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We previously reported a model of progressive retinal degeneration resulting from the knockout of the retina-specific riboflavin binding protein, retbindin (Rtbdn-/-). We also demonstrated a reduction in neural retinal flavins as a result of the elimination of RTBDN. Given the role of flavins in metabolism, herein we investigated the underlying mechanism of this retinal degeneration by performing metabolomic analyses on predegeneration at postnatal day (P) 45 and at the onset of functional degeneration in the P120 retinas. Metabolomics of hydrophilic metabolites revealed that individual glycolytic products accumulated in the P45 Rtbdn-/- neural retinas along with the elevation of pentose phosphate pathway, while TCA cycle intermediates remained unchanged. This was confirmed by using 13C-labeled flux measurements and immunoblotting, revealing that the key regulatory step of phosphoenolpyruvate to pyruvate was inhibited via down-regulation of the tetrameric pyruvate kinase M2 (PKM2). Separate metabolite assessments revealed that almost all intermediates of acylcarnitine fatty acid oxidation, ceramides, sphingomyelins, and multiple toxic metabolites were significantly elevated in the predegeneration Rtbdn-/- neural retina. Our data show that lack of RTBDN, and hence reduction in flavins, forced the neural retina into repurposing glucose for free-radical mitigation over ATP production. However, such sustained metabolic reprogramming resulted in an eventual metabolic collapse leading to neurodegeneration. [ABSTRACT FROM AUTHOR]

Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulationmechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit. [ABSTRACT FROM AUTHOR]

Flavin-binding LOV domains are blue-light photosensory modules that are conserved in a number of developmental and circadian regulatory proteins in plants, algae, and fungi. LOV domains are also present in bacterial genomes, and are commonly located at the amino termini of sensor histidine kinases. Genes predicted to encode LOV-histidine kinases are conserved across a broad range of bacterial taxa, from aquatic oligotrophs to plant and mammalian pathogens. However, the function of these putative prokaryotic photoreceptors remains largely undefined. The differentiating bacterium, Caulobactercrescentus, contains an operon encoding a two-component signaling system consisting of a LOV-histidine kinase, LovK, and a single-domain response regulator, LovR. LovK binds a flavin cofactor, undergoes a reversible photocycle, and displays increased ATPase and autophosphorylation activity in response to visible light. Deletion of the response regulator gene, IovR, results in severe attenuation of cell attachment to a glass surface under laminar flow, whereas coordinate, low-level over- expression of IovK and IovR results in a light-independent increase in cell-cell attachment, a response that requires both the con- served histidine phosphorylation site in LovK and aspartate phos- phorylation site in LovR. Growing C. crescentus in the presence of blue light dramatically enhances cell-cell attachment in the IovK- IovR overexpression background. A conserved cysteine residue in the LOV domain of LovK, which forms a covalent adduct with the flavin cofactor upon absorption of visible light, is necessary for the light-dependent regulation of LovK enzyme activity and is required for the light-dependent enhancement of intercellular attachment. [ABSTRACT FROM AUTHOR]

Plants sense their environmental light conditions by using three photoreceptors that absorb in the UV, blue/near UV, and red/far-red spectral ranges. These photoreceptors have specific chromophore components corresponding to their absorption spectra. Phytochrome, a red/far-red light receptor, has phytochromobilin as its chromophore, whereas the blue/near UV photoreceptors cryptochrome and phototropin have a pair of flavin derivatives. Plants use these various photoreceptors to assess the surrounding light environment. Phytochrome 3 (PHY3) is a red light receptor found in some ferns, which preferentially grow under weak light. PHY3 is composed of a phytochrome chromophore-binding domain in its N-terminal portion and an almost full-length phototropin in its C-terminal half. This unusual domain organization implies that two different light-sensing systems coexist in this single photoreceptor, although these light-sensing systems usually reside in independent photoreceptors. Here, we show that PHY3 acts as a dual-channel photoreceptor that possesses both the red light-sensing system of phytochrome and the blue light-sensing system of phototropin. Furthermore, red- and blue-light signals perceived by PHY3 are processed synergistically within this single chromoprotein. These unusual properties might confer an enhanced light sensitivity on PHY3, allowing ferns to grow under a low-light canopy. [ABSTRACT FROM AUTHOR]