Molecular evolution and functional divergence of the bestrophin protein family

<p>Abstract</p> <p>Background</p> <p>Mutations in human bestrophin 1 are associated with at least three autosomal-dominant macular dystrophies including Best disease, adult onset vitelliform macular dystrophy and autosomal dominant vitreo-retinochoroidopathy. The protein is integral to the membrane and is likely involved in Ca²⁺-dependent transport of chloride ions across cellular membranes. Bestrophin 1 together with its three homologues forms a phylogenetically highly conserved family of proteins.</p> <p>Results</p> <p>A bioinformatics study was performed to investigate the phylogenetic relationship among the bestrophin family members and to statistically evaluate sequence conservation and functional divergence. Phylogenetic tree assembly with all available eukaryotic bestrophin sequences suggests gene duplication events in the lineage leading to the vertebrates. A common N-terminal topology which includes four highly conserved transmembrane domains is shared by the members of the four paralogous groups of vertebrate bestrophins and has been constrained by purifying selection. Pairwise comparison shows that altered functional constraints have occurred at specific amino acid positions after phylogenetic diversification of the paralogues. Most notably, significant functional divergence was found between bestrophin 4 and the other family members, as well as between bestrophin 2 and bestrophin 3. Site-specific profiles were established by posterior probability analysis revealing significantly divergent clusters mainly in two hydrophilic loops and a region immediately adjacent to the last predicted transmembrane domain. Strikingly, codons 279 and 347 of human bestrophin 4 reveal high divergence when compared to the paralogous positions strongly indicating the functional importance of these residues for the bestrophin 4 protein. None of the functionally divergent amino acids were found to reside within obvious sequences patterns or motifs.</p> <p>Conclusion</p> <p>Our study highlights the molecular evolution of the bestrophin family of transmembrane proteins and indicates amino acid residues likely relevant for distinct functional properties of the paralogues. These findings may provide a starting point for further experimental verifications.</p

Evolution and functional divergence of the anoctamin family of membrane proteins

Our study suggests that anoctamins have evolved by series of duplication events, and that they are constrained by purifying selection. In addition we identified a number of protein domains, and amino acid residues which contribute to predicted functional divergence. Hopefully, this work will facilitate future functional characterization of the anoctamin membrane protein family

Microglial Pro-Inflammatory and Anti-Inflammatory Phenotypes Are Modulated by Translocator Protein Activation

A key role of the mitochondrial Translocator Protein 18 KDa (TSPO) in neuroinflammation has been recently proposed. However, little is known about TSPO-activated pathways underlying the modulation of reactive microglia. In the present work, the TSPO activation was explored in an in vitro human primary microglia model (immortalized C20 cells) under inflammatory stimulus. Two different approaches were used with the aim to (i) pharmacologically amplify or (ii) silence, by the lentiviral short hairpin RNA, the TSPO physiological function. In the TSPO pharmacological stimulation model, the synthetic steroidogenic selective ligand XBD-173 attenuated the activation of microglia. Indeed, it reduces and increases the release of pro-inflammatory and anti-inflammatory cytokines, respectively. Such ligand-induced effects were abolished when C20 cells were treated with the steroidogenesis inhibitor aminoglutethimide. This suggests a role for neurosteroids in modulating the interleukin production. The highly steroidogenic ligand XBD-173 attenuated the neuroinflammatory response more effectively than the poorly steroidogenic ones, which suggests that the observed modulation on the cytokine release may be influenced by the levels of produced neurosteroids. In the TSPO silencing model, the reduction of TSPO caused a more inflamed phenotype with respect to scrambled cells. Similarly, during the inflammatory response, the TSPO silencing increased and reduced the release of pro-inflammatory and anti-inflammatory cytokines, respectively. In conclusion, the obtained results are in favor of a homeostatic role for TSPO in the context of dynamic balance between anti-inflammatory and pro-inflammatory mediators in the human microglia-mediated inflammatory response. Interestingly, our preliminary results propose that the TSPO expression could be stimulated by NF-kappa B during activation of the inflammatory response

Disease-associated missense mutations in bestrophin-1 affect cellular trafficking and anion conductance

Bestrophin-1, an integral membrane protein encoded by the BEST1 gene, is localized predominantly to the basolateral membrane of the retinal pigment epithelium. Mutations in the BEST1 gene have been associated with Best vitelliforme macular dystrophy (BMD), a central retinopathy with autosomal dominant inheritance and variable penetrance. Over 120 disease-causing mutations are known, the majority of which result in amino acid substitutions within four mutational hot-spot regions in the highly conserved N-terminal half of the protein. Although initially thought to impair Cl⁻ channel function, the molecular pathology of BEST1 mutations is still controversial. We have analyzed the subcellular localization of 13 disease-associated BEST1 mutant proteins in polarized MDCK II cells, an established model of apical to basolateral protein sorting. Immunostaining demonstrated that nine of the 13 mutant proteins failed to integrate into the cell membrane. The defective proteins were predominantly retained in the cytoplasm, whereas wild-type bestrophin-1 revealed cell membrane localization. Functional analysis of I⁻ fluxes in HEK-293 cells showed that all mutants exhibited a significant reduction in anion conductance. Our data indicate that defective intracellular trafficking could be a common cause of BMD accompanied by impaired anion conductance, representing a loss of anion channel function that is probably due to mistargeting of mutant protein

Impact of Translocator Protein 18 kDa (TSPO) Deficiency on Mitochondrial Function and the Inflammatory State of Human C20 Microglia Cells

Abstract Microglia are the resident immune cells of the central nervous system. Upon stimulus presentation, microglia polarize from a resting to an activated state. Microglial translocator protein 18 kDa (TSPO) is considered a marker of inflammation. Here, we characterized the role of TSPO by investigating the impact of TSPO deficiency on human microglia. We used TSPO knockout (TSPO−/−) variants of the human C20 microglia cell line. We found a significant reduction in the TSPO-associated protein VDAC1 in TSPO−/− cells compared to control cells. Moreover, we assessed the impact of TSPO deficiency on calcium levels and the mitochondrial membrane potential. Cytosolic and mitochondrial calcium concentrations were increased in TSPO−/− cell lines, whereas the mitochondrial membrane potential tended to be lower. Assessment of the mitochondrial DNA copy number via RT-PCR revealed a decreased amount of mtDNA in the TSPO−/− cells when compared to controls. Moreover, the metabolic profiles of C20 cells were strongly dependent on the glycolytic pathway. However, TSPO depletion did not affect the cellular metabolic profile. Measurement of the mRNA expression levels of the pro-inflammatory mediators revealed an attenuated response to pro-inflammatory stimuli in TSPO-depleted cells, implying a role for the TSPO protein in the process of microglial polarization

In-Depth Characterisation of Retinal Pigment Epithelium (RPE) Cells Derived from Human Induced Pluripotent Stem Cells (hiPSC)

Induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) has widely been appreciated as a promising tool to model human ocular disease emanating from primary RPE pathology. Here, we describe the successful reprogramming of adult human dermal fibroblasts to iPSCs and their differentiation to pure expandable RPE cells with structural and functional features characteristic for native RPE. Fibroblast cultures were established from skin biopsy material and subsequently reprogrammed following polycistronic lentiviral transduction with OCT4, SOX2, KLF4 and L-Myc. Fibroblast-derived iPSCs showed typical morphology, chromosomal integrity and a distinctive stem cell marker profile. Subsequent differentiation resulted in expandable pigmented hexagonal RPE cells. The cells revealed stable RNA expression of mature RPE markers RPE65, RLBP and BEST1. Immunolabelling verified localisation of BEST1 at the basolateral plasma membrane, and scanning electron microscopy showed typical microvilli at the apical side of iPSC-derived RPE cells. Transepithelial resistance was maintained at high levels during cell culture indicating functional formation of tight junctions. Secretion capacity was demonstrated for VEGF-A. Feeding of porcine photoreceptor outer segments revealed the proper ability of these cells for phagocytosis. IPSC-derived RPE cells largely maintained these properties after cryopreservation. Together, our study underlines that adult dermal fibroblasts can serve as a valuable resource for iPSC-derived RPE with characteristics highly reminiscent of true RPE cells. This will allow its broad application to establish cellular models for RPE-related human diseases

Differential effects of the translocator protein 18 kDa (TSPO) ligand etifoxine and the benzodiazepine alprazolam on startle response to predictable threat in a NPU-threat task after acute and short-term treatment

Rationale Benzodiazepines have been extensively investigated in experimental settings especially after single administration, which mostly revealed effects on unpredictable threat (U-threat) rather than predictable threat (P-threat). Given the need for pharmacological alternatives with a preferable side-effect profile and to better represent clinical conditions, research should cover also other anxiolytics and longer application times. Objectives The present study compared the acute and short-term effects of the translocator protein 18 kDa (TSPO) ligand etifoxine and the benzodiazepine alprazolam on P-threat and U-threat while controlling for sedation. Methods Sixty healthy male volunteers, aged between 18 and 55 years, were randomly assigned to receive a daily dose of either 150 mg etifoxine, 1.5 mg alprazolam, or placebo for 5 days. On days 1 and 5 of intake, they performed a NPU-threat task including neutral (N), predictable (P), and unpredictable (U) conditions, while startle responsivity and self-reports were studied. Sedative effects were assessed using a continuous performance test. Results Neither alprazolam nor etifoxine affected startle responsivity to U-threat on any of the testing days. While etifoxine reduced the startle response to P-threat on day 1 of treatment for transformed data, a contrary effect of alprazolam was found for raw values. No effects on self-reports and no evidence of sedation could be observed for either drug. Conclusions None of the anxiolytic substances had an impact on startle potentiation to U-threat even after several days of intake. The effects of the anxiolytics on startle responsivity to P-threat as well as implications for future studies are discussed

Major Depressive Disorder is Associated with Impaired Mitochondrial Function in Skin Fibroblasts

Mitochondrial malfunction is supposed to be involved in the etiology and pathology of major depressive disorder (MDD). Here, we aimed to identify and characterize the molecular pathomechanisms related to mitochondrial dysfunction in adult human skin fibroblasts, which were derived from MDD patients or non-depressive control subjects. We found that MDD fibroblasts showed significantly impaired mitochondrial functioning: basal and maximal respiration, spare respiratory capacity, non-mitochondrial respiration and adenosine triphosphate (ATP)-related oxygen consumption was lower. Moreover, MDD fibroblasts harbor lower ATP levels and showed hyperpolarized mitochondrial membrane potential. To investigate cellular resilience, we challenged both groups of fibroblasts with hormonal (dexamethasone) or metabolic (galactose) stress for one week, and found that both stressors increased oxygen consumption but lowered ATP content in MDD as well as in non-depressive control fibroblasts. Interestingly, the bioenergetic differences between fibroblasts from MDD or non-depressed subjects, which were observed under non-treated conditions, could not be detected after stress. Our findings support the hypothesis that altered mitochondrial function causes a bioenergetic imbalance, which is associated with the molecular pathophysiology of MDD. The observed alterations in the oxidative phosphorylation system (OXPHOS) and other mitochondria-related properties represent a basis for further investigations of pathophysiological mechanisms and might open new ways to gain insight into antidepressant signaling pathways

CRISPR-Cas9 Mediated TSPO Gene Knockout alters Respiration and Cellular Metabolism in Human Primary Microglia Cells

The 18 kDa translocator protein (TSPO) is an evolutionary conserved cholesterol binding protein localized in the outer mitochondrial membrane. It has been implicated in the regulation of various cellular processes including oxidative stress, proliferation, apoptosis, and steroid hormone biosynthesis. Since the expression of TSPO in activated microglia is upregulated in various neuroinflammatory and neurodegenerative disorders, we set out to examine the role of TSPO in an immortalized human microglia C20 cell line. To this end, we performed a dual approach and used (i) lentiviral shRNA silencing to reduce TSPO expression, and (ii) the CRISPR/Cas9 technology to generate complete TSPO knockout microglia cell lines. Functional characterization of control and TSPO knockdown as well as knockout cells, revealed only low de novo steroidogenesis in C20 cells, which was not dependent on the level of TSPO expression or influenced by the treatment with TSPO-specific ligands. In contrast to TSPO knockdown C20 cells, which did not show altered mitochondrial function, the TSPO deficient knockout cells displayed a significantly decreased mitochondrial membrane potential and cytosolic Ca2+ levels, as well as reduced respiratory function. Performing the rescue experiment by lentiviral overexpression of TSPO in knockout cells, increased oxygen consumption and restored respiratory function. Our study provides further evidence for a significant role of TSPO in cellular and mitochondrial metabolism and demonstrates that different phenotypes of mitochondrial function are dependent on the level of TSPO expression

Effects of genetic variants in the TSPO gene on protein structure and stability

The 18 kDa translocator protein (TSPO) is an evolutionary conserved cholesterol binding protein localized in the outer mitochondrial membrane. Expression of TSPO is upregulated in activated microglia in various neuroinflammatory, neurodegenerative, and neoplastic disorders. Therefore, TSPO radioligands are used as biomarkers in positron emission tomography (PET) studies. In particular, a common A147T polymorphism in the TSPO gene affects binding of several high affinity TSPO radioligands. Given the relevance of TSPO as a diagnostic biomarker in disease processes, we systematically searched for mutations in the human TSPO gene by a wide array of evolution and structure based bioinformatics tools and identified potentially deleterious missense mutations. The two most frequently observed missense mutations A147T and R162H were further analysed in structural models of human wildtype and mutant TSPO proteins. The effects of missense mutations were studied on the atomic level using molecular dynamics simulations. To analyse putative effects of A147T and R162H variants on protein stability we established primary dermal fibroblast cultures from wt and homozygous A147T and R162H donors. Stability of endogenous TSPO protein, which is abundantly expressed in fibroblasts, was studied using cycloheximide protein degradation assay. Our data show that the A147T mutation significantly alters the flexibility and stability of the mutant protein. Furthermore both A147T and R162H mutations decreased the half-life of the mutant proteins by about 25 percent, which could in part explain its effect on reduced pregnenolone production and susceptibility to neuropsychiatric disorders. The present study is the first comprehensive bioinformatic analysis of genetic variants in the TSPO gene, thereby extending the knowledge about the clinical relevance of TSPO nsSNPs