Distinct oligomerization and NADPH binding modes observed between L. donovani and human quinone oxidoreductases.

Electron-driven process helps the living organism in the generations of energy, biomass production and detoxification of synthetic compounds. Soluble quinone oxidoreductases (QORs) mediate the transfer of an electron from NADPH to various quinone and other compounds, helping in the detoxification of quinones. QORs play a crucial role in cellular metabolism and are thus potential targets for drug development. Here we report the crystal structure of the NADPH-dependent QOR from Leishmania donovani (LdQOR) at 2.05 Å. The enzyme exists as a homo-dimer, with each protomer consisting of two domains, responsible for binding NADPH cofactor and the substrate. Interestingly, the human QOR exists as a tetramer. Comparative analysis of the oligomeric interfaces of LdQOR with HsQOR shows no significant differences in the protomer/dimer assembly. The tetrameric interface of HsQOR is stabilized by salt bridges formed between Arg 169 and Glu 271 which is non-existent in LdQOR, with an Alanine replacing the glutamate. This distinct feature is conserved across other dimeric QORs, indicating the importance of this interaction for tetramer association. Among the homologs, the sequences of the loop region involved in the stabilization and binding of the adenine ring of the NADPH shows significant differences except for an Arginine & glycine residues. In dimer QORs, this Arginine acts as a gate to the co-factor, while the NADPH binding mode in the human homolog is distinct, stabilized by His 200 and Asn 229, which are not conserved in LdQOR. These distinct features have the potential to be utilized for therapeutic interventions.

The Candida albicans ζ-crystallin homolog Zta1 promotes resistance to oxidative stress.

IMPORTANCE: Candida albicans is an important human pathogen that can cause lethal systemic infections. The ability of C. albicans to colonize and establish infections is closely tied to its highly adaptable nature and capacity to resist various types of stress, including oxidative stress. Previous studies showed that four C. albicans proteins belonging to the flavodoxin-like protein family of quinone reductases are needed for resistance to quinones and virulence. Therefore, in this study, we examined the role of a distinct type of quinone reductase, Zta1, and found that it acts in conjunction with the flavodoxin-like proteins to protect against oxidative stress.

The Candida albicans ζ-crystallin homolog Zta1 promotes resistance to oxidative stress.

The fungal pathogen Candida albicans is capable of causing lethal infections in humans. Its pathogenic potential is due in part to the ability to resist various stress conditions in the host, including oxidative stress. Recent studies showed that a family of four flavodoxin-like proteins (Pst1, Pst2, Pst3, Ycp4) that function as quinone reductases promotes resistance to oxidation and is needed for virulence. Therefore, in this study Zta1 was examined because it belongs to a structurally distinct family of quinone reductases that are highly conserved in eukaryotes and have been called the ζ-crystallins. The levels of Zta1 in C. albicans rapidly increased after exposure to oxidants, consistent with a role in resisting oxidative stress. Accumulation of reactive oxygen species was significantly higher in cells lacking ZTA1 upon exposure to quinones and other oxidants. Furthermore, deletion of ZTA1 in a mutant lacking the four flavodoxin-like proteins, resulted in further increased susceptibility to quinones, indicating that these distinct quinone reductases work in combination. These results demonstrate that Zta1 contributes to C. albicans survival after exposure to oxidative conditions, which increases the understanding of how C. albicans resists stressful conditions in the host.

Elucidation of kinetic and structural properties of eye lens ζ-crystallin: an in vitro and in silico approach.

The Arabian Camelus dromedarius contains significant concentration of eye lens ζ-crystallin. This enzyme is also present in other life forms including humans, however in lower catalytic amounts. The recombinant camel ζ-crystallin was expressed in the E. coli BL21 (DE3) pLysS strain and purified using HisTrap column. The Km of the enzyme for 9,10-phenanthrenequinone (9,10-PQ) substrate and NADPH cofactor was determined to be 11.66 and 50.93 µM, respectively. The Vmax for 9,10-PQ and NADPH was obtained as 23.19 and 19.98 µM min-1, respectively. The optimum activity of the purified enzyme was found to be at pH 6.0 and at 55 °C. Different physico-chemical parameters were analysed including instability index (II), aliphatic index (AI) and the GRAVY index to establish proper characterization. The sequence of the recombinant ζ-crystallin was subjected to homology modelling using SWISS-MODEL webserver followed by validation of the modelled target structure. The evaluation of the modelled ζ-crystallin was performed by several parameters including Ramachandran plot, Z-score values followed by molecular dynamics (MD) simulation. The cumulative analysis of the physico-chemical, quantitative, qualitative and the essential dynamics of simulation of ζ-crystallin and its complexes with 9,10-PQ and NADPH helped in verifying the acceptable quality and stability of the ζ-crystallin structure.Communicated by Ramaswamy H. Sarma.

An insight into the binding and inhibition of eye ζ-crystallin by the environmental toxin arsenic: implications in eye diseases.

Arsenic contamination is highly prevalent in food chain, soil and groundwater. Continuous exposure to elevated levels of this environmental toxin is a global concern. Studies have reported enriched accumulation of arsenic in the eyes compared to other body organs leading to various eye diseases. Here, the impact of arsenic exposure on the enzymatic eye ζ-crystallin has been investigated. Arsenic inhibited the activity of the enzyme with an IC50 value of 35 µM. It decreased the free thiol group content of ζ-crystallin due to protein oxidation. The binding of arsenic with ζ-crystallin was explored using biophysical and computational tools. The enzyme undergoes some conformational changes upon arsenic binding. The binding constant (Kb) was determined to be 1.2 × 102 M-1. Thermodynamic parameters were determined by isothermal titration calorimetry (ITC) and the binding energy (ΔG) was calculated to be -3.52 kcal/mol. Molecular docking studies helped in visualizing the amino acid residues (especially Cys165) of the enzyme involved in binding with arsenic. Continuous arsenic exposure is expected to increase the eye crystallin-related abnormalities, elevating the risk of cataractogenesis. Therefore, proper measures need to be taken by authorities to control the contamination of arsenic in the environment and groundwater.Communicated by Ramaswamy H. Sarma.

Protection of ζ-crystallin by α-crystallin under thermal stress.

Cataract is one of the major causes of blindness worldwide. Several factors including post-translational modification, thermal and solar radiations promote cataractogenesis. The camel lens proteins survive very harsh desert conditions and resist cataractogenesis. The folding and aggregation mechanism of camel lens proteins are poorly characterized. The camel lens contains three ubiquitous crystallins (α-, ß-, and γ-crystallin) and a novel protein (ζ-crystallin) in large amounts. In this study, a sequence similarity search of camel α-crystallin with that of other organisms showed that the camel αB-crystallin consists of an extended N-terminal domain. Our results indicate that camel α-crystallin efficiently prevented aggregation of ζ-crystallin, with or without an obligate cofactor up to 89 °C. It performed a quick and efficient holdase function irrespective of the unfolding stage or aggregation. Camel α-crystallin exhibits approximately 20% chaperone activity between 30 and 40 °C and is completely activated above 40 °C. Camel α-crystallin underwent a single reversible thermal transition without loss of ß-sheet secondary structure. Intrinsic tryptophan fluorescence and ANS binding experiments revealed two transitions which corresponded to activation of its chaperone function. In contrast to earlier studies, camel α-crystallin completely protected lens proteins during thermal stress.

Structural stability and solubility of glycated camel lens ζ-crystallin.

The camel has several biochemical, physiological, and anatomical features to withstand the harsh desert climate. Camel eye lens contains a novel protein (ζ-crystallin) in bulk quantity. Previous reports suggest that non-enzymatic glycation of eye lens proteins plays an important role in the etiology of cataract. In this study, we have characterized the role of glucose, fructose, and methylglyoxal (MGO) in the glycation of camel lens ζ-crystallin. From the results obtained, it was found that MGO reacted rapidly, fructose reacted moderately, and glucose was the least reactive even after prolonged incubation (>100 days). Glycation with MGO and fructose led to changes in the structure of ζ-crystallin, while glucose had no remarkable effect. The surface hydrophobicity did not change and no aggregates or amyloid fibrils were observed in the glycated ζ-crystallin. Moreover, the secondary structure of glycated ζ-crystallin remained similar after glycation. Our results suggested that due to natural adaptation, the camel lens protein ζ-crystallin retained its structure and solubility even after glycation to perform the single known function of the lens proteins: to focus unscattered light on the retina.

Spectral and thermal properties of novel eye lens ζ-crystallin.

Eye lenses are exposed to thermal, solar radiations, dryness that enhances cataractogenesis. Some animal lenses contain novel proteins in bulk quantities. ζ-crystallin occurred in three ecologically divergent species, but it's physiological role not known. The truncated variant of ζ-crystallin causes hereditary cataract. Guinea pig ζ-crystallin is temperature-sensitive and rapidly aggregates at 41°C. Camels adopted to survive above 50°C, which raises an interesting question about how it retains lens proteins in the soluble state? Here, we have optimized expression and purification of recombinant camel ζ-crystallin. We have studied thermodynamic and spectroscopic properties using orthogonal techniques. Dynamic multimode spectroscopy results showed that camel ζ-crystallin unfolds via single transition with Tm value of 60.8±0.1°C and van't Hoff enthalpy of 714.7±7.1kJ/mol. Thermal-shift assay calculates Tm value of 62°C at pH 7. Additionally, the conformational stability of ζ-crystallin increases with ionic-strength. The influence of pH on ζ-crystallin was evaluated where the protein was found to be stable in the pH range of 6-9, but its stability drastically decreases below pH 6. Our results also showed that quaternary structure of ζ-crystallin drastically changed as a result of lowering pH. This study provides significant understandings onto the conformational, thermodynamic and unfolding pathway of camel ζ-crystallin.

Simultaneous separation of taxon-specific crystallins from Mule duck and characterization of their enzymatic activities and structures.

Methods to obtain pure proteins in large amounts are indispensible in protein research. We report here a large-scale/simultaneous isolation of taxon-specific crystallins (ɛ- and δ-crystallin) from the eye lenses of Mule duck. We also investigate the compositions, enzymatic activities, and structures of these purified taxon-specific proteins. A relatively mild method of ion-exchange chromatography was developed to fractionate ɛ-crystallin and δ-crystallin in large amount, ca. ∼6.60mg/g-lens and ∼41.0mg/g-lens, respectively. Both crystallins were identified by electrophoresis, HPLC, and MALDI-TOF-MS. ɛ-Crystallin, with native composition of Mr 142kDa, consisted of two subunits of 35kDa and 36kDa, while δ-Crystallin, with native molecular mass of 200kDa, comprised single subunit of Mr ∼50kDa. Both ɛ- and δ-crystallin were tetramers. The former showed lactate dehydrogenase (LDH) activity, while the latter appeared slightly active in an argininosuccinate lyase (ASL) assay. Raman spectroscopic results indicated that the secondary structures of ɛ- and δ-crystallin were predominantly α-helix as evidenced by the vibrational stretching of amide III over 1260cm-1 and amide I at 1255cm-1, in greatly contrast to the anti-parallel ß-sheet of α- and ß-crystallin as demonstrated by amide III at 1238cm-1 and amide I at 1672cm-1. The microenvironments of aromatic amino acids and the status of thiol groups also vary in different crystallins. The compositions, enzyme activities, and structures of the ɛ- and δ-crystalline of Mule duck are different from those of Muscovy duck (Cairina moschata) or Kaiya duck (Anas Platyrhynchos var. domestica), which reflect faithfully species specificity.

Dual Roles for Epithelial Splicing Regulatory Proteins 1 (ESRP1) and 2 (ESRP2) in Cancer Progression.

Epithelial splicing regulatory protein 1 (ESRP1) and 2 (ESRP2) are members of the hnRNP family of RNA binding proteins that regulate alternative splicing events associated with epithelial phenotypes. These proteins play crucial roles during organogenesis, including craniofacial and epidermal development as well as branching morphogenesis in the lungs and salivary glands. Recent reports have also addressed their roles during cancer progression. Expression of ESRP proteins is low in normal epithelium but upregulated in carcinoma in situ and advanced carcinomas. Intriguingly, they are downregulated in invasive fronts. The plastic nature of ESRP expression suggests dual roles for them in cancer progression. Consistently, it has been shown that ESRPs suppress motility and anchorage-independent growth of cancer cells while supporting cell survival by enhancing resistance to reactive oxygen species. Regulatory circuits that fine-tune ESRP gene expression have recently emerged. Here, we summarize recent findings on the molecular mechanisms by which ESRPs exert positive as well as negative effects on cancer progression.

Crystallization and preliminary X-ray diffraction analysis of the Pax9 paired domain bound to a DC5 enhancer DNA element.

Pax genes belong to a family of metazoan transcription factors that are known to play a critical role in eye, ear, kidney and neural development. The mammalian Pax family of transcription factors is characterized by a ∼128-amino-acid DNA-binding paired domain that makes sequence-specific contacts with DNA. The diversity in Pax gene activities emerges from complex modes of interaction with enhancer regions and heterodimerization with multiple interaction partners. Based on in vitro optimal binding-site selection studies and enhancer identification assays, it has been suggested that Pax proteins may recognize and bind their target DNA elements with different binding modes/topologies, however this hypothesis has not yet been structurally explored. One of the most extensively studied DNA target elements of the Pax6 paired domain is the eye-lens specific DC5 (δ-crystallin) enhancer element. In order to shed light on Pax6-DC5 DNA interactions, the related paired-domain prototype Pax9 was crystallized with the minimal δ-crystallin DC5 enhancer element and preliminary X-ray diffraction analysis was attempted. A 3.0 Šresolution native data set was collected at the National Synchrotron Light Source (NSLS), Brookhaven from crystals grown in a solution consisting of 10%(w/v) PEG 20K, 20%(v/v) PEG 550 MME, 0.03 M NaNO3, 0.03 M Na2HPO4, 0.03 M NH2SO4, 0.1 M MES/imidazole pH 6.5. The data set was indexed and merged in space group C2221, with unit-cell parameters a = 75.74, b = 165.59, c = 70.14 Å, α = ß = γ = 90°. The solvent content in the unit cell is consistent with the presence of one Pax9 paired domain bound to duplex DNA in the asymmetric unit.

δEF1 upregulates CDK4 transcription via the E2-box element on the CDK4 promoter.

The zinc finger-homeodomain transcription factor, δ-crystallin enhancer factor 1 (δEF1) has been identified as a regulatory factor involved in the promotion of breast cancer cell proliferation via the downregulation of p21 and the upregulation of cyclin-dependent kinase-2 (CDK2) and CDK4 expression. However, the molecular mechanisms underlying the regulation of CDK4 expression by δEF1 have not yet been elucidated. The present study demonstrated that the ectopic expression of δEF1 in MDA-MB-231 breast cancer cells significantly increased the activity of the CDK4 promoter. Deletion of the E2-box (CACGTG), which is located at position -197/-191 on the human CDK4 promoter, significantly attenuated the activation of CDK4 transcription by δEF1. In addition, a CDK4 promoter-M construct was generated via site-directed mutagenesis of the E2-box on the human CDK4 promoter. Luciferase assay showed that the activation of CDK4 promoter-M activity by δEF1 was markedly decreased compared with the CDK4-promoter-0.4k promoter. Knockdown of δEF1 using RNA interference resulted in the inhibition of CDK4 promoter activity. These observations suggest that δEF1 upregulates CDK4 transcription via the E2-box element on the CDK4 promoter.

Stimulation of crystallin synthesis in embryonic brain cells in culture.

Expression of δ-crystallin, a lens-specific protein, in 6-day-old chick embryonic brain cells was examined in situ and in vitro. The presence of minute amounts of δ-crystallin and its mRNA (δ-mRNA) in brain cells in situ was demonstrated by immunoblot and Northern blot analysis. In spreading cultures of the brain cells, δ-crystallin and δ-mRNA showed a significant increase from their in situ level. Immunohistological staining (peroxidase antiperoxidase) with monospecific anti-serum against δ-crystallin revealed that δ-producers were both epithelial cells and dendritic cells. Neither lentoidogenesis nor α-crystallin expression was observed. Stimulation of δ-crystallin synthesis in cultured brain cells differed when compared with transdifferentiating cultures of neural retina cells. In the latter, δ-crystallin synthesis occurred concomitantly with differentiation of morphologically distinct lens cells containing α-crystallin.