Aquaporin 9 regulates Leydig cell steroidogenesis in diabetes.

Diabetes mellitus induced hyperglycemia increases oxidative stress, which contributes to impairment of male reproductive function. Aquaporins (AQPs) belong to a transmembrane protein superfamily containing 13 isoforms (AQP0-12), differentially expressed in various organs, and play a pivotal role in male reproductive function. In the current study, we investigated the relationship between AQPs and testicular steroidogenesis under hyperglycemia in vivo and in vitro. The effect of high glucose on the role of AQPs in Leydig cell steroidogenesis was analyzed in diabetic rats (in-vivo) and LC540 rat Leydig cells (in vitro) via enzyme assays, quantitative RT-PCR, siRNA knock down and western blotting. AQP 9 was significantly up-regulated in STZ-induced diabetic rat testis and high glucose treated LC540 cells. Further, oxidative stress marker nuclear factor erythroid 2-related factor 2 (Nrf2) expression was decreased with impaired testicular steroidogenesis under hyperglycemia. Knock-down of AQP 9 resulted in increased Nrf2 expression and thus increased testicular steroidogenesis in hyperglycemia. Diabetes-associated hyperglycemia induced oxidative stress is a widely proven cause for diabetes-related male infertility. Our results collectively suggest that AQP 9 impairs testicular steroidogenesis via the regulation of oxidative stress in diabetes.

Construction of Genomic Library and Screening of Edwardsiella tarda Immunogenic Proteins for Their Protective Efficacy Against Edwardsiellosis.

Edwardsiella tarda is a severe aquaculture pathogen that can infect many hosts including humans, animals, and fish. Timely diagnosis and treatment are crucial for the control of edwardsiellosis in the aqua industry. By using rabbit polyclonal antibody, an expression gene library of virulent Edwardsiella tarda strain ED-BDU 1 isolated in south India was constructed and screened. The identified immune expressive proteins were characterized, and the corresponding coding sequences were cloned, expressed, and the purified recombinant proteins were used as antigens. The identified immunoreactive proteins namely HflC, HflK, and YhcI were studied for their immune protective potential in vivo by challenge experiments. The protective efficacy of HflC, HflK, and YhcI showed that the clearance of Edwardsiella from the host with ~ 60% survivability. Further, the immunoreactive proteins induce a strong immune response upon infection and elicit the significant production of IL-10, IFN-γ, Th1, and Th2 mediated mRNA expression and were therefore effective in vaccine production for edwardsiellosis.

The possible role of sirtuins in male reproduction.

Global influence of male infertility is increasing in recent decades. Proper understanding of genetics, anatomy, physiology and the intricate interrelation of male reproductive system are much needed for explaining the etiology of male infertility; and a detailed study on the epigenetics, indeed, will reveal the molecular mechanism behind its etiology. Sirtuins, the molecular sensors, are NAD+ dependent histone deacetylases and ADP- ribosyl transferases, participate in the chief events of epigenetics. In mammals, sirtuin family comprises seven members (SIRT1-SIRT7), and they all possess a conserved NAD+ binding catalytic domain, termed the sirtuin core domain which is imperative for their activity. Sirtuins exert a pivotal role in cellular homeostasis, energy metabolism, apoptosis, age-related disorders and male reproductive system. However, their exact role in male reproduction is still obscure. This article specifically reviews the role of mammalian sirtuins in male reproductive function, thereby, prompting further research to discover the restorative methods and its implementation in reproductive medicine.

Could aquaporin modulators be employed as prospective drugs for COVID-19 related pulmonary comorbidity?

COVID-19 initially an epidemic caused by SARS-CoV-2 has turned out to be a life- threatening global pandemic with increased morbidity and mortality. The presence of cytokine storm has been linked with the pathogenesis of severe lung injury as evinced in COVID-19. Aquaporins (AQPs) are molecular water channels, facilitating water transport across the cell membrane in response to osmotic gradients. Impairment in alveolar fluid clearance due to altered functional expression of respiratory AQPs highlight their pathophysiological significance in pulmonary edema associated respiratory illness. Therefore, we hypothesize that targeted modulation of AQPs in lungs in the intervening period of time, could diminish the dreadful effects of inflammation- induced comorbidity in COVID-19.

Role of Aquaporins in Inflammation-a Scientific Curation.

Inflammation is a universal response mechanism existing as inter-communicator of biological systems. Uncontrolled or dysregulated inflammation addresses chronic low-grade effects eventually resulting in multimorbidity. Active solute transport across the membrane establishes varying osmotic gradients. Aquaporins (AQPs) are a class of critical ubiquitously expressed transmembrane proteins that aid in fluid and small solute transport via facilitated diffusion over established osmotic gradients. Numerous significant data features the biological functions of AQPs rendering them as an appropriate biomarker of health and diseases. Besides their physiological role in well-balanced inflammatory responses, it is worth noting the dysregulation of AQPs during any undesirable inflammatory event. Most literature to date clearly sets out AQPs as potential drug targets instigating AQP-based therapies. In light of this conception, the current review provides a compendious overview on the propitious and portentous out-turns of AQPs under inflammation.

Multidimensional significance of crystallin protein-protein interactions and their implications in various human diseases.

BACKGROUND: Crystallins are the important structural and functional proteins in the eye lens responsible for refractive index. Post-translational modifications (PTMs) and mutations are major causative factors that affect crystallin structural conformation and functional characteristics thus playing a vital role in the etiology of cataractogenesis. SCOPE OF REVIEW: The significance of crystallin protein-protein interactions (PPIs) in the lens and non-lenticular tissues is summarized. MAJOR CONCLUSIONS: Aberrancy of PPIs between crystallin, its associated protein and metal ions has been accomplished in various human diseases including cataract. A detailed account on multidimensional structural and functional significance of crystallin PPI in humans must be brought into limelight, in order to understand the biochemical and molecular basis augmenting the aberrancies of such interaction. In this scenario, the present review is focused to shed light on studies which will aid to expand our present understanding on disease pathogenesis related to loss of PPI thereby paving the way for putative future therapeutic targets to curb such diseases. GENERAL SIGNIFICANCE: The interactions with α-crystallins always aid to protect their structural and functional characteristics. The up-regulation of αB-crystallin in the non-lenticular tissues always decodes as biomarker for various stress related disorders. For better understanding and treatment of various diseases, PPI studies provide overall outline about the structural and functional characteristics of the proteins. This information not only helps to find out the route of cataractogenesis but also aid to identify potential molecules to inhibit/prevent the further development of such complicated phenomenon. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Bacteriocinogenic potential of a probiotic strain Bacillus coagulans [BDU3] from Ngari.

Bacteriocin producing strain BDU3 was isolated from a traditional fermented fish of Manipur Ngari. The strain BDU3 was identified as Bacillus coagulans by phenotypic and genotypic characterization. The BDU3 produced novel bacteriocin, which showed an antimicrobial spectrum toward a wide spectrum of food borne, and closely related pathogens with a MIC that ranged between 0.5 and 2.5 µg/mL. The isolate was able to tolerate pH as low as 2.0 and up to 0.2% bile salt concentration. Three step purification was employed to increase the specific activity of the antimicrobial compound. The fractions were further chromatographed by Rp-HPLC C-18 column and the purified bacteriocin had a specific activity of ∼8500 AU/mg. However, the potency of bacteriocin was susceptible to digestion with Proteinase K, Pepsin, SDS, EDTA and Urea. Molecular mass of purified bacteriocin was found to be 1.4 kDa using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). The functional group was revealed by FTIR analysis. The cytotoxicity assay (MTT) using purified bacteriocin showed 2 times lower EC50 values compared to SDS. This is the smaller bacteriocin ever reported before from B. coagulans with greater antimicrobial potency with lower cytotoxicity. This bacteriocin raises the possibilities to be used as a biopreservative in food industries.

Real-time heterogeneous protein-protein interaction between αA-crystallin N-terminal mutants and αB-crystallin using quartz crystal microbalance (QCM).

The lens transparency depends on higher concentration of lens proteins and their interactions. α-Crystallin is one of the predominant lens proteins, responsible for proper structural and functional architecture of the lens microenvironment, and any alteration of which results in cataract formation. The R12C, R21L, R49C and R54C are the most significant and prevalent αA-crystallin congenital cataract-causing mutants worldwide. Protein-protein interaction, crucial for lens proper structure and function, was posited to be lost due to point mutation and the elucidation of which could shed light on the molecular basis of cataract. In this conjuncture, we report quartz crystal microbalance (QCM) as a warranted technique for real-time analysis of protein-protein interaction between the N-terminal mutants of αA-crystallin and αB-crystallin. The biophysical characteristics of the mutated proteins were determined by size-exclusion HPLC, far-UV circular dichroism and fluorescence studies. Far-UV circular dichroism spectral analysis displayed slight modifications in ß-sheet of R54C mutant. Altered intrinsic tryptophan fluorescence and decreased bis-ANS fluorescence were observed in all the N-terminal mutations revealing the tertiary structural changes and decreased exposure of surface hydrophobicity. An emphatic fall in the chaperone activity was observed in the N-terminal mutants, R12C, R21L and R54C. QCM analysis revealed the occurrence of strong heterogeneous interaction between αA-crystallin and αB-crystallin. Nevertheless, decreased interactions were observed with the N-terminal mutants. In summary, the present study concludes that the loss of interactions between αA-crystallin N-terminal mutants and αB-crystallin signifies quaternary structural alterations due to mutation in the arginine residues.

Interaction of αA-crystallin F71L mutant with wild type αA- and αB-crystallins by mammalian two hybrid assay.

Incidence of age related cataract (ARC) increases by a variety of factors including metabolic and environmental factors. Nonetheless, genetic mutations are responsible for the altered structural stability of the proteins, especially; the F71L mutation in αA-crystallin has been shown to be responsible for the incidence of cataracts. However, structural characteristics and chaperone function of this mutant and its interaction with wild type (WT) crystallins may aid to decipher its role in cataractogenesis. The aim of the present study is to show the interaction of F71L mutant protein with the WT α-crystallins. The F71L mutant used in this study was created by site-directed mutagenesis, overexpressed and purified. Biophysical characteristics determined by size exclusion HPLC, DLS, CD spectrometry, tryptophan fluorescence and surface hydrophobicity did not show significant structural changes in the mutant protein compared to WT counterpart. Interestingly, the F71L mutant displayed a significant loss in homogenous interaction with WT αA-crystallin and F71L mutant as well as heterogeneous interaction with αB-crystallin as evaluated by mammalian two hybrid system. Our findings suggest that F71L loses the ability to form homo and hetero-oligomers seems to result in the loss in chaperone like activity (CLA) and refractive index resulting in the development of cataracts.

Transcriptional regulation of crystallin, redox, and apoptotic genes by C-Phycocyanin in the selenite-induced cataractogenic rat model.

PURPOSE: This study was designed to examine the constrictive potential of C-Phycocyanin (C-PC) in regulating changes imposed on gene expression in the selenite-induced cataract model. METHODS: Wistar rat pups were divided into three groups of eight each. On P10, Group I received an intraperitoneal injection of normal saline. Groups II and III received a subcutaneous injection of sodium selenite (19 µmol/kg bodyweight); Group III also received an intraperitoneal injection of C-PC (200 mg/kg bodyweight) on P9-14. Total RNA was isolated on P16, and the relative abundance of mRNA of the crystallin structural genes, redox components, and apoptotic cascade were ascertained with real-time PCR with reference to the internal control ß-actin. RESULTS: Real-time PCR analysis showed the crystallin genes (αA-, ßB1-, γD-) and redox cycle components (Cat, SOD-1, Gpx) were downregulated, the apoptotic components were upregulated, and antiapoptotic Bcl-2 was downregulated in Group II. Treatment with 200 mg/kg bodyweight C-PC (Group III) transcriptionally regulated the instability of the expression of these genes, thus ensuring C-PC is a prospective anticataractogenic agent that probably delays the onset and progression of cataractogenesis induced by sodium selenite. CONCLUSIONS: C-PC treatment possibly prevented cataractogenesis triggered by sodium selenite, by regulating the lens crystallin, redox genes, and apoptotic cascade mRNA expression and thus maintains lens transparency. C-PC may be developed as a potential antioxidant compound applied in the future to prevent and treat age-related cataract.

Characterization of Bacillus spp. from the gastrointestinal tract of Labeo rohita--towards to identify novel probiotics against fish pathogens.

The aim of the present study is to screen and characterize endogenous microbiota Bacillus spp. from the gastrointestinal (GI) tract of Labeo rohita in order to evaluate their probiotic attributes. A total of 74 isolates from the GI of L. rohita were evaluated for their antimicrobial properties by agar well-diffusion method against fish pathogens. Based on the better antibacterial features, three isolates (KADR1, KADR3, and KADR4) were selected for further delineation. The three selected isolates exhibited higher tolerance to bile salt, moderate tolerance to low pH, high surface hydrophobicity to solvents, and capable to autoaggregate. All three isolates demonstrated notable proteolytic, catalase activity and susceptibility to various antibiotics. Partial 16S rRNA sequencing revealed that the isolates exhibited 99 % sequence homology with Bacillus subtilis, Bacillus aerophilus, and Bacillus firmus of the database substantiating morphological and physiological characterization. Survivability in low pH and bile salt ensures their adaptability in the fish intestinal microenvironment. The ability to autoaggregate reveals colonization potential in the GI of the fish. Absence of hemolytic activity, antibiotic susceptibility to certain antibiotics, presence of protease and catalase activity, and non-pathogenic caliber of the above-mentioned isolates could be feasible characteristics when considering them as probiotics in the aquaculture industry.

Comparison of effect of gamma ray irradiation on wild-type and N-terminal mutants of αA-crystallin.

PURPOSE: To study the comparative structural and functional changes between wild-type (wt) and N-terminal congenital cataract causing αA-crystallin mutants (R12C, R21L, R49C, and R54C) upon exposure to different dosages of gamma rays. METHODS: Alpha A crystallin N-terminal mutants were created with the site-directed mutagenesis method. The recombinantly overexpressed and purified wt and mutant proteins were used for further studies. A (60)Co source was used to generate gamma rays to irradiate wild and mutant proteins at dosages of 0.5, 1.0, and 2.0 kGy. The biophysical property of the gamma irradiated (GI) and non-gamma irradiated (NGI) αA-crystallin wt and N-terminal mutants were determined. Oligomeric size was determined by size exclusion high-performance liquid chromatography (HPLC), the secondary structure with circular dichroism (CD) spectrometry, conformation of proteins with surface hydrophobicity, and the functional characterization were determined regarding chaperone activity using the alcohol dehydrogenase (ADH) aggregation assay. RESULTS: αA-crystallin N-terminal mutants formed high molecular weight (HMW) cross-linked products as well as aggregates when exposed to GI compared to the NGI wt counterparts. Furthermore, all mutants exhibited changed ß-sheet and random coil structure. The GI mutants demonstrated decreased surface hydrophobicity when compared to αA-crystallin wt at 0, 1.0, and 1.5 kGy; however, at 2.0 kGy a drastic increase in hydrophobicity was observed only in the mutant R54C, not the wt. In contrast, chaperone activity toward ADH was gradually elevated at the minimum level in all GI mutants, and significant elevation was observed in the R12C mutant. CONCLUSIONS: Our findings suggest that the N-terminal mutants of αA-crystallin are structurally and functionally more sensitive to GI when compared to their NGI counterparts and wt. Protein oxidation as a result of gamma irradiation drives the protein to cross-link and aggregate culminating in cataract formation.

Antioxidant activity of exopolysaccharide from probiotic strain Enterococcus faecium (BDU7) from Ngari.

"Ngari" is a traditional fermented fish of Manipur and considered for its therapeutic value in healing stomach ulcers. In the present study, an attempt was made to isolate and identify an efficient antioxidant probiotic isolate from Ngari. BDU7 with potent antioxidant property was isolated and characterized. The isolate was identified by 16S rRNA genotyping as Enterococcus faecium. E. faecium showed auto aggregation and hydrophobicity of 72.7 and 54.8% respectively. The extrapolysaccharide (EPS) was extracted from the culture free supernatant and assayed for its radical scavenging activity. The EPS showed significant 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (63.5%), superoxide (77.3%) and hydroxyl (38.4%) radical scavenging ability. The structural analysis of the extracted and purified EPS was performed by FTIR and NMR analysis. From the present study E. faecium BDU7 can be claimed as a promising and an efficient probiotic candidate. The present study evidenced that EPS from E. faecium BDU7 showed strong DPPH and superoxide radical scavenging ability in vitro. Considering its potency as a potential antioxidant the extracted EPS can find wide application in functional food and pharmaceutical formulations.

Protective role of C-phycocyanin against secondary changes during sodium selenite mediated cataractogenesis.

Age related cataract is the leading cause of blindness associated with accumulation of oxidative stress in the eye lens. The present investigation reveals the rational of the beneficial effects of the natural compound C-phycocyanin (C-PC) is beneficial when administered to rat pups to protect against the secondary effects of sodium selenite induced cataractogenesis. A single subcutaneous dose of sodium selenite (19 µmol/kg body weight) on the 10th day of postpartum is adequate to induce cataract in rat pups. Serum biochemical parameters, such as the level of electrolytes, mean activities of anti-oxidant enzymes i.e. superoxide dismutase, catalase and reduced glutathione were observed to be significantly altered during selenite induced cataractogenic process. Histopathological examination revealed signs of degradation of normal cell architecture in the liver, kidney and eye lens. Interestingly, the deleterious effects of sodium selenite toxicity were restored with the simultaneous treatment with C-PC. The results suggest that an administration of 200 mg/kg body weight of C-PC has the ability to prevent/alter the secondary changes reflected in the serum biochemical and histological modifications in rats exposed to sodium selenite. These results complement the beneficial role of C-PC of cyanobacterial origin as a efficacious anti-cataractogenic agent against sodium selenite toxicity.

B-cell-specific peptides of leptospira interrogans LigA for diagnosis of patients with acute leptospirosis.

Leptospirosis is a reemerging infectious disease that is underdiagnosed and under-recognized due to low-sensitivity and cumbersome serological tests. Rapid reliable alternative tests are needed for early diagnosis of the disease. Considering the importance of the pathogenesis-associated leptospiral LigA protein expressed in vivo, we have evaluated its application in the diagnosis of the acute form of leptospirosis. The C-terminal coding sequence of ligA (ligA-C) was cloned into pET15b and expressed in Escherichia coli. Furthermore, the B-cell-specific epitopes were predicted and were synthesized as peptides for evaluation along with recombinant LigA-C. Epitope 1 (VVIENTPGK), with a VaxiJen score of 1.3782, and epitope 2 (TALSVGSSK), with a score of 1.2767, were utilized. A total of 140 serum samples collected from leptospirosis cases during the acute stage of the disease and 138 serum samples collected from normal healthy controls were utilized for evaluation. The sensitivity, specificity, positive predictive value, and negative predictive value were calculated for the recombinant LigA-C-specific IgM enzyme-linked immunosorbent assay (ELISA) and were found to be 92.1%, 97.7%, 92.8%, and 97.5%, respectively. Epitopes 1 and 2 used in the study showed 5.1 to 5.8% increased sensitivity over recombinant LigA-C in single and combination assays for IgM antibody detection. These findings suggest that these peptides may be potential candidates for the early diagnosis of leptospirosis.

Antimicrobial and antibiofilm activity of designed and synthesized antimicrobial peptide, KABT-AMP.

Lysine-rich peptide, designated as KABT-AMP, was designed and synthesized to supersede the irrational use of chemical antibiotics as standard therapy. KABT-AMP is a 22-amino acid helical cationic peptide (+10) and amphipathic in nature. The antimicrobial kinetics of the peptide was ascertained in the representative strains of gram-positive, gram-negative, and fungal strains, viz., Staphylococcus aureus MTCC 2940, Escherichia coli MTCC 2939, and Candida albicans MTCC 227, respectively. KABT-AMP was synthesized by solid-phase synthesis and purified using reverse-phase high-performance liquid chromatography which resulted in >95 % purity, and matrix-assisted laser desorption/ionization time of flight revealed the mass of the peptide to be 2.8 kDa. KABT-AMP showed significant broad-spectrum antimicrobial activity against the bacterial and fungal strains analyzed in the present study with survivability of 30.8, 30.6, and 31.7 % in E. coli, S. aureus, and C. albicans, respectively, at 6 h. KABT-AMP also demonstrated antibiofilm activity against the tested biofilm forming clinical isolate, Candida tropicalis. The putative membranolytic activity of the peptide was substantiated by electron microscopic analysis. Results reveal that KABT-AMP will exhibit noteworthy antimicrobial activity against multidrug-resistant bacteria and fungus at micromolar concentrations with minimal cytotoxicity and thus could be conceived for biomedical application.

C-phycocyanin modulates selenite-induced cataractogenesis in rats.

The present investigation is aimed to evaluate the anticataractogenic potential of C-phycocyanin (C-PC), extracted and purified from Spirulina platensis. Enucleated rat lenses were maintained in vitro in Dulbecco's modified Eagle medium (DMEM). Group I contained DMEM, Group II and Group III contained 100 µM of sodium selenite, Group III was subdivided into three viz IIIa, IIIb, IIIc supplemented with 100, 150, 200 µg of C-PC respectively. In the in vivo study, on tenth day post partum: Group I rat pups received an intraperitoneal injection of saline, Group II, IIIa, IIIb, and IIIc rat pups received a subcutaneous injection of sodium selenite (19 µmol/kg bodyweight) Group IIIa, IIIb, IIIc also received an intraperitoneal injection of 100, 150, 200 mg/kg body weight of C-PC, respectively, from postpartum days 9-14. On termination of the experiment, the lenses from both in vitro and in vivo studies were subjected to morphological examination and subsequently processed to estimate the activities of antioxidant enzymes namely superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, levels of reduced glutathione and lipid peroxidation products. Sodium selenite-exposed, C-PC-treated rat lenses (Group IIIc), showed significant restoration of antioxidant enzyme activity (p < 0.05) when compared to their counterpart Group II. Group IIIc conserved the levels of GSH and lipid peroxidation products at near to normal levels as compared with Group II. Results conclude the possible role of C-PC in modulating the antioxidant enzyme status, thereby retarding sodium selenite-induced cataract incidence both in vitro and in vivo.

Mangrove Streptomyces sp. BDUKAS10 as nanofactory for fabrication of bactericidal silver nanoparticles.

Biosynthesis has led to the development of various biomimetic approaches for the fabrication of nanoscale materials. The present study reveals a unique procedure for the biosynthesis of bactericidal silver nanoparticles (AgNPs) using a novel Streptomyces sp. BDUKAS10, an isolate of mangrove sediment. Aqueous silver nitrate (AgNO(3)) solution was treated with cell free supernatant (CFS) of the isolate to synthesize bactericidal silver nanoparticles. Initial characterization was performed by visual observation for color change to intense brown color. UV-visible spectrophotometry (UV-vis) for measuring surface plasmon resonance indicated a maximum absorption peak at 441 nm. Fourier Transform Infrared Spectroscopy (FTIR) analysis provides evidence for proteins as possible reducing, and capping agents. Energy dispersive X-ray (EDAX) spectroscopy analysis showed elemental silver as major signal. Transmission Electron Microscopy (TEM) study indicated spherical silver nanoparticles in the size range of 21-48 nm. Compared to the CFS, the biosynthesized AgNPs exemplified superior bactericidal efficacy towards the tested bacterial strains. Results from this study suggested that Streptomyces sp. BDUKAS10 can be advantageous for the synthesis of AgNPs by extracellular method in the view of sustainable and ecofriendly approach.

Comparative evaluation of antibacterial activity of silver nanoparticles synthesized using Rhizophora apiculata and glucose.

The focus of the study is to compare the antibacterial efficacy of silver nanoparticles (AgNPs) fabricated by exploiting biological (a mangrove plant, Rhizophora apiculata) and chemical means (Glucose). The synthesized nanoparticles were characterised using UV-visible absorption spectrophotometry (UV-vis), Fourier transform Infra-red Spectroscopy (FTIR) and Transmission electron microscopy (TEM). Biologically synthesized silver nanoparticles (BAgNPs) were observed at 423 nm with particle sizes of 19-42 nm. The chemically synthesized silver nanoparticles (CAgNPs) showed a maximum peak at 422 nm with particle sizes of 13-19 nm. An obvious superiority of the antibacterial potency of BAgNPs compared to the CAgNPs as denoted by the zone of inhibition (ZoI) was noted when the nanoparticles were treated against seven different Microbial Type Culture Collection (MTCC) strains. The current study therefore elucidates that the synthesized AgNPs were efficient against the bacterial strains tested.

Confocal fluorescence resonance energy transfer microscopy study of protein-protein interactions of lens crystallins in living cells.

PURPOSE: To determine protein-protein interactions among lens crystallins in living cells. METHODS: Fluorescence resonance energy transfer (FRET) microscopy was used to visualize interactions in living cells directly. Two genes, one (alphaA-crystallin) fused with green fluorescence protein (GFP) and the other (each of the following genes: alphaB-, betaB2-, gammaC-crystallin, and R120G alphaB-crystallin mutant) fused with GFP variant red fluorescence protein (RED), were cotransfected into HeLa cells. After culture, confocal microscopy images were taken and FRET values were calculated. RESULTS: FRET occurs when the two proteins interact. The data show strong interactions between alphaA- and alphaB-crystallin and weak interactions between alphaA- and betaB2- or gammaC-crystallin, which is consistent with our previous two-hybrid system study. The R120G alphaB-crystallin mutant, however, showed significantly less FRET than wild-type alphaB-crystallin. There are also more R120G alphaB-crystallin transfected cells with protein aggregates than wild-type alphaB-crystallin transfected cells. Cotransfection with alphaA-crystallin could not rescue R120G alphaB-crystallin from aggregation. CONCLUSIONS: FRET microscopy gave excellent results on the protein-protein interactions among crystallins. It supports many previous studies and provides a novel technique for further study of protein-protein interactions among lens proteins including membrane and cytoskeletal proteins.