Phosphorylation Code of Human Nucleophosmin Includes Four Cryptic Sites for Hierarchical Binding of 14-3-3 Proteins.

Nucleophosmin (NPM1) is the 46th most abundant human protein with many functions whose dysregulation leads to various cancers. Pentameric NPM1 resides in the nucleolus but can also shuttle to the cytosol. NPM1 is regulated by multisite phosphorylation, yet molecular consequences of site-specific NPM1 phosphorylation remain elusive. Here we identify four 14-3-3 protein binding sites in NPM1 concealed within its oligomerization and α-helical C-terminal domains that are found phosphorylated in vivo. By combining mutagenesis, in-cell phosphorylation and PermaPhos technology for site-directed incorporation of a non-hydrolyzable phosphoserine mimic, we show how phosphorylation promotes NPM1 monomerization and partial unfolding, to recruit 14-3-3 dimers with low-micromolar affinity. Using fluorescence anisotropy we quantified pairwise interactions of all seven human 14-3-3 isoforms with four recombinant NPM1 phosphopeptides and assessed their druggability by fusicoccin. This revealed a complex hierarchy of 14-3-3 affinities toward the primary (S48, S293) and secondary (S106, S260) sites, differentially modulated by the small molecule. As three of these 14-3-3 binding phosphosites in NPM1 reside within signal sequences, this work suggests a mechanism of NPM1 regulation by which NPM1 phosphorylation can promote 14-3-3 binding to affect NPM1 shuttling between cell compartments. It also provides further evidence that phosphorylation-induced structural rearrangements of globular proteins serve to expose otherwise cryptic 14-3-3-binding sites that are important for cellular function.

Multimodal non-invasive probing of stress-induced carotenogenesis in the cells of microalga Bracteacoccus aggregatus.

Microalgae are the richest source of natural carotenoids-accessory photosynthetic pigments used as natural antioxidants, safe colorants, and nutraceuticals. Microalga Bracteacoccus aggregatus IPPAS C-2045 responds to stresses, including high light, with carotenogenesis-gross accumulation of secondary carotenoids (the carotenoids structurally and energetically uncoupled from photosynthesis). Precise mechanisms of cytoplasmic transport and subcellular distribution of the secondary carotenoids under stress are still unknown. Using multimodal imaging combining micro-Raman imaging (MRI), fluorescent lifetime (τ) imaging (FLIM), and transmission electron microscopy (TEM), we monitored ultrastructural and biochemical rearrangements of B. aggregatus cells during the stress-induced carotenogenesis. MRI revealed a decline in the diversity of molecular surrounding of the carotenoids in the cells compatible with the relocation of the bulk of the carotenoids in the cell from functionally and structurally heterogeneous photosynthetic apparatus to the more homogenous lipid matrix of the oleosomes. Two-photon FLIM highlighted the pigment transformation in the cell during the stress-induced carotenogenesis. The structures co-localized with the carotenoids with shorter τ (mainly chloroplast) shrunk, whereas the structures harboring secondary carotenoids with longer τ (mainly oleosomes) expanded. These changes were in line with the ultrastructural data (TEM). Fluorescence of B. aggregatus carotenoids, either in situ or in acetone extracts, possessed a surprisingly long lifetime. We hypothesize that the extension of τ of the carotenoids is due to their aggregation and/or association with lipids and proteins. The propagation of the carotenoids with prolonged τ is considered to be a manifestation of the secondary carotenogenesis suitable for its non-invasive monitoring with multimodal imaging.

Insights into the molecular mechanism of yellow cuticle coloration by a chitin-binding carotenoprotein in gregarious locusts.

Carotenoids are hydrophobic pigments binding to diverse carotenoproteins, many of which remain unexplored. Focusing on yellow gregarious locusts accumulating cuticular carotenoids, here we use engineered Escherichia coli cells to reconstitute a functional water-soluble ß-carotene-binding protein, BBP. HPLC and Raman spectroscopy confirmed that recombinant BBP avidly binds ß-carotene, inducing the unusual vibronic structure of its absorbance spectrum, just like native BBP extracted from the locust cuticles. Bound to recombinant BBP, ß-carotene exhibits pronounced circular dichroism and allows BBP to withstand heating (T0.5 = 68 °C), detergents and pH variations. Using bacteria producing distinct xanthophylls we demonstrate that, while ß-carotene is the preferred carotenoid, BBP can also extract from membranes ketocarotenoids and, very poorly, hydroxycarotenoids. We show that BBP-carotenoid complex reversibly binds to chitin, but not to chitosan, implying the role for chitin acetyl groups in cuticular BBP deposition. Reconstructing such locust coloration mechanism in vitro paves the way for structural studies and BBP applications.

DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity.

Two prokaryotic defence systems, prokaryotic Argonautes (pAgos) and CRISPR-Cas, detect and cleave invader nucleic acids using complementary guides and the nuclease activities of pAgo or Cas proteins. However, not all pAgos are active nucleases. A large clade of short pAgos bind nucleic acid guides but lack nuclease activity, suggesting a different mechanism of action. Here we investigate short pAgos associated with a putative effector nuclease, NbaAgo from Novosphingopyxis baekryungensis and CmeAgo from Cupriavidus metallidurans. We show that these pAgos form a heterodimeric complex with co-encoded effector nucleases (short prokaryotic Argonaute, DNase and RNase associated (SPARDA)). RNA-guided target DNA recognition unleashes the nuclease activity of SPARDA leading to indiscriminate collateral cleavage of DNA and RNA. Activation of SPARDA by plasmids or phages results in degradation of cellular DNA and cell death or dormancy, conferring target-specific population protection and expanding the range of known prokaryotic immune systems.

Elements of the C-terminal tail of a C-terminal domain homolog of the Orange Carotenoid Protein determining xanthophyll uptake from liposomes.

Carotenoids perform multifaceted roles in life ranging from coloration over light harvesting to photoprotection. The Orange Carotenoid Protein (OCP), a light-driven photoswitch involved in cyanobacterial photoprotection, accommodates a ketocarotenoid vital for its function. OCP extracts its ketocarotenoid directly from membranes, or accepts it from homologs of its C-terminal domain (CTDH). The CTDH from Anabaena (AnaCTDH) was shown to be important for carotenoid transfer and delivery from/to membranes. The C-terminal tail of AnaCTDH is a critical structural element likely serving as a gatekeeper and facilitator of carotenoid uptake from membranes. We investigated the impact of amino acid substitutions within the AnaCTDH-CTT on echinenone and canthaxanthin uptake from DOPC and DMPG liposomes. The transfer rate was uniformly reduced for substitutions of Arg-137 and Arg-138 to Gln or Ala, and depended on the lipid type, indicating a weaker interaction particularly with the lipid head group. Our results further suggest that Glu-132 has a membrane-anchoring effect on the PC lipids, specifically at the choline motif as inferred from the strongly different effects of the CTT variants on the extraction from the two liposome types. The substitution of Pro-130 by Gly suggests that the CTT is perpendicular to both the membrane and the main AnaCTDH protein during carotenoid extraction. Finally, the simultaneous mutation of Leu-133, Leu-134 and Leu-136 for alanines showed that the hydrophobicity of the CTT is crucial for carotenoid uptake. Since some substitutions accelerated carotenoid transfer into AnaCTDH while others slowed it down, carotenoprotein properties can be engineered toward the requirements of applications.

Phosphorylation code of human nucleophosmin includes four cryptic sites for hierarchical binding of 14-3-3 proteins.

Nucleophosmin (NPM1) is the 46th most abundant human protein with many functions whose dysregulation leads to various cancers. Pentameric NPM1 resides in the nucleolus but can also shuttle to the cytosol. NPM1 is regulated by multisite phosphorylation, yet molecular consequences of site-specific NPM1 phosphorylation remain elusive. Here we identify four 14-3-3 protein binding sites in NPM1 concealed within its oligomerization and α-helical C-terminal domains that are found phosphorylated in vivo. By combining mutagenesis, in-cell phosphorylation and PermaPhos technology for site-directed incorporation of a non-hydrolyzable phosphoserine mimic, we show how phosphorylation promotes NPM1 monomerization and partial unfolding, to recruit 14-3-3 dimers with low-micromolar affinity. Using fluorescence anisotropy we quantified pairwise interactions of all seven human 14-3-3 isoforms with four recombinant NPM1 phosphopeptides and assessed their druggability by fusicoccin. This revealed a complex hierarchy of 14-3-3 affinities toward the primary (S48, S293) and secondary (S106, S260) sites, differentially modulated by the small molecule. As three of these 14-3-3 binding phospho-sites in NPM1 reside within signal sequences, this work highlights a key mechanism of NPM1 regulation by which NPM1 phosphorylation promotes 14-3-3 binding to control nucleocytoplasmic shuttling. It also provides further evidence that phosphorylation-induced structural rearrangements of globular proteins serve to expose otherwise cryptic 14-3-3-binding sites that are important for cellular function.

Structural framework for the understanding spectroscopic and functional signatures of the cyanobacterial Orange Carotenoid Protein families.

The Orange Carotenoid Protein (OCP) is a unique photoreceptor crucial for cyanobacterial photoprotection. Best studied Synechocystis sp. PCC 6803 OCP belongs to the large OCP1 family. Downregulated by the Fluorescence Recovery Protein (FRP) in low-light, high-light-activated OCP1 binds to the phycobilisomes and performs non-photochemical quenching. Recently discovered families OCP2 and OCP3 remain structurally and functionally underexplored, and no systematic comparative studies have ever been conducted. Here we present two first crystal structures of OCP2 from morphoecophysiologically different cyanobacteria and provide their comprehensive structural, spectroscopic and functional comparison with OCP1, the recently described OCP3 and all-OCP ancestor. Structures enable correlation of spectroscopic signatures with the effective number of hydrogen and discovered here chalcogen bonds anchoring the ketocarotenoid in OCP, as well as with the rotation of the echinenone's ß-ionone ring in the CTD. Structural data also helped rationalize the observed differences in OCP/FRP and OCP/phycobilisome functional interactions. These data are expected to foster OCP research and applications in optogenetics, targeted carotenoid delivery and cyanobacterial biomass engineering.

Interaction of the C-terminal immunoglobulin-like domains (Ig 22-24) of filamin C with human small heat shock proteins.

Small heat shock proteins are the well-known regulators of the cytoskeleton integrity, yet their complexes with actin-binding proteins are underexplored. Filamin C, a dimeric 560 kDa protein, abundant in cardiac and skeletal muscles, crosslinks actin filaments and contributes to Z-disc formation and membrane-cytoskeleton attachment. Here, we analyzed the interaction of a human filamin C fragment containing immunoglobulin-like domains 22-24 (FLNC22-24) with five small heat shock proteins (HspB1, HspB5, HspB6, HspB7, HspB8) and their α-crystallin domains. On size-exclusion chromatography, only HspB7 or its α-crystallin domain formed complexes with FLNC22-24. Despite similar isoelectric points of the small heat shock proteins analyzed, only HspB7 and its α-crystallin domain interacted with FLNC22-24 on native gel electrophoresis. Crosslinking with glutaraldehyde confirmed the formation of complexes between HspB7 (or its α-crystallin domain) and the filamin С fragment, inhibiting intersubunit FLNC crosslinking. These data are consistent with the structure modeling using Alphafold. Thus, the C-terminal fragment (immunoglobulin-like domains 22-24) of filamin C contains the site for HspB7 (or its α-crystallin domain) interaction, which competes with FLNC22-24 dimerization and its probable interaction with different target proteins.

Anti-stokes fluorescence of phycobilisome and its complex with the orange carotenoid protein.

Phycobilisomes (PBSs) are giant water-soluble light-harvesting complexes of cyanobacteria and red algae, consisting of hundreds of phycobiliproteins precisely organized to deliver the energy of absorbed light to chlorophyll chromophores of the photosynthetic electron-transport chain. Quenching the excess of excitation energy is necessary for the photoprotection of photosynthetic apparatus. In cyanobacteria, quenching of PBS excitation is provided by the Orange Carotenoid Protein (OCP), which is activated under high light conditions. In this work, we describe parameters of anti-Stokes fluorescence of cyanobacterial PBSs in quenched and unquenched states. We compare the fluorescence readout from entire phycobilisomes and their fragments. The obtained results revealed the heterogeneity of conformations of chromophores in isolated phycobiliproteins, while such heterogeneity was not observed in the entire PBS. Under excitation by low-energy quanta, we did not detect a significant uphill energy transfer from the core to the peripheral rods of PBS, while the one from the terminal emitters to the bulk allophycocyanin chromophores is highly probable. We show that this direction of energy migration does not eliminate fluorescence quenching in the complex with OCP. Thus, long-wave excitation provides new insights into the pathways of energy conversion in the phycobilisome.

Lipid composition and properties affect protein-mediated carotenoid uptake efficiency from membranes.

Carotenoids are pigments of diverse functions ranging from coloration over light-harvesting to photoprotection. Yet, the number of carotenoid-binding proteins, which mobilize these pigments in physiological media, is limited, and the mechanisms of carotenoid mobilization are still not well understood. The same applies for the determinants of carotenoid uptake from membranes into carotenoproteins, especially regarding the dependence on the chemical properties of membrane lipids. Here, we investigate xanthophyll uptake capacity and kinetics of a paradigmatic carotenoid-binding protein, the homolog of the Orange Carotenoid Protein's C-terminal domain from Anabaena sp. PCC 7120 (AnaCTDH), using liposomes formed from defined lipid species and loaded with canthaxanthin (CAN) and echinenone (ECN), respectively. Phospholipids with different chain length and degree of saturation were investigated. The composition of carotenoid-loaded liposomes directly affected the incorporation yield and storage ratio of CAN and ECN as well as the rate of carotenoid uptake by AnaCTDH. Generally, saturated PC lipids were identified as unsuitable, and a high phase transition temperature of the lipids negatively affected the carotenoid incorporation and storage yield. For efficient carotenoid transfer, the velocity increases with increasing chain length or membrane thickness. An average transfer yield of 93 % and 43 % were obtained for the formation of AnaCTDH(CAN) and AnaCTDH(ECN) holoproteins, respectively. In summary, the most suitable lipids for the formation of AnaCTDH(CAN/ECN) holoproteins by carotenoid transfer from artificial liposomes are phosphatidylcholine (18:1) and phosphatidylglycerol (14:0). Thus, these two lipids provide the best conditions for further investigation of lipid-protein interaction and the carotenoid uptake process.

Crystal structure reveals canonical recognition of the phosphorylated cytoplasmic loop of human alpha7 nicotinic acetylcholine receptor by 14-3-3 protein.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels composed of five homologous subunits. The homopentameric α7-nAChR, abundantly expressed in the brain, is involved in the regulation of the neuronal plasticity and memory and undergoes phosphorylation by protein kinase A (PKA). Here, we extracted native α7-nAChR from murine brain, validated its assembly by cryo-EM and showed that phosphorylation by PKA in vitro enables its interaction with the abundant human brain protein 14-3-3ζ. Bioinformatic analysis narrowed the putative 14-3-3-binding site down to the fragment of the intracellular loop (ICL) containing Ser365 (Q361RRCSLASVEMS372), known to be phosphorylated in vivo. We reconstructed the 14-3-3ζ/ICL peptide complex and determined its structure by X-ray crystallography, which confirmed the Ser365 phosphorylation-dependent canonical recognition of the ICL by 14-3-3. A common mechanism of nAChRs' regulation by ICL phosphorylation and 14-3-3 binding that potentially affects nAChR activity, stoichiometry, and surface expression is suggested.

Intrinsic tryptophan fluorescence quenching by iodine in non-canonical amino acid reveals alteration of the hydrogen bond network in the photoactive orange carotenoid protein.

The Orange Carotenoid Protein (OCP) regulates cyanobacterial photosynthetic activity through photoactivation in intense light. A hydrogen bonding network involving the keto-carotenoid oxygen and Y201 and W288 residues prevents the spontaneous activation of dark-adapted OCP. To investigate the role of the hydrogen bonds in OCP photocycling, we introduced non-canonical amino acids near the keto-carotenoid, particularly iodine at the meta-position of Y201. This modification significantly increased the yield of red OCP photoproducts, albeit with a shorter lifetime. Changes in tryptophan fluorescence during photocycling influenced by the presence of iodine near W288 revealed interactions between Y201 and W288 in the absence of the carotenoid in the C-domain. We propose that upon the relaxation of red states, a ternary complex with the carotenoid is formed. Analysis of spectral signatures and interaction energies indicates that the specific iodo-tyrosine configuration enhances interactions between the carotenoid and W288.

Development of Heterologous Expression System and Optimization of the Method of Cholera Toxin ß-Subunit Production in E. coli.

Cholera is a deadly infection disease, which is usually associated with low hygiene levels and limited access to high-quality drinking water. An effective way to prevent cholera is the use of vaccines. Among active vaccine components there is the CtxB protein (cholera toxin ß-subunit). In the current work, we have developed a genetic system for production of the recombinant CtxB in E. coli cells and studied conditions for synthesis and purification of the target product at the laboratory scale. It has been found that the optimal algorithm for isolation of the recombinant protein is to grow E. coli culture in the synthetic M9 medium with glycerol, followed by CtxB purification out of the spent culture medium using Ni2+-chelate affinity chromatography techniques. Forty-eight hours after induction of CtxB expression, concentration of the target product could be up to 50 mg/liter in the culture medium. The CtxB protein retains its pentameric structure during expression and through purification. The latter makes it possible to consider the developed system as a promising tool for the industrial-level production of recombinant CtxB for medical and research purposes.

Autonomous Synthesis of Functional, Permanently Phosphorylated Proteins for Defining the Interactome of Monomeric 14-3-3ζ.

14-3-3 proteins are dimeric hubs that bind hundreds of phosphorylated "clients" to regulate their function. Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study 14-3-3 function in cellular-like environments, but a previous genetic code expansion (GCE) system to translationally install nonhydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with CH2, site-specifically into proteins has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into Escherichia coli a six-step biosynthetic pathway that produces nhpSer from phosphoenolpyruvate. Using this autonomous "PermaPhos" expression system, we produce three biologically relevant proteins with nhpSer and confirm that nhpSer mimics the effects of phosphoserine for activating GSK3ß phosphorylation of the SARS-CoV-2 nucleocapsid protein, promoting 14-3-3/client complexation, and monomerizing 14-3-3 dimers. Then, to understand the biological function of these phosphorylated 14-3-3ζ monomers (containing nhpSer at Ser58), we isolate its interactome from HEK293T lysates and compare it with that of wild-type 14-3-3ζ. These data identify two new subsets of 14-3-3 client proteins: (i) those that selectively bind dimeric 14-3-3ζ and (ii) those that selectively bind monomeric 14-3-3ζ. We discover that monomeric-but not dimeric-14-3-3ζ interacts with cereblon, an E3 ubiquitin-ligase adaptor protein of pharmacological interest.

Structural basis for the ligand promiscuity of the neofunctionalized, carotenoid-binding fasciclin domain protein AstaP.

Fasciclins (FAS1) are ancient adhesion protein domains with no common small ligand binding reported. A unique microalgal FAS1-containing astaxanthin (AXT)-binding protein (AstaP) binds a broad repertoire of carotenoids by a largely unknown mechanism. Here, we explain the ligand promiscuity of AstaP-orange1 (AstaPo1) by determining its NMR structure in complex with AXT and validating this structure by SAXS, calorimetry, optical spectroscopy and mutagenesis. α1-α2 helices of the AstaPo1 FAS1 domain embrace the carotenoid polyene like a jaw, forming a hydrophobic tunnel, too short to cap the AXT ß-ionone rings and dictate specificity. AXT-contacting AstaPo1 residues exhibit different conservation in AstaPs with the tentative carotenoid-binding function and in FAS1 proteins generally, which supports the idea of AstaP neofunctionalization within green algae. Intriguingly, a cyanobacterial homolog with a similar domain structure cannot bind carotenoids under identical conditions. These structure-activity relationships provide the first step towards the sequence-based prediction of the carotenoid-binding FAS1 members.

Stages of OCP-FRP Interactions in the Regulation of Photoprotection in Cyanobacteria, Part 1: Time-Resolved Spectroscopy.

Most cyanobacteria utilize a water-soluble Orange Carotenoid Protein (OCP) to protect their light-harvesting complexes from photodamage. The Fluorescence Recovery Protein (FRP) is used to restore photosynthetic activity by inactivating OCP via dynamic OCP-FRP interactions, a multistage process that remains underexplored. In this work, applying time-resolved spectroscopy, we demonstrate that the interaction of FRP with the photoactivated OCP begins early in the photocycle. Interacting with the compact OCP state, FRP completely prevents the possibility of OCP domain separation and formation of the signaling state capable of interacting with the antenna. The structural element that prevents FRP binding and formation of the complex is the short α-helix at the beginning of the N-terminal domain of OCP, which masks the primary site in the C-terminal domain of OCP. We determined the rate of opening of this site and show that it remains exposed long after the relaxation of the red OCP states. Observations of the OCP transitions on the ms time scale revealed that the relaxation of the orange photocycle intermediates is accompanied by an increase in the interaction of the carotenoid keto group with the hydrogen bond donor tyrosine-201. Our data refine the current model of photoinduced OCP transitions and the interaction of its intermediates with FRP.

Parameterization of a single H-bond in Orange Carotenoid Protein by atomic mutation reveals principles of evolutionary design of complex chemical photosystems.

Introduction: Dissecting the intricate networks of covalent and non-covalent interactions that stabilize complex protein structures is notoriously difficult and requires subtle atomic-level exchanges to precisely affect local chemical functionality. The function of the Orange Carotenoid Protein (OCP), a light-driven photoswitch involved in cyanobacterial photoprotection, depends strongly on two H-bonds between the 4-ketolated xanthophyll cofactor and two highly conserved residues in the C-terminal domain (Trp288 and Tyr201). Method: By orthogonal translation, we replaced Trp288 in Synechocystis OCP with 3-benzothienyl-L-alanine (BTA), thereby exchanging the imino nitrogen for a sulphur atom. Results: Although the high-resolution (1.8 Å) crystal structure of the fully photoactive OCP-W288_BTA protein showed perfect isomorphism to the native structure, the spectroscopic and kinetic properties changed distinctly. We accurately parameterized the effects of the absence of a single H-bond on the spectroscopic and thermodynamic properties of OCP photoconversion and reveal general principles underlying the design of photoreceptors by natural evolution. Discussion: Such "molecular surgery" is superior over trial-and-error methods in hypothesis-driven research of complex chemical systems.

Molecular insight into the specific interactions of the SARS-Coronavirus-2 nucleocapsid with RNA and host protein.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) nucleocapsid protein is the most abundantly expressed viral protein during infection where it targets both RNA and host proteins. However, identifying how a single viral protein interacts with so many different targets remains a challenge, providing the impetus here for identifying the interaction sites through multiple methods. Through a combination of nuclear magnetic resonance (NMR), electron microscopy, and biochemical methods, we have characterized nucleocapsid interactions with RNA and with three host proteins, which include human cyclophilin-A, Pin1, and 14-3-3τ. Regarding RNA interactions, the nucleocapsid protein N-terminal folded domain preferentially interacts with smaller RNA fragments relative to the C-terminal region, suggesting an initial RNA engagement is largely dictated by this N-terminal region followed by weaker interactions to the C-terminal region. The nucleocapsid protein forms 10 nm ribonuclear complexes with larger RNA fragments that include 200 and 354 nucleic acids, revealing its potential diversity in sequestering different viral genomic regions during viral packaging. Regarding host protein interactions, while the nucleocapsid targets all three host proteins through its serine-arginine-rich region, unstructured termini of the nucleocapsid protein also engage host cyclophilin-A and host 14-3-3τ. Considering these host proteins play roles in innate immunity, the SARS-CoV-2 nucleocapsid protein may block the host response by competing interactions. Finally, phosphorylation of the nucleocapsid protein quenches an inherent dynamic exchange process within its serine-arginine-rich region. Our studies identify many of the diverse interactions that may be important for SARS-CoV-2 pathology during infection.

Protein-Mediated Carotenoid Delivery Suppresses the Photoinducible Oxidation of Lipofuscin in Retinal Pigment Epithelial Cells.

Lipofuscin of retinal pigment epithelium (RPE) cells is a complex heterogeneous system of chromophores which accumulates as granules during the cell's lifespan. Lipofuscin serves as a source of various cytotoxic effects linked with oxidative stress. Several age-related eye diseases such as macular degeneration of the retina, as well as some severe inherited eye pathologies, are accompanied by a significant increase in lipofuscin granule concentration. The accumulation of carotenoids in the RPE could provide an effective antioxidant protection against lipofuscin cytotoxic manifestations. Given the highly lipophilic nature of carotenoids, their targeted delivery to the vulnerable tissues can potentially be assisted by special proteins. In this study, we demonstrate how protein-mediated delivery of zeaxanthin using water-soluble Bombyx mori carotenoid-binding protein (BmCBP-ZEA) suppresses the photoinducible oxidative stress in RPE cells caused by irradiation of lipofuscin with intense white light. We implemented fluorescence lifetime imaging of the RPE cell culture ARPE-19 fed with lipofuscin granules and then irradiated by white light with and without the addition of BmCBP-ZEA. We demonstrate that after irradiation the mean fluorescence lifetime of lipofuscin significantly increases, while the presence of BmCBP-ZEA at 200 nM concentration suppresses the increase in the average lifetime of lipofuscin fluorescence, indicating an approx. 35% inhibition of the oxidative stress. This phenomenon serves as indirect yet important evidence of the efficiency of the protein-mediated carotenoid delivery into pigment epithelium cells.

Genetic encoding of 3-nitro-tyrosine reveals the impacts of 14-3-3 nitration on client binding and dephosphorylation.

14-3-3 proteins are central hub regulators of hundreds of phosphorylated "client" proteins. They are subject to over 60 post-translational modifications (PTMs), yet little is known how these PTMs alter 14-3-3 function and its ability to regulate downstream signaling pathways. An often neglected, but well-documented 14-3-3 PTM found under physiological and immune-stimulatory conditions is the conversion of tyrosine to 3-nitro-tyrosine at several Tyr sites, two of which are located at sites considered important for 14-3-3 function: Y130 (ß-isoform numbering) is located in the primary phospho-client peptide-binding groove, while Y213 is found on a secondary binding site that engages with clients for full 14-3-3/client complex formation and client regulation. By genetically encoding 3-nitro-tyrosine, we sought to understand if nitration at Y130 and Y213 effectively modulated 14-3-3 structure, function, and client complexation. The 1.5 Å resolution crystal structure of 14-3-3 nitrated at Y130 showed the nitro group altered the conformation of key residues in the primary binding site, while functional studies confirmed client proteins failed to bind this variant of 14-3-3. But, in contrast to other client-binding deficient variants, it did not localize to the nucleus. The 1.9 Å resolution structure of 14-3-3 nitrated at Y213 revealed unusual flexibility of its C-terminal α-helix resulting in domain swapping, suggesting additional structural plasticity though its relevance is not clear as this nitrated form retained its ability to bind clients. Collectively, our data suggest that nitration of 14-3-3 will alter downstream signaling systems, and if uncontrolled could result in global dysregulation of the 14-3-3 interactome.