2+ ions on actin filaments

“…A non-GIF mutant (Pro7Ser/Pro9Ala) and a phospho-mimetic mutant (Ser33Asp) were both constructed by PCR-based mutagenesis [41] (DNA sequences in SI) and purified using the same protocol as WT MT-3 [34]. Rabbit skeletal muscle actin from acetone powder ( Pel-Freez Biologicals ) and recombinant S. pombe profilin were purified, and actin was labeled as previously described [42].…”

“…Bulk actin assembly assays were carried out as described [42], with a few changes. EGTA was excluded from the polymerization assays to avoid metal chelation.…”