Restoration of mutant bestrophin-1 expression, localisation and function in a polarised epithelial cell model

Abstract

Autosomal recessive bestrophinopathy (ARB) is a retinopathy caused by mutations in the bestrophin-1 protein, which is thought to function as a Ca2+-gated Cl− channel in the basolateral surface of the retinal pigment epithelium (RPE). Using a stably transfected polarised epithelial cell model, we show that four ARB mutant bestrophin-1 proteins were mislocalised and subjected to proteasomal degradation. In contrast to the wild-type bestrophin-1, each of the four mutant proteins also failed to conduct Cl− ions in transiently transfected cells as determined by whole-cell patch clamp. We demonstrate that a combination of two clinically approved drugs, bortezomib and 4-phenylbutyrate (4PBA), successfully restored the expression and localisation of all four ARB mutant bestrophin-1 proteins. Importantly, the Cl− conductance function of each of the mutant bestrophin-1 proteins was fully restored to that of wild-type bestrophin-1 by treatment of cells with 4PBA alone. The functional rescue achieved with 4PBA is significant because it suggests that this drug, which is already approved for long-term use in infants and adults, might represent a promising therapy for the treatment of ARB and other bestrophinopathies resulting from missense mutations in BEST1.

Bestrophin 1 and retinal disease

“…Contemporary discoveries and fresh avenues of research, however, have created some excitingly tangible clinical possibilities. A recent study from the Forbes Manson laboratory reported that four ARB mutants (L41P, R141H, R202W, M325T) were mislocalized to the cytoplasm, underwent proteasomal degradation, and failed to conduct Cl − anions in transfected HEK293 cells (Uggenti et al, 2016). The authors found that treatment with proteasome inhibitor4-phenylbutyrate restored the chloride conductances of these mutant proteins and that a combination treatment with 4-phenylbutyrate and a second proteasome inhibitor, bortezomib, rescued their localization back to the basolateral plasma membrane.…”

Mutation-Dependent Pathomechanisms Determine the Phenotype in the Bestrophinopathies

“…The discovery of the crystal structure of BEST1 orthologues [18,19], and the availability of electron microscopy and electrophysiological recordings revealed that the introduction of various mutations into the chicken BEST1 produces channels with dramatically altered gating properties and diminishes channel inactivation [20]. In cell culture model systems disease-causing BEST1 mutations have been reported to affect protein localization [21][22][23], stability [24][25][26], and ion gating properties of the forming anion channel [27][28][29][30][31]. Likewise, stress responses upon proteasomal or endo-lysosomal dysfunction [24,32] and dysregulation of calcium homeostasis [33] are postulated to play a role in the pathogenesis of the bestrophinopathies.…”

Familial autosomal recessive bestrophinopathy: identification of a novel variant in BEST1 gene and the specific metabolomic profile

“…Although the disease-associated nonsense and frameshift mutations predict a truncated and thus likely inactive protein, the molecular mechanism underlying the effect of missense mutation on protein function and the pathology of disease has not been well elucidated yet. Uggenti et al [12] showed that ARB-associated BEST1 protein were degradated via the ubiquitin-proteasome pathway using a stably transfected polarised epithelial cell model. Milenkovic et al [13] further proved that the ARB-associated missense mutations triggered a strong and fast protein degradation process in the endoplasmic reticulum, thereby favouring a decreased stoichiometry of mutant versus normal BEST1 subunits in the assembly of the homo-pentameric BEST1 chloride channel.…”