Peropsin modulates transit of vitamin A from retina to retinal pigment epithelium.

Peropsin is a non-visual opsin in both vertebrate and invertebrate species. In mammals, peropsin is present in the apical microvilli of retinal pigment epithelial (RPE) cells. These structures interdigitate with the outer segments of rod and cone photoreceptor cells. RPE cells play critical roles in the maintenance of photoreceptors, including the recycling of visual chromophore for the opsin visual pigments. Here, we sought to identify the function of peropsin in the mouse eye. To this end, we generated mice with a null mutation in the peropsin gene (Rrh). These mice exhibited normal retinal histology, normal morphology of outer segments and RPE cells, and no evidence of photoreceptor degeneration. Biochemically, Rrh-/- mice had ∼2-fold higher vitamin A (all-trans-retinol (all-trans-ROL)) in the neural retina following a photobleach and 5-fold lower retinyl esters in the RPE. This phenotype was similar to those reported in mice that lack interphotoreceptor retinoid-binding protein (IRBP) or cellular retinol-binding protein, suggesting that peropsin plays a role in the movement of all-trans-ROL from photoreceptors to the RPE. We compared the phenotypes in mice lacking both peropsin and IRBP with those of mice lacking peropsin or IRBP alone and found that the retinoid phenotype was similarly severe in each of these knock-out mice. We conclude that peropsin controls all-trans-ROL movement from the retina to the RPE or may regulate all-trans-ROL storage within the RPE. We propose that peropsin affects light-dependent regulation of all-trans-ROL uptake from photoreceptors into RPE cells through an as yet undefined mechanism.

Identification of the 11-cis-specific retinyl-ester synthase in retinal Müller cells as multifunctional O-acyltransferase (MFAT).

Absorption of a photon by a rhodopsin or cone-opsin pigment isomerizes its 11-cis-retinaldehyde (11-cis-RAL) chromophore to all-trans-retinaldehyde (all-trans-RAL), which dissociates after a brief period of activation. Light sensitivity is restored to the resulting apo-opsin when it recombines with another 11-cis-RAL. Conversion of all-trans-RAL to 11-cis-RAL is carried out by an enzyme pathway called the visual cycle in cells of the retinal pigment epithelium. A second visual cycle is present in Müller cells of the retina. The retinol isomerase for this noncanonical pathway is dihydroceramide desaturase (DES1), which catalyzes equilibrium isomerization of retinol. Because 11-cis-retinol (11-cis-ROL) constitutes only a small fraction of total retinols in an equilibrium mixture, a subsequent step involving selective removal of 11-cis-ROL is required to drive synthesis of 11-cis-retinoids for production of visual chromophore. Selective esterification of 11-cis-ROL is one possibility. Crude homogenates of chicken retinas rapidly convert all-trans-ROL to 11-cis-retinyl esters (11-cis-REs) with minimal formation of other retinyl-ester isomers. This enzymatic activity implies the existence of an 11-cis-specific retinyl-ester synthase in Müller cells. Here, we evaluated multifunctional O-acyltransferase (MFAT) as a candidate for this 11-cis-RE-synthase. MFAT exhibited much higher catalytic efficiency as a synthase of 11-cis-REs versus other retinyl-ester isomers. Further, we show that MFAT is expressed in Müller cells. Finally, homogenates of cells coexpressing DES1 and MFAT catalyzed the conversion of all-trans-ROL to 11-cis-RP, similar to what we observed with chicken-retina homogenates. MFAT is therefore an excellent candidate for the retinyl-ester synthase that cooperates with DES1 to drive synthesis of 11-cis-retinoids by mass action.

Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly.

Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone in the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to improve our understanding of the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry. Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into an Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly.

Membrane lipids influence protein complex assembly-disassembly.

Approximately 11% smaller t-/v-SNARE ring complexes are generated using 50 nm cholesterol-associated vesicles as opposed to vesicles containing L-alpha-lysophosphatidylcholine (LPC), as observed using atomic force microscopy. Circular dichroism spectroscopy demonstrated that in the presence of LPC as opposed to cholesterol, N-ethylmaleimide-sensitive factor + adenosine triphosphate induces disassembly of beta-sheet structures but not the alpha-helical contents within the t-/v-SNARE complex.

Evolution of metal(loid) binding sites in transcriptional regulators.

Expression of the genes for resistance to heavy metals and metalloids is transcriptionally regulated by the toxic ions themselves. Members of the ArsR/SmtB family of small metalloregulatory proteins respond to transition metals, heavy metals, and metalloids, including As(III), Sb(III), Cd(II), Pb(II), Zn(II), Co(II), and Ni(II). These homodimeric repressors bind to DNA in the absence of inducing metal(loid) ion and dissociate from the DNA when inducer is bound. The regulatory sites are often three- or four-coordinate metal binding sites composed of cysteine thiolates. Surprisingly, in two different As(III)-responsive regulators, the metalloid binding sites were in different locations in the repressor, and the Cd(II) binding sites were in two different locations in two Cd(II)-responsive regulators. We hypothesize that ArsR/SmtB repressors have a common backbone structure, that of a winged helix DNA-binding protein, but have considerable plasticity in the location of inducer binding sites. Here we show that an As(III)-responsive member of the family, CgArsR1 from Corynebacterium glutamicum, binds As(III) to a cysteine triad composed of Cys(15), Cys(16), and Cys(55). This binding site is clearly unrelated to the binding sites of other characterized ArsR/SmtB family members. This is consistent with our hypothesis that metal(loid) binding sites in DNA binding proteins evolve convergently in response to persistent environmental pressures.

Structure and dynamics of metalloproteins in live cells.

X-ray absorption spectroscopy (XAS) has emerged as one of the premier tools for investigating the structure and dynamic properties of metals in cells and in metal containing biomolecules. Utilizing the high flux and broad energy range of X-rays supplied by synchrotron light sources, one can selectively excite core electronic transitions in each metal. Spectroscopic signals from these electronic transitions can be used to dissect the chemical architecture of metals in cells, in cellular components, and in biomolecules at varying degrees of structural resolution. With the development of ever-brighter X-ray sources, X-ray methods have grown into applications that can be utilized to provide both a cellular image of the relative distribution of metals throughout the cell as well as a high-resolution picture of the structure of the metal. As these techniques continue to grow in their capabilities and ease of use, so too does the demand for their application by chemists and biochemists interested in studying the structure and dynamics of metals in cells, in cellular organelles, and in metalloproteins.

Circular dichroism (CD) spectroscopy of the assembly and disassembly of SNAREs: The proteins involved in membrane fusion in cells.

In this study, we report for the first time that both t-SNAREs and v-SNARE and their complexes in buffered suspension, exhibit defined peaks at CD signals of 208 and 222 nm wavelengths, consistent with a higher degree of helical secondary structure. Surprisingly, when incorporated in lipid membrane, both SNAREs and their complexes exhibit reduced folding. In presence of NSF-ATP, the SNARE complex disassembles, as reflected from the CD signals demonstrating elimination of α-helices within the structure.

The structure and function of frataxin.

Frataxin, a highly conserved protein found in prokaryotes and eukaryotes, is required for efficient regulation of cellular iron homeostasis. Humans with a frataxin deficiency have the cardio- and neurodegenerative disorder Friedreich's ataxia, commonly resulting from a GAA trinucleotide repeat expansion in the frataxin gene. While frataxin's specific function remains a point of controversy, the general consensus is that the protein assists in controlling cellular iron homeostasis by directly binding iron. This review focuses on the structural and biochemical aspects of iron binding by the frataxin orthologs and outlines molecular attributes that may help explain the protein's role in different cellular pathways.

Monomeric yeast frataxin is an iron-binding protein.

Friedreich's ataxia, an autosomal cardio- and neurodegenerative disorder that affects 1 in 50,000 humans, is caused by decreased levels of the protein frataxin. Although frataxin is nuclear-encoded, it is targeted to the mitochondrial matrix and necessary for proper regulation of cellular iron homeostasis. Frataxin is required for the cellular production of both heme and iron-sulfur (Fe-S) clusters. Monomeric frataxin binds with high affinity to ferrochelatase, the enzyme involved in iron insertion into porphyrin during heme production. Monomeric frataxin also binds to Isu, the scaffold protein required for assembly of Fe-S cluster intermediates. These processes (heme and Fe-S cluster assembly) share requirements for iron, suggesting that monomeric frataxin might function as the common iron donor. To provide a molecular basis to better understand frataxin's function, we have characterized the binding properties and metal-site structure of ferrous iron bound to monomeric yeast frataxin. Yeast frataxin is stable as an iron-loaded monomer, and the protein can bind two ferrous iron atoms with micromolar binding affinity. Frataxin amino acids affected by the presence of iron are localized within conserved acidic patches located on the surfaces of both helix-1 and strand-1. Under anaerobic conditions, bound metal is stable in the high-spin ferrous state. The metal-ligand coordination geometry of both metal-binding sites is consistent with a six-coordinate iron-(oxygen/nitrogen) based ligand geometry, surely constructed in part from carboxylate and possibly imidazole side chains coming from residues within these conserved acidic patches on the protein. On the basis of our results, we have developed a model for how we believe yeast frataxin interacts with iron.