Structural Basis for DNA Recognition by FoxO1 and Its Regulation by Posttranslational Modification

SummaryFoxO transcription factors regulate the transcription of genes that control metabolism, cellular proliferation, stress tolerance, and possibly life span. A number of posttranslational modifications within the forkhead DNA-binding domain regulate FoxO-mediated transcription. We describe the crystal structures of FoxO1 bound to three different DNA elements and measure the change in FoxO1-DNA affinity with acetylation and phosphorylation. The structures reveal additional contacts and increased DNA distortion for the highest affinity DNA site. The flexible wing 2 region of the forkhead domain was not observed in the structures but is necessary for DNA binding, and we show that p300 acetylation in wing 2 reduces DNA affinity. We also show that MST1 phosphorylation of FoxO1 prevents high-affinity DNA binding. The observation that FoxO-DNA affinity varies between response elements and with posttranslational modifications suggests that modulation of FoxO-DNA affinity is an important component of FoxO regulation in health and misregulation in disease

Bestrophin1: A Gene that Causes Many Diseases

Bestrophinopathies are a group of clinically distinct inherited retinal dystrophies that lead to the gradual loss of vision in and around the macular area. There are no treatments for patients suffering from bestrophinopathies, and no measures can be taken to prevent visual deterioration in those who have inherited disease-causing mutations. Bestrophinopathies are caused by mutations in the Bestrophin1 gene (BEST1), a protein found exclusively in the retinal pigment epithelial (RPE) cells of the eye. Mutations in BEST1 affect the function of the RPE leading to the death of overlying retinal cells and subsequent vision loss. The pathogenic mechanisms arising from BEST1 mutations are still not fully understood, and it is not clear how mutations in BEST1 lead to diseases with distinct clinical features. This chapter discusses BEST1, the use of model systems to investigate the effects of mutations and the potential to investigate individual bestrophinopathies using induced pluripotent stem cells

Sweden: Political Developments and Data in 2019

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Underdeveloped RPE Apical Domain Underlies Lesion Formation in Canine Bestrophinopathies

Canine bestrophinopathy (cBest) is an important translational model for BEST1-associated maculopathies in man that recapitulates the broad spectrum of clinical and molecular disease aspects observed in patients. Both human and canine bestrophinopathies are characterized by focal to multifocal separations of the retina from the RPE. The lesions can be macular or extramacular, and the specific pathomechanism leading to formation of these lesions remains unclear. We used the naturally occurring canine BEST1 model to examine factors that underlie formation of vitelliform lesions and addressed the susceptibility of the macula to its primary detachment in BEST1-linked maculopathies

Mislocalisation of BEST1 in iPSC-derived retinal pigment epithelial cells from a family with autosomal dominant vitreoretinochoroidopathy (ADVIRC)

Autosomal dominant vitreoretinochoroidopathy (ADVIRC) is a rare, early-onset retinal dystrophy characterised by distinct bands of circumferential pigmentary degeneration in the peripheral retina and developmental eye defects. ADVIRC is caused by mutations in the Bestrophin1 (BEST1) gene, which encodes a transmembrane protein thought to function as an ion channel in the basolateral membrane of retinal pigment epithelial (RPE) cells. Previous studies suggest that the distinct ADVIRC phenotype results from alternative splicing of BEST1 pre-mRNA. Here, we have used induced pluripotent stem cell (iPSC) technology to investigate the effects of an ADVIRC associated BEST1 mutation (c.704T > C, p.V235A) in patient-derived iPSC-RPE. We found no evidence of alternate splicing of the BEST1 transcript in ADVIRC iPSC-RPE, however in patient-derived iPSC-RPE, BEST1 was expressed at the basolateral membrane and the apical membrane. During human eye development we show that BEST1 is expressed more abundantly in peripheral RPE compared to central RPE and is also expressed in cells of the developing retina. These results suggest that higher levels of mislocalised BEST1 expression in the periphery, from an early developmental stage, could provide a mechanism that leads to the distinct clinical phenotype observed in ADVIRC patients

The copper chaperone CCS facilitates copper binding to MEK1/2 to promote kinase activation

Normal physiology relies on the precise coordination of intracellular signaling pathways that respond to nutrient availability to balance cell growth and cell death. The canonical mitogen-activated protein kinase pathway consists of the RAFMEK- ERK signaling cascade and represents one of the most well-defined axes within eukaryotic cells to promote cell proliferation, which underscores its frequent mutational activation in human cancers. Our recent studies illuminated a function for the redox-active micronutrient copper (Cu) as an intracellular mediator of signaling by connecting Cu to the amplitude of mitogen-activated protein kinase signaling via a direct interaction between Cu and the kinases MEK1 and MEK2. Given the large quantities of molecules such as glutathione and metallothionein that limit cellular toxicity from free Cu ions, evolutionarily conserved Cu chaperones facilitate efficient delivery of Cu to cuproenzymes. Thus, a dedicated cellular delivery mechanism of Cu to MEK1/2 likely exists. Using surface plasmon resonance and proximity-dependent biotin ligase studies, we report here that the Cu chaperone for superoxide dismutase (CCS) selectively bound to and facilitated Cu transfer to MEK1. Mutants of CCS that disrupt Cu(I) acquisition and exchange or a CCS small-molecule inhibitor were used and resulted in reduced Cu-stimulated MEK1 kinase activity. Our findings indicate that the Cu chaperone CCS provides fidelity within a complex biological system to achieve appropriate installation of Cu within the MEK1 kinase active site that in turn modulates kinase activity and supports the development of novel MEK1/2 inhibitors that target the Cu structural interface or blunt dedicated Cu delivery mechanisms via CCS

Interaction of Bestrophin-1 and Ca2+ Channel β-Subunits: Identification of New Binding Domains on the Bestrophin-1 C-Terminus

Bestrophin-1 modulates currents through voltage-dependent L-type Ca2+ channels by physically interacting with the β-subunits of Ca2+ channels. The main function of β-subunits is to regulate the number of pore-forming CaV-subunits in the cell membrane and modulate Ca2+ channel currents. To understand the influence of full-length bestrophin-1 on β-subunit function, we studied binding and localization of bestrophin-1 and Ca2+ channel subunits, together with modulation of CaV1.3 Ca2+ channels currents. In heterologeous expression, bestrophin-1 showed co-immunoprecipitation with either, β3-, or β4-subunits. We identified a new highly conserved cluster of proline-rich motifs on the bestrophin-1 C-terminus between amino acid position 468 and 486, which enables possible binding to SH3-domains of β-subunits. A bestrophin-1 that lacks these proline-rich motifs (ΔCT-PxxP bestrophin-1) showed reduced efficiency to co-immunoprecipitate with β3 and β4-subunits. In the presence of ΔCT-PxxP bestrophin-1, β4-subunits and CaV1.3 subunits partly lost membrane localization. Currents from CaV1.3 subunits were modified in the presence of β4-subunit and wild-type bestrophin-1: accelerated time-dependent activation and reduced current density. With ΔCTPxxP bestrophin-1, currents showed the same time-dependent activation as with wild-type bestrophin-1, but the current density was further reduced due to decreased number of Ca2+ channels proteins in the cell membrane. In summary, we described new proline-rich motifs on bestrophin-1 C-terminus, which help to maintain the ability of β-subunits to regulate surface expression of pore-forming CaV Ca2+-channel subunits

EMSY links breast cancer gene 2 to the 'Royal Family'

Although the role of the breast cancer gene 2 (BRCA2) tumor suppressor gene is well established in inherited breast and ovarian carcinomas, its involvement in sporadic disease is still uncertain. The recent identification of a novel BRCA2 binding protein, EMSY, as a putative oncogene implicates the BRCA2 pathway in sporadic tumors. Furthermore, EMSY's binding to members of the 'Royal Family' of chromatin remodeling proteins may lead to a better understanding of the physiological function of BRCA2 and its role in chromatin remodeling

Knockout mice: Is it just genetics? Effect of enriched housing on fibulin-4+/- mice

Background. Fibulin-4 is an extracellular matrix protein expressed by vascular smooth muscle cells that is essential for maintaining arterial integrity. Fibulin-4-/- mice die just before birth due to arterial hemorrhage, but fibulin-4+/- mice appear to be outwardly normal. Experiments were therefore performed to determine whether fibulin-4+/- mice display arterial pathologies on a microscopic scale. After preliminary experiments were performed, a second purpose developed, which was to test the hypothesis that any observed pathologies would be ameliorated by housing the animals in enriched cages. Methodology. Fibulin-4+/- and wild-type mice were housed either four/cage in standard cages or two per cage in larger cages, each cage containing a tunnel and a wheel. After three weeks the mice were sacrificed, and the aortas perfusion-fixed and excised for light and electron microscopy. Principle Findings. When the mice were in standard cages, localized regions of disorganized extracellular matrix and collagen fibers consistently appeared between some of the medial smooth muscle cells in the fibulin-4+/- mice. In the wild-type mice, the smooth muscle cells were closely connected to each other and the media was more compact. The number of disorganized regions per square mm was significantly greater for fibulin-4+/- mice (172±43 (SEM)) than for wild-type mice (15±8) (p<0.01, n = 8). When the mice were in enriched cages, the fibulin-4+/- mice showed significantly fewer disorganized regions than those in standard cages (35±12) (p<0.05, n = 8). The wild type mice also showed fewer disorganized regions (3±2), but this difference was not significant. Conclusions. These results indicate that arterial pathologies manifested in fibulin-4+/- mice can be reduced by enriching the housing conditions, and imply that appropriate environments may counteract the effects of some genetic deficiencies

A mitotic function for the high-mobility group protein HMG20b regulated by its interaction with the BRC repeats of the BRCA2 tumor suppressor.

The inactivation of BRCA2, a suppressor of breast, ovarian and other epithelial cancers, triggers instability in chromosome structure and number, which are thought to arise from defects in DNA recombination and mitotic cell division, respectively. Human BRCA2 controls DNA recombination via eight BRC repeats, evolutionarily conserved motifs of ∼35 residues, that interact directly with the recombinase RAD51. How BRCA2 controls mitotic cell division is debated. Several studies by different groups report that BRCA2 deficiency affects cytokinesis. Moreover, its interaction with HMG20b, a protein of uncertain function containing a promiscuous DNA-binding domain and kinesin-like coiled coils, has been implicated in the G2-M transition. We show here that HMG20b depletion by RNA interference disturbs the completion of cell division, suggesting a novel function for HMG20b. In vitro, HMG20b binds directly to the BRC repeats of BRCA2, and exhibits the highest affinity for BRC5, a motif that binds poorly to RAD51. Conversely, the BRC4 repeat binds strongly to RAD51, but not to HMG20b. In vivo, BRC5 overexpression inhibits the BRCA2-HMG20b interaction, recapitulating defects in the completion of cell division provoked by HMG20b depletion. In contrast, BRC4 inhibits the BRCA2-RAD51 interaction and the assembly of RAD51 at sites of DNA damage, but not the completion of cell division. Our findings suggest that a novel function for HMG20b in cytokinesis is regulated by its interaction with the BRC repeats of BRCA2, and separate this unexpected function for the BRC repeats from their known activity in DNA recombination. We propose that divergent tumor-suppressive pathways regulating chromosome segregation as well as chromosome structure may be governed by the conserved BRC motifs in BRCA2