Nano-assemblies enhance chaperone activity, stability, and delivery of alpha B-crystallin-D3 (αB-D3).

Crystallins, small heat shock chaperone proteins that prevent protein aggregation, are of potential value in treating protein aggregation disorders. However, their therapeutic use is limited by their low potency and poor intracellular delivery. One approach to facilitate the development of crystallins is to improve their activity, stability, and delivery. In this study, zinc addition to αB-crystallin-D3 (αB-D3) formed supramolecular nano- and micro- assemblies, induced dose-dependent changes in structure (beta-sheet to alpha-helix) and increased surface hydrophobicity and chemical stability. Further, crystallin assemblies exhibited a size-dependent chaperone activity, with the nano-assemblies being superior to micro-assemblies and 4.3-fold more effective than the native protein in preventing ß-mercaptoethanol induced aggregation of insulin. Insulin rescued by crystallin assemblies retained the activity as evidenced by glucose uptake in 3T3-L1 cells. The most active nano-assemblies enhanced protein stability, in the presence of urea, by 1.6-fold, whereas intracellular delivery was enhanced by 3.0-fold. The αB-D3 crystallin nano-assemblies exhibit uniquely enhanced stability, activity, and delivery compared to the native protein.

Varicella Zoster Virus Induces Differential Cell-Type Specific Responses in Human Corneal Epithelial Cells and Keratocytes.

Purpose: While VZV DNA and antigen have been detected in acute and chronic VZV keratitis, it is unclear whether productive infection of corneal cells is ongoing or whether residual, noninfectious VZV antigens elicit inflammation. Herein, we examined VZV-infected primary human corneal epithelial cells (HCECs) and keratocytes (HKs) to elucidate the pathogenesis of VZV keratitis. Methods: HCECs and HKs were mock- or VZV infected. Seven days later, cells were examined for morphology, proinflammatory cytokine and matrix metalloproteinase (MMP) release, ability to recruit peripheral blood mononuclear cells (PBMCs) and neutrophils, and MMP substrate cleavage. Results: Both cell types synthesized infectious virus. VZV-infected HCECs proliferated, whereas VZV-infected HKs died. Compared to mock-infected cells, VZV-infected HCECs secreted significantly more IL-6, IL-8, IL-10, and IL-12p70 that were confirmed at the transcript level, and MMP-1 and MMP-9; conditioned supernatant attracted PBMCs and neutrophils and cleaved MMP substrates. In contrast, VZV-infected HKs suppressed cytokine secretion except for IL-8, which attracted neutrophils, and suppressed MMP release and substrate cleavage. Conclusions: Overall, VZV-infected HCECs recapitulate findings of VZV keratitis with respect to epithelial cell proliferation, pseudodendrite formation and creation of a proinflammatory environment, providing an in vitro model for VZV infection of corneal epithelial cells. Furthermore, the proliferation and persistence of VZV-infected HCECs suggest that these cells may serve as viral reservoirs if immune clearance is incomplete. Finally, the finding that VZV-infected HKs die and suppress most proinflammatory cytokines and MMPs may explain the widespread death of these cells with unchecked viral spread due to ineffective recruitment of PBMCs.

Changes in function but not oligomeric size are associated with αB-crystallin lysine substitution.

αB-Crystallin, ubiquitously expressed in many tissues including the ocular lens, is a small heat shock protein that can prevent protein aggregation. A number of post-translation modifications are reported to modify αB-crystallin function. Recent studies have identified αB-crystallin lysine residues are modified by acetylation and ubiquitination. Therefore, we sought to determine the effects of lysine to alanine substitution on αB-crystallin functions including chaperone activity and modulation of actin polymerization. Analysis of the ten substitution mutants as recombinant proteins indicated all the proteins were soluble and formed oligomeric complexes similar to wildtype protein. Lysozyme aggregation induced by chemical treatment indicated that K82, K90, K121, K166 and K174/K175 were required for efficient chaperone activity. Thermal induction of γ-crystallin aggregation could be prevented by all αB-crystallin substitution mutants. These αB-crystallin mutants also were able to mediate wildtype levels of actin polymerization. Further analysis of two clones with either enhanced or reduced chaperone activity on individual client substrates or actin polymerization indicated both retained broad chaperone activity and anti-apoptotic activity. Collectively, these studies show the requirements for lysine residues in αB-crystallin function.

Therapeutic potential of α-crystallin.

BACKGROUND: The findings that α-crystallins are multi-functional proteins with diverse biological functions have generated considerable interest in understanding their role in health and disease. Recent studies have shown that chaperone peptides of α-crystallin could be delivered into cultured cells and in experimental animals with beneficial effects against protein aggregation, oxidation, inflammation and apoptosis. SCOPE OF REVIEW: In this review, we will summarize the latest developments on the therapeutic potential of α-crystallins and their functional peptides. MAJOR CONCLUSIONS: α-Crystallins and their functional peptides have shown significant favorable effects against several diseases. Their targeted delivery to tissues would be of great therapeutic benefit. However, α-crystallins can also function as disease-causing proteins. These seemingly contradictory functions must be carefully considered prior to their therapeutic use. GENERAL SIGNIFICANCE: αA and αB-Crystallin are members of the small heat shock protein family. These proteins exhibit molecular chaperone and anti-apoptotic activities. The core crystallin domain within these proteins is largely responsible for these prosperities. Recent studies have identified peptides within the crystallin domain of both α- and αB-crystallins with remarkable chaperone and anti-apoptotic activities. Administration of α-crystallin or their functional peptides has shown substantial inhibition of pathologies in several diseases. However, α-crystallins have been shown to promote disease-causing pathways. These two sides of the proteins are discussed in this review. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Impact of Subunit Composition on the Uptake of α-Crystallin by Lens and Retina.

Misfolded protein aggregation, including cataract, cause a significant amount of blindness worldwide. α-Crystallin is reported to bind misfolded proteins and prevent their aggregation. We hypothesize that supplementing retina and lens with α-crystallin may help to delay disease onset. The purpose of this study was to determine if αB-crystallin subunits containing a cell penetration peptide (gC-tagged αB-crystallin) facilitate the uptake of wild type αA-crystallin (WT-αA) in lens and retina. Recombinant human αB-crystallin was modified by the addition of a novel cell penetration peptide derived from the gC gene product of herpes simplex virus (gC-αB). Recombinant gC-αB and wild-type αA-crystallin (WT-αA) were purified from E. coli over-expression cultures. After Alexa-labeling of WT-αA, these proteins were mixed at ratios of 1:2, 1:5 and 1:10, respectively, and incubated at 37°C for 4 hours to allow for subunit exchange. Mixed oligomers were subsequently incubated with tissue culture cells or mouse organ cultures. Similarly, crystallin mixtures were injected into the vitreous of rat eyes. At various times after exposure, tissues were harvested and analyzed for protein uptake by confocal microscopy or flow cytometry. Chaperone-like activity assays were performed on α-crystallins ratios showing optimal uptake using chemically-induced or heat induced substrate aggregation assays. As determined by flow cytometry, a ratio of 1:5 for gC-αB to WT-αA was found to be optimal for uptake into retinal pigmented epithelial cells (ARPE-19). Chaperone-like activity assays demonstrated that hetero-oligomeric complex of gC-αB to WT-αA (in 1:5 ratio) retained protein aggregation protection. We observed a significant increase in protein uptake when optimized (gC-αB to WT-αA (1:5 ratio)) hetero-oligomers were used in mouse lens and retinal organ cultures. Increased levels of α-crystallin were found in lens and retina following intravitreal injection of homo- and hetero-oligomers in rats.

Alpha-crystallin-mediated protection of lens cells against heat and oxidative stress-induced cell death.

In addition to their key role as structural lens proteins, α-crystallins also appear to confer protection against many eye diseases, including cataract, retinitis pigmentosa, and macular degeneration. Exogenous recombinant α-crystallin proteins were examined for their ability to prevent cell death induced by heat or oxidative stress in a human lens epithelial cell line (HLE-B3). Wild type αA- or αB-crystallin (WT-αA and WT-αB) and αA- or αB-crystallins, modified by the addition of a cell penetration peptide (CPP) designed to enhance the uptake of proteins into cells (gC-αB, TAT-αB, gC-αA), were produced by recombinant methods. In vitro chaperone-like assays were used to assay the ability of α-crystallins to protect client proteins from chemical or heat induced aggregation. In vivo viability assays were performed in HLE-B3 to determine whether pre-treatment with α-crystallins reduced death after exposure to oxidative or heat stress. Most of the five recombinant α-crystallin proteins tested conferred some in vitro protection from protein aggregation, with the greatest effect seen with WT-αB and gC-αB. All α-crystallins displayed significant protection to oxidative stress induced cell death, while only the αB-crystallins reduced cell death induced by thermal stress. Our findings indicate that the addition of the gC tag enhanced the protective effect of αB-crystallin against oxidative but not thermally-induced cell death. In conclusion, modifications that increase the uptake of α-crystallin proteins into cells, without destroying their chaperone-like activity and anti-apoptotic functions, create the potential to use these proteins therapeutically.

Varicella-zoster virus open reading frame 48 encodes an active nuclease.

Based on a DNA sequence and relative genomic position similar to those other herpesviruses, varicella-zoster virus (VZV) open reading frame 48 (ORF48) is predicted to encode an alkaline nuclease. Here we report the cloning, expression, purification, and characterization of recombinant VZV ORF48 protein and a VZV ORF48 point mutation (T172P). Protein encoded by wild-type ORF48, but not mutant protein, displayed both endo- and exonuclease activity, identifying ORF48 as a potential therapeutic target in VZV disease since efficient viral replication requires viral nuclease activity.

Cell penetration peptides for enhanced entry of αB-crystallin into lens cells.

PURPOSE: The prevalence of cataract increases with age. Conversely, the abundance of native α-crystallin diminishes with age and cataract development. We hypothesize replenishing lens α-crystallin may delay or prevent cataract. Herein we investigated the ability of cell penetration peptides (CPP) to enhance entry of α-crystallins into lens-derived cells. METHODS: Recombinant αB-crystallins were modified by the addition of CPPs. Candidate CPP were designed with reference to the HSV-1 glycoprotein C gene (gC) or the HIV-1 TAT peptide. αB-crystallins produced by fusing gC or TAT were over-expressed in E. coli. Purified proteins were subjected to size exclusion chromatography (SEC) to characterize oligomeric complexes (OC). Chaperone-like activity (CLA) was evaluated by measuring the ability of α-crystallins to suppress chemically-induced protein aggregation. To evaluate protein uptake, labeled α-crystallins were incubated with HLE B3 cells and monitored by fluorescence microscopy for 48 hours. RESULTS: We examined the effects of the addition of CPP on the structure, CLA, and cell transduction properties of αB-crystallins. C-terminal CPP fused crystallins had poor solubility. In contrast, N-terminal tagged αB-crystallins were soluble. These modified αB-crystallins formed OC that were larger than wild-type based on SEC. Wild-type and gC tagged αB-crystallin displayed robust CLA. Subunit exchange was observed when gC-fused αB-crystallin was mixed with αA. In contrast to wild-type, modified α-crystallins accumulated in HLE B3 cells. CONCLUSIONS: Addition of CPP improves the uptake of αB-crystallins into HLE B3 cells. No undesirable changes to the chaperone-like abilities of α-crystallins were observed in αB-crystallin modified by the addition of the gC-derived CPP.

Recombinant monoclonal antibody recognizes a unique epitope on varicella-zoster virus immediate-early 63 protein.

We previously constructed a recombinant monoclonal antibody (rec-MAb 63P4) that detects immediate-early protein IE63 encoded by varicella-zoster virus (VZV) in the cytoplasm of productively infected cells. Here, we used ORF63 truncation mutants to map the rec-MAb 63P4 binding epitope to amino acids 141 to 150 of VZV IE63, a region not shared with other widely used anti-IE63 antibodies, and found that the recombinant antibody does not bind to the simian IE63 counterpart.

Characterization of the 8-hydroxyquinoline scaffold for inhibitors of West Nile virus serine protease.

West Nile virus (WNV) is a mosquito-borne member of flaviviruses that causes significant morbidity and mortality especially among children. There is currently no approved vaccine or antiviral therapeutic for human use. In a previous study, we described compounds containing the 8-hydroxyquinoline (8-HQ) scaffold as inhibitors of WNV serine protease (NS2B/NS3pro) in a high throughput screen (HTS) using the purified WNV NS2B/NS3pro as the target. In this study, we analyzed potencies of some commercially available as well as chemically synthesized derivatives of 8-HQ by biochemical assays. An insight into the contribution of various substitutions of 8-HQ moiety for inhibition of the protease activity was revealed. Most importantly, the substitution of the N1 of the 8-HQ ring by -CH- in compound 26 significantly reduced the inhibition of the viral protease by this naphthalen-1-ol derivative. The kinetic constant (K(i)) for the most potent 8-HQ inhibitor (compound 14) with an IC(50) value of 2.01 ± 0.08 µM using the tetra-peptide substrate was determined to be 5.8 µM. This compound inhibits the WNV NS2B/NS3pro by a competitive mode of inhibition which is supported by molecular modeling.

Temperature-dependent structural and functional properties of a mutant (F71L) αA-crystallin: molecular basis for early onset of age-related cataract.

Previously we identified a novel mutation (F71L) in the αA-crystallin gene associated with early onset of age-related cataract. However, it is not known how the missense substitution translates into reduced chaperone-like activity (CLA), and how the structural and functional changes lead to early onset of the disease. Herein, we show that under native conditions the F71L-mutant is not significantly different from wild-type with regard to secondary and tertiary structural organization, hydrophobicity and the apparent molecular mass of oligomer but has substantial differences in structural and functional properties following a heat treatment. Wild-type αA-crystallin demonstrated increased CLA, whereas the F71L-mutant substantially lost its CLA upon heat treatment. Further, unlike the wild-type αA-subunit, F71L-subunit did not protect the αB-subunit in hetero-oligomeric complex from heat-induced aggregation. Moreover, hetero-oligomer containing F71L and αB in 3:1 ratio had significantly lower CLA upon thermal treatment compared to its unheated control. These results indicate that α-crystallin complexes containing F71L-αA subunits are less stable and have reduced CLA. Therefore, F71L may lead to earlier onset of cataract due to interaction with several environmental factors (e.g., temperature in this case) along with the aging process.

Simian varicella virus open reading frame 63/70 expression is required for efficient virus replication in culture.

Simian varicella virus (SVV) open reading frame (ORF) 63, duplicated in the virus genome as ORF 70, is homologous to varicella zoster virus ORF 63/70. Transfection of bacterial artificial chromosome clones containing the wild-type SVV genome and mutants with stop codons in ORF 70, in both ORFs 63 and 70 and the repaired virus DNA sequences into Vero cells produced a cytopathic effect (CPE). The onset of CPE was much slower with the double-mutant transfectants (10 days vs. 3 days) and plaques were smaller. While SVV ORF 63 is not required for replication in culture, its expression leads to robust virus replication.

Identification of phosphorylated residues on varicella-zoster virus immediate-early protein ORF63.

Efficient replication of varicella-zoster virus (VZV) in cell culture requires expression of protein encoded by VZV open reading frame 63 (ORF63p). Two-dimensional gel analysis demonstrates that ORF63p is extensively modified. Mass spectroscopy analysis of ORF63p isolated from transiently transfected HEK 293 and stably transfected MeWo cells identified 10 phosphorylated residues. In VZV-infected MeWo cells, only six phosphorylated residues were detected. This report identifies phosphorylation of two previously uncharacterized residues (Ser5 and Ser31) in ORF63p extracted from cells infected with VZV or transfected with an ORF63p expression plasmid. Computational analysis of ORF63p for known kinase substrates did not identify Ser5 or Ser31 as candidate phosphorylation sites, suggesting that either atypical recognition sequences or novel cellular kinases are involved in ORF63p post-translational modification.

Clinical and molecular aspects of varicella zoster virus infection.

A declining cell-mediated immunity to varicella zoster virus (VZV) with advancing age or immunosuppression results in virus reactivation from latently infected human ganglia anywhere along the neuraxis. Virus reactivation produces zoster, often followed by chronic pain (postherpetic neuralgia or PHN) as well as vasculopathy, myelopathy, retinal necrosis and cerebellitis. VZV reactivation also produces pain without rash (zoster sine herpete). Vaccination after age 60 reduces the incidence of shingles by 51%, PHN by 66% and the burden of illness by 61%. However, even if every healthy adult over age 60 years is vaccinated, there would still be about 500,000 zoster cases annually in the United States alone, about 200,000 of whom will experience PHN. Analyses of viral nucleic acid and gene expression in latently infected human ganglia and in an animal model of varicella latency in primates are serving to determine the mechanism(s) of VZV reactivation with the aim of preventing reactivation and the clinical sequelae.

Phosphorylation of the nuclear form of varicella-zoster virus immediate-early protein 63 by casein kinase II at serine 186.

Varicella-zoster virus (VZV) open reading frame (ORF) 63 is abundantly transcribed in latently infected human ganglia and encodes a 278-amino-acid protein, IE63, with immediate-early kinetics. IE63 is expressed in the cytoplasm of neurons during VZV latency and in both the cytoplasm and the nucleus during productive infection; however, the mechanism(s) involved in IE63 nuclear import and retention has remained unclear. We constructed and identified a recombinant monoclonal antibody to detect a posttranslationally modified form of IE63. Analysis of a series of IE63 truncation and substitution mutants showed that amino acids 186 to 195 are required for antibody binding. Synthetic peptides corresponding to this region identified IE63 S186 as a target for casein kinase II phosphorylation. In addition, acidic charges supplied by E194 and E195 were required for antibody binding. Immunofluorescence analysis of VZV-infected MeWo cells using the recombinant monoclonal antibody detected IE63 exclusively in the nuclei of infected cells, indicating that casein kinase II phosphorylation of S186 occurs in the nucleus and possibly identifying an initial molecular event operative in VZV reactivation.

Effects of detergents on the West Nile virus protease activity.

Detergents such as Triton X-100 are often used in drug discovery research to weed out small molecule promiscuous and non-specific inhibitors which act by aggregation in solution and undesirable precipitation in aqueous assay buffers. We evaluated the effects of commonly used detergents, Triton X-100, Tween-20, Nonidet-40 (NP-40), Brij-35, and CHAPS, on the enzymatic activity of West Nile virus (WNV) protease. Unexpectedly, Triton X-100, Tween-20, and NP-40 showed an enhancement of in vitro WNV protease activity from 2 to 2.5-fold depending on the detergent and its concentration. On the other hand, Brij-35, at 0.001% enhanced the protease activity by 1.5-fold and CHAPS had the least enhancing effect. The kinetic analysis showed that the increase in protease activity by Triton X-100 was dose-dependent. Furthermore, at Triton X-100 and Tween-20 concentrations higher than 0.001%, the inhibition of compound B, one of the lead compounds against WNV protease identified in a high throughput screen (IC(50) value of 5.7+/-2.5 microM), was reversed. However, in the presence of CHAPS, compound B still showed good inhibition of WNV protease. Our results, taken together, indicate that nonionic detergents, Triton X-100, Tween, and NP-40 are unsuitable for the purpose of discrimination of true versus promiscuous inhibitors of WNV protease in high throughput assays.

Identification and biochemical characterization of small-molecule inhibitors of west nile virus serine protease by a high-throughput screen.

West Nile virus and dengue virus are mosquito-borne flaviviruses that cause a large number of human infections each year. No vaccines or chemotherapeutics are currently available. These viruses encode a serine protease that is essential for polyprotein processing, a required step in the viral replication cycle. In this study, a high-throughput screening assay for the West Nile virus protease was employed to screen approximately 32,000 small-molecule compounds for identification of inhibitors. Lead inhibitor compounds with three distinct core chemical structures (1 to 3) were identified. In a secondary screening of selected compounds, two compounds, belonging to the 8-hydroxyquinoline family (compounds A and B) and containing core structure 1, were identified as potent inhibitors of the West Nile virus protease, with K(i) values of 3.2 +/- 0.3 microM and 3.4 +/- 0.6 microM, respectively. These compounds inhibited the dengue virus type 2 protease with K(i) values of 28.6 +/- 5.1 microM and 30.2 +/- 8.6 microM, respectively, showing some selectivity in the inhibition of these viral proteases. However, the compounds show no inhibition of cellular serine proteases, trypsin, or factor Xa. Kinetic analysis and molecular docking of compound B onto the known crystal structure of the West Nile virus protease indicate that the inhibitor binds in the substrate-binding cleft. Furthermore, compound B was capable of inhibiting West Nile virus RNA replication in cultured Vero cells (50% effective concentration, 1.4 +/- 0.4 microM; selectivity index, 100), presumably by inhibition of polyprotein processing.

Varicella zoster virus infection: clinical features, molecular pathogenesis of disease, and latency.

Varicella zoster virus (VZV) is an exclusively human neurotropic alphaherpesvirus. Primary infection causes varicella (chickenpox), after which virus becomes latent in cranial nerve ganglia, dorsal root ganglia, and autonomic ganglia along the entire neuraxis. Years later, in association with a decline in cell-mediated immunity in elderly and immunocompromised individuals, VZV reactivates and causes a wide range of neurologic disease. This article discusses the clinical manifestations, treatment, and prevention of VZV infection and reactivation; pathogenesis of VZV infection; and current research focusing on VZV latency, reactivation, and animal models.

Construction of recombinant mouse IgG1 antibody directed against varicella zoster virus immediate early protein 63.

Five varicella zoster virus (VZV) genes are known to be transcribed in latently infected human ganglia. Transcripts from VZV gene 63, which encodes an immediate early (IE) protein, are the most prevalent and abundant. To obtain a reagent that might facilitate studies of the role of the IE63 protein in latency and reactivation, we selected an IE63-specific Fab fragment from a phage library and used it to prepare a recombinant mouse IgG1 antibody that detects IE63 and functions in Western blot, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence assays.