Chaperone-like Function Research Articles

Analysis of two domains with novel RNA-processing activities throws light on the complex evolution of ribosomal RNA biogenesis.

Ribosomal biogenesis has been extensively investigated, especially to identify the elusive nucleases and cofactors involved in the complex rRNA processing events in eukaryotes. Large-scale screens in yeast identified two biochemically uncharacterized proteins, TSR3 and TSR4, as being key players required for rRNA maturation. Using multiple computational approaches we identify the conserved domains comprising these proteins and establish sequence and structural features providing novel insights regarding their roles. TSR3 is unified with the DTW domain into a novel superfamily of predicted enzymatic domains, with the balance of the available evidence pointing toward an RNase role with the archaeo-eukaryotic TSR3 proteins processing rRNA and the bacterial versions potentially processing tRNA. TSR4, its other eukaryotic homologs PDCD2/rp-8, PDCD2L, Zfrp8, and trus, the predominantly bacterial DUF1963 proteins, and other uncharacterized proteins are unified into a new domain superfamily, which arose from an ancient duplication event of a strand-swapped, dimer-forming all-beta unit. We identify conserved features mediating protein-protein interactions (PPIs) and propose a potential chaperone-like function. While contextual evidence supports a conserved role in ribosome biogenesis for the eukaryotic TSR4-related proteins, there is no evidence for such a role for the bacterial versions. Whereas TSR3-related proteins can be traced to the last universal common ancestor (LUCA) with a well-supported archaeo-eukaryotic branch, TSR4-related proteins of eukaryotes are derived from within the bacterial radiation of this superfamily, with archaea entirely lacking them. This provides evidence for “systems admixture,” which followed the early endosymbiotic event, playing a key role in the emergence of the uniquely eukaryotic ribosome biogenesis process.

Glycolipozyme membrane protein integrase (MPIase): recent data.

A novel factor for membrane protein integration, from the cytoplasmic membrane of Escherichia coli, named MPIase (membrane protein integrase), has recently been identified and characterized. MPIase was revealed to be essential for the membrane integration of a subset of membrane proteins, despite that such integration reactions have been, thus far, thought to occur spontaneously. The structure determination study revealed that MPIase is a novel glycolipid comprising a glycan chain with three N-acetylated amino sugars connected to diacylglycerol through a pyrophosphate linker. As MPIase catalyzes membrane protein integration, we propose that MPIase is a glycolipozyme on the basis of its enzyme-like function. The glycan chain exhibits a molecular chaperone-like function by directly interacting with substrate membrane proteins. Moreover, MPIase also affects the dimer structure of SecYEG, a translocon, thereby significantly stimulating preprotein translocation. The molecular mechanisms of MPIase functions will be outlined.

A heroin addiction severity-associated intronic single nucleotide polymorphism modulates alternative pre-mRNA splicing of the μ opioid receptor gene OPRM1 via hnRNPH interactions.

Single nucleotide polymorphisms (SNPs) in the OPRM1 gene have been associated with vulnerability to opioid dependence. The current study identifies an association of an intronic SNP (rs9479757) with the severity of heroin addiction among Han-Chinese male heroin addicts. Individual SNP analysis and haplotype-based analysis with additional SNPs in the OPRM1 locus showed that mild heroin addiction was associated with the AG genotype, whereas severe heroin addiction was associated with the GG genotype. In vitro studies such as electrophoretic mobility shift assay, minigene, siRNA, and antisense morpholino oligonucleotide studies have identified heterogeneous nuclear ribonucleoprotein H (hnRNPH) as the major binding partner for the G-containing SNP site. The G-to-A transition weakens hnRNPH binding and facilitates exon 2 skipping, leading to altered expressions of OPRM1 splice-variant mRNAs and hMOR-1 proteins. Similar changes in splicing and hMOR-1 proteins were observed in human postmortem prefrontal cortex with the AG genotype of this SNP when compared with the GG genotype. Interestingly, the altered splicing led to an increase in hMOR-1 protein levels despite decreased hMOR-1 mRNA levels, which is likely contributed by a concurrent increase in single transmembrane domain variants that have a chaperone-like function on MOR-1 protein stability. Our studies delineate the role of this SNP as a modifier of OPRM1 alternative splicing via hnRNPH interactions, and suggest a functional link between an SNP-containing splicing modifier and the severity of heroin addiction.

60S ribosome biogenesis requires rotation of the 5S ribonucleoprotein particle

During eukaryotic ribosome biogenesis, nascent ribosomal RNA (rRNA) forms pre-ribosomal particles containing ribosomal proteins and assembly factors. Subsequently, these immature rRNAs are processed and remodelled. Little is known about the premature assembly states of rRNAs and their structural rearrangement during ribosome biogenesis. Using cryo-EM we characterize a pre-60S particle, where the 5S rRNA and its associated ribosomal proteins L18 and L5 (5S ribonucleoprotein (RNP)) are rotated by almost 180° when compared with the mature subunit. Consequently, neighbouring 25S rRNA helices that protrude from the base of the central protuberance are deformed. This altered topology is stabilized by nearby assembly factors (Rsa4 and Nog1), which were identified by fitting their three-dimensional structures into the cryo-EM density. We suggest that the 5S RNP performs a semicircular movement during 60S biogenesis to adopt its final position, fulfilling a chaperone-like function in guiding the flanking 25S rRNA helices of the central protuberance to their final topology.

Mechanosensitive Liposomes as Artificial Chaperones for Shear-Driven Acceleration of Enzyme-Catalyzed Reaction

Mechanosensitive liposomes were prepared and applied to continuously accelerate the glucose oxidase (GO) reaction in shear flow. The liposome membrane was composed of a ternary lipid mixture containing 20 mol % negatively charged lipid and 30 mol % cholesterol. The liposomes encapsulating GO and catalase were passed through microtubes with inner diameter of 190 or 380 μm at 25 °C to induce the catalytic oxidation of 10 mM glucose with simultaneous decomposition of H2O2 produced. The liposomal GO showed significantly low reactivity in the static liquid system because of the permeation resistance of lipid membranes to glucose. On the other hand, the enzyme activity of liposomal GO observed at the average shear rate of 7.8 × 10(3) s(-1) was significantly larger than its intrinsic activity free of mass transfer effect in the static liquid system. The structure of liposomes was highly shear-sensitive as elucidated on the basis of shear rate-dependent physical stability of liposomes and membrane permeability to 5(6)-carboxyfluorescein as well as to GO. Thus, the above shear-driven acceleration of GO reaction was indicated to be caused by the free GO molecules released from the structurally altered liposomes at high shear rates. Moreover, the shear-induced denaturation of free GO was completely depressed by the interaction with the sheared liposomes with the chaperone-like function. The shear-sensitive liposomal GO system can be a unique catalyst that continuously accelerates and also decelerates the oxidation reaction depending on the applied shear rate.

Spectroscopic studies of the unfolding of a multimeric protein α‐crystallin

α-Crystallin is a multimeric eye lens protein having molecular chaperone-like function which is crucial for lens transparency. The stability and unfolding-refolding properties of α-crystallin plays important roles for its function. We undertook a multi probe based fluorescence spectroscopic approach to explore the changes in the various levels of organization of this protein at different urea concentration. Steady state fluorescence studies reveal that at 0.6M urea a compact structural intermediate is formed which has a native-like secondary structure with enhanced surface exposure of hydrophobic groups. At 2.8M urea the tertiary interactions are largely collapsed with partial collapse of secondary and quaternary structure. The surface solvation probed by picosecond time resolved fluorescence of acrylodan labeled α-crystallin revealed dry native-like core of α-crystallin at 0.6M urea compared to enhanced water penetration at 2.8M urea and extensive solvation at 6M urea. Activation energy for the subunit exchange decreased by 22 kJ mol(-1) on changing urea concentration from 0 to 0.6M compared with over 75 kJ mol(-1) on changing urea concentration from 0 to 2.8M. Light scattering and analytical ultracentrifugation techniques were used to determine size and oligomerization of the unfolding intermediates. The data indicated swelling but no oligomer breakdown at 0.6M urea. At 2.8M urea the oligomeric size is considerably reduced and a monomer is produced at 6M urea. The data clearly reveals that structural breakdown of α-crystallin does not follow hierarchical sequence as tertiary structure dissolution takes place before complete oligomeric dissociation.

The Intrinsically Disordered Sem1 Protein Functions as a Molecular Tether during Proteasome Lid Biogenesis

The intrinsically disordered yeast protein Sem1 (DSS1 in mammals) participates in multiple protein complexes, including the proteasome, but its role(s) within these complexes is uncertain. We report that Sem1 enforces the ordered incorporation of subunits Rpn3 and Rpn7 into the assembling proteasome lid. Sem1 uses conserved acidic segments separated by a flexible linker to grasp Rpn3 and Rpn7. The same segments are used for protein binding in other complexes, but in the proteasome lid they are uniquely deployed for recognizing separate polypeptides. We engineered TEV protease-cleavage sites into Sem1 to show that the tethering function of Sem1 is important for the biogenesis and integrity of the Rpn3-Sem1-Rpn7 ternary complex but becomes dispensable once the ternary complex incorporates into larger lid precursors. Thus, although Sem1 is a stoichiometric component of the mature proteasome, it has a distinct, chaperone-like function specific to early stages of proteasome assembly.

Revisiting Structural Hieracrchy: A Fluorescence Investigation of Unfolding of an Oligomeric Protein

α-crystallin is a multimeric lens protein with chaperone-like function and is responsible for maintaining lens transparency. The structural stability and unfolding-refolding properties of this protein are believed to be important for its function. We undertook a multi-probe based fluorescence approach to explore the changes in the various levels of organization of α-crystallin at different urea concentration region. Steady-state fluorescence emission and quenching studies using extrinsic and intrinsic fluorescence probes reveal that at 0.6 M urea a compact structural intermediate is formed which has a native-like secondary structure with minimal yet detectable tertiary structure perturbation associated with enhanced surface exposure of hydrophobic groups, possibly arising from interfacial structure meltdown. At 2.8 M urea the tertiary interactions undergo almost complete collapse with partial disintegration of secondary and quaternary structure. Investigation of surface solvation with picosecond resolved fluorescence transients of acrylodan covalently tagged to α-crystallin reveals a dry native-like core of α-crystallin at 0.6 M urea compared to enhanced water penetration at 2.8 M urea and extensive solvation at 6 M urea. Temperature dependent subunit exchange kinetics reveal decrease of activation energy for the subunit exchange process by 22 kJ mol-1 on changing urea concentration from 0 to 0.6 M compared to over 75 kJ mol-1 on changing urea concentration from 0 to 2.8 M. Dynamic light scattering study indicates swelling at 0.6 M urea, however oligomerization is retained as observed from sedimentation equilibrium experiment. At 2.8 M urea the oligomeric size is reduced and a monomer is produced at 6 M urea. These data clearly demonstrates that tertiary structure dissolution precedes oligomeric degradation. Such non-hierarchical structure dissolution indicates the possibility of tertiary contact formation to be a rather later folding event in case of large oligomeric proteins likes α-Crystallin.

Stabilization of the μ-Opioid Receptor by Truncated Single Transmembrane Splice Variants through a Chaperone-like Action

The μ-opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, as illustrated by the identification of an array of splice variants generated by both 5' and 3' alternative splicing. The current study reports the identification of another set of splice variants conserved across species that are generated through exon skipping or insertion that encodes proteins containing only a single transmembrane (TM) domain. Using a Tet-Off system, we demonstrated that the truncated single TM variants can dimerize with the full-length 7-TM μ-opioid receptor (MOR-1) in the endoplasmic reticulum, leading to increased expression of MOR-1 at the protein level by a chaperone-like function that minimizes endoplasmic reticulum-associated degradation. In vivo antisense studies suggested that the single TM variants play an important role in morphine analgesia, presumably through modulation of receptor expression levels. Our studies suggest the functional roles of truncated receptors in other G protein-coupled receptor families.

Chaperone-like effects of a scFv antibody on the folding of human muscle creatine kinase

Molecular chaperones play an essential role in assisting the folding of a myriad of nascent peptides to form different biologically active proteins. Therefore, their low substrate specificity is important for the functions of these housekeeping proteins. However, discovering chaperones which assist the folding of a particular protein can shed new light on the folding pathway of the protein, offering an interesting approach for developing specific therapeutic agents to treat protein-misfolding diseases. Screening of antibodies with chaperone-like function represents a novel strategy to meet the challenges. In this study, some single-chain variable fragment (scFv) antibodies were selected from a high-capacity phage antibody library using human muscle creatine kinase (HCK) as antigen. A scFv antibody (scFv A4) was determined to inhibit aggregation and favor recovery of the native conformation of HCK during its refolding. This antibody also increased the stability of HCK during its heat-induced unfolding process. Our findings demonstrate that scFv A4 has dual-chaperone-like activities: assisting in correct protein folding as well as protecting the native protein from unfolding. A molecular mechanism by which scFv A4 exhibits chaperone-like effects on HCK was proposed. This study demonstrates that phage antibody libraries can lead to chaperone-like proteins, and the specificity of the resulting antibody toward its antigen could provide new molecular details regarding how the chaperone interacts with the protein's unfolding and folding pathways.

A study of the mechanism of the chaperone-like function of an scFv of human creatine kinase by computer simulation.

A new application of antibodies is to use them as macromolecular chaperones. Protein antigens usually have multiple epitopes, thus, there may be a plurality of antibodies binding to one antigen. However, not all antibodies that bind to one antigen could act as a chaperone. Experiments show that some screened anti-human creatine kinase single chain antibodies (scFV) could assist in the folding and stabilizing of the enzyme, while others could not. We built the model of the single chain antibody (scFv-A4) that increased the stability of human creatine kinase (HCK) by the homology modeling method. Epitopes of human creatine kinase were predicted by computer and then the binding of scFv-A4 and HCK was modeled with computer. The calculation results were further combined with the peptide array membrane experiment results to obtain reliable models for the scFv-A4-HCK complex. Based on the above study we gave an explanation about how scFv-A4 could act as a macromolecular chaperone assisting the folding of HCK. This study provides an approach for predicting antigen-antibody binding mode and also a useful theoretical guidance for the study of antibodies' chaperone-like function.

Proteolytically Inactive Insulin-Degrading Enzyme Inhibits Amyloid Formation Yielding Non-Neurotoxic Aβ Peptide Aggregates

Insulin-degrading enzyme (IDE) is a neutral Zn2+ peptidase that degrades short peptides based on substrate conformation, size and charge. Some of these substrates, including amyloid β (Aβ) are capable of self-assembling into cytotoxic oligomers. Based on IDE recognition mechanism and our previous report of the formation of a stable complex between IDE and intact Aβ in vitro and in vivo, we analyzed the possibility of a chaperone-like function of IDE. A proteolytically inactive recombinant IDE with Glu111 replaced by Gln (IDEQ) was used. IDEQ blocked the amyloidogenic pathway of Aβ yielding non-fibrillar structures as assessed by electron microscopy. Measurements of the kinetics of Aβ aggregation by light scattering showed that 1) IDEQ effect was promoted by ATP independent of its hydrolysis, 2) end products of Aβ-IDEQ co-incubation were incapable of “seeding” the assembly of monomeric Aβ and 3) IDEQ was ineffective in reversing Aβ aggregation. Moreover, Aβ aggregates formed in the presence of IDEQ were non-neurotoxic. IDEQ had no conformational effects upon insulin (a non-amyloidogenic protein under physiological conditions) and did not disturb insulin receptor activation in cultured cells. Our results suggest that IDE has a chaperone-like activity upon amyloid-forming peptides. It remains to be explored whether other highly conserved metallopeptidases have a dual protease-chaperone function to prevent the formation of toxic peptide oligomers from bacteria to mammals.

Evolution of crystallins for a role in the vertebrate eye lens.

The camera eye lens of vertebrates is a classic example of the re-engineering of existing protein components to fashion a new device. The bulk of the lens is formed from proteins belonging to two superfamilies, the α-crystallins and the βγ-crystallins. Tracing their ancestry may throw light on the origin of the optics of the lens. The α-crystallins belong to the ubiquitous small heat shock proteins family that plays a protective role in cellular homeostasis. They form enormous polydisperse oligomers that challenge modern biophysical methods to uncover the molecular basis of their assembly structure and chaperone-like protein binding function. It is argued that a molecular phenotype of a dynamic assembly suits a chaperone function as well as a structural role in the eye lens where the constraint of preventing protein condensation is paramount. The main cellular partners of α-crystallins, the β- and γ-crystallins, have largely been lost from the animal kingdom but the superfamily is hugely expanded in the vertebrate eye lens. Their structures show how a simple Greek key motif can evolve rapidly to form a complex array of monomers and oligomers. Apart from remaining transparent, a major role of the partnership of α-crystallins with β- and γ-crystallins in the lens is to form a refractive index gradient. Here, we show some of the structural and genetic features of these two protein superfamilies that enable the rapid creation of different assembly states, to match the rapidly changing optical needs among the various vertebrates.

PPIase independent chaperone-like function of recombinant human Cyclophilin A during arginine kinase refolding

Whether Cyclophilin A (CyPA) functions as a foldase or a chaperone when assisting protein folding has long been argued. In this study, we engineered four variants of recombinant human Cyclophilin A (rhCyPA), all of which were inactive to tetrapeptide substrate Suc-AAPF-pNA. However, these variants were able to suppress aggregation during arginine kinase (AK) refolding as efficient as wild-type rhCyPA, especially, variant Q63A had even more efficiency to suppress aggregation and improve reactivation yields of AK. These results indicate that rhCyPA have peptidyl–prolyl cis–trans isomerase (PPIase) independent chaperone-like activity during AK folding. In addition, results suggest that surface hydrophobicity of rhCyPA can suppress AK aggregation and binding to rhCyPA hydrophobic active pocket is a prerequisite for chaperoning AK folding.

Lipid Rafts Alter the Stability and Activity of the Cholera Toxin A1 Subunit

Cholera toxin (CT) travels from the cell surface to the endoplasmic reticulum (ER) as an AB holotoxin. ER-specific conditions then promote the dissociation of the catalytic CTA1 subunit from the rest of the toxin. CTA1 is held in a stable conformation by its assembly in the CT holotoxin, but the dissociated CTA1 subunit is an unstable protein that spontaneously assumes a disordered state at physiological temperature. This unfolding event triggers the ER-to-cytosol translocation of CTA1 through the quality control mechanism of ER-associated degradation. The translocated pool of CTA1 must regain a folded, active structure to modify its G protein target which is located in lipid rafts at the cytoplasmic face of the plasma membrane. Here, we report that lipid rafts place disordered CTA1 in a functional conformation. The hydrophobic C-terminal domain of CTA1 is essential for binding to the plasma membrane and lipid rafts. These interactions inhibit the temperature-induced unfolding of CTA1. Moreover, lipid rafts could promote a gain of structure in the disordered, 37 °C conformation of CTA1. This gain of structure corresponded to a gain of function: whereas CTA1 by itself exhibited minimal in vitro activity at 37 °C, exposure to lipid rafts resulted in substantial toxin activity at 37 °C. In vivo, the disruption of lipid rafts with filipin substantially reduced the activity of cytosolic CTA1. Lipid rafts thus exhibit a chaperone-like function that returns disordered CTA1 to an active state and is required for the optimal in vivo activity of CTA1.

WhiB2/Rv3260c, a cell division‐associated protein of Mycobacterium tuberculosis H37Rv, has properties of a chaperone

whiB-like genes have been found in all actinomycetes sequenced so far. The amino-acid sequences of WhiB proteins of Mycobacterium tuberculosis H37Rv are highly conserved and participate in several cellular functions. Unlike other WhiB proteins of M. tuberculosis that have properties of protein disulfide reductases, WhiB2 showed properties like a chaperone as it suppressed the aggregation of several model substrates (e.g. citrate synthase, rhodanese and luciferase). Suppression of aggregation of the model substrates did not require ATP. Four cysteine residues of WhiB2 form two intramolecular disulfide bonds; however, chaperone function was unaffected by the redox state of the cysteines. WhiB2 also restored the activity of chemically denatured citrate synthase and did not require either ATP or a co-chaperone for refolding. The results indicate that WhiB2, which has been shown to be associated with cell division in mycobacteria and streptomyces, has evolved independently of other WhiBs, although it retains basic properties of this group of proteins. This is the first report to show that a WhiB protein has chaperone-like function; therefore, this report will have major implications in attempts to understand the role of WhiB proteins in mycobacteria, particularly in cell division.

α2δ expression sets presynaptic calcium channel abundance and release probability

Synaptic neurotransmitter release is driven by Ca(2+) influx through active zone voltage-gated calcium channels (VGCCs). Control of active zone VGCC abundance and function remains poorly understood. Here we show that a trafficking step probably sets synaptic VGCC levels in rats, because overexpression of the pore-forming α1(A) VGCC subunit fails to change synaptic VGCC abundance or function. α2δs are a family of glycosylphosphatidylinositol (GPI)-anchored VGCC-associated subunits that, in addition to being the target of the potent neuropathic analgesics gabapentin and pregabalin (α2δ-1 and α2δ-2), were also identified in a forward genetic screen for pain genes (α2δ-3). We show that these proteins confer powerful modulation of presynaptic function through two distinct molecular mechanisms. First, α2δ subunits set synaptic VGCC abundance, as predicted from their chaperone-like function when expressed in non-neuronal cells. Second, α2δs configure synaptic VGCCs to drive exocytosis through an extracellular metal ion-dependent adhesion site (MIDAS), a conserved set of amino acids within the predicted von Willebrand A domain of α2δ. Expression of α2δ with an intact MIDAS motif leads to an 80% increase in release probability, while simultaneously protecting exocytosis from blockade by an intracellular Ca(2+) chelator. α2δs harbouring MIDAS site mutations still drive synaptic accumulation of VGCCs; however, they no longer change release probability or sensitivity to intracellular Ca(2+) chelators. Our data reveal dual functionality of these clinically important VGCC subunits, allowing synapses to make more efficient use of Ca(2+) entry to drive neurotransmitter release.

Novel structural and functional insights into the MoxR family of AAA+ ATPases

The MoxR family of AAA+ ATPases is widespread among bacteria and archaea, although their cellular functions are not well characterized. Based on recent studies, MoxR ATPases are proposed to have chaperone-like function for the maturation of specific protein complexes or for the insertion of cofactors into proteins. MoxR proteins have been found to be important modulators of multiple stress response pathways in different organisms. For example, the respective MoxR proteins have been found to play important roles in the cell envelope stress response in Rhizobium leguminosarum, in the oxidative stress, acid stress, and heat stress responses in Francisella tularensis, in the acid stress and stringent responses in Escherichia coli, in viral tail formation in the crenarchaeal Acidianus two-tailed virus, and in the utilization of carbon monoxide as the sole carbon source by the Gram-negative chemolithoautotrophe Oligotropha carboxidovorans. Recent structural studies on the MoxR proteins from E. coli and Cytophaga hutchinsonii show the unique spatial arrangement of the αβα and all-α subdomains of the AAA+ domain in these proteins compared to the typical arrangement found in canonical AAA+ proteins such as HslU. The spatial organization of the subdomains in the AAA+ domain of MoxR proteins is similar to that found in the ATPase component of the magnesium chelatase complexes, possibly suggesting a similar mechanism of function. In this review, we provide an overview of the newly identified functions and the newly obtained structures of MoxR AAA+ ATPases.

The neuroendocrine peptide 7B2 prevents neurodegenerative disease‐related protein aggregation

Neurodegenerative diseases like Alzheimer's (AD) and Parkinson's (PD) are characterized by an abnormal aggregation of misfolded beta‐sheet rich proteins such as β‐amyloid (Aβ), tau (in AD) and α‐synuclein (in PD). In healthy cells, the correct folding of proteins is controlled by ubiquitously expressed molecular chaperones, which prevent the accumulation of harmful protein species. Our understanding of this quality control machinery and its involvement in the pathogenesis of neurodegenerative diseases is, however, very limited. We here describe a novel chaperone‐like function for the neuroendocrine protein 7B2, a highly conserved protein that is widely expressed in the nervous and endocrine systems. In vitro, 7B2 efficiently prevented the fibrillation of Aβ(1–42), Aβ(1–40), tau and α‐synuclein at a molar ratio of 1:10. This anti‐aggregation effect was dose‐dependent and most effective when 7B2 was present as the full‐length (27 kDa) form. Both exogenous application of 7B2 into the medium of Neuro2A cells, as well as endogenous adenoviral‐mediated overexpression of 7B2 blocked the neurocytotoxic effect of Aβ(1–42) and significantly increased cell viability. In the substantia nigra of a human PD‐affected brain, 7B2 was highly colocalized with α‐synuclein, indicating a physiological association. Taken together our results suggest that 7B2 may play a role in the etiology of neurodegenerative diseases.

Cloning of crystallin from orange-spotted grouper and characterization of its activity as potential protective agent

Oxidative stress associated with nodavirus infection is poorly understood, especially pertaining to infection-mediated brain injury. Indirect evidence indicates that infection increases cellular abundance of reactive oxygen species (ROS) with consequent increase in cellular dityrosine production. The detection of dityrosine in nodavirus-infected grouper was demonstrated using immunohistochemical (IHC) staining. Proteomic analyses with eye tissues of healthy grouper revealed more abundant expression of crystallin protein in the eye than in various tissues, which was confirmed by real-time polymerase chain reaction and IHC analyses. Grouper crystallin belongs to a small heat shock protein family with chaperone-like function that prevents heat-induced and oxidative stress-induced protein aggregation. Recombinant crystallin induced nitric oxide (NO) production in RAW 264.7 cells after treatment. The results provide new insight into the pathogenesis of nodavirus and demonstrate an experimental rationale for antioxidant therapy research.