Ekta Tiwary

Interaction among crystallins is required for the maintenance of lens transparency. Deamidation is one of the most common post-translational modifications in crystallins, which results in incorrect interaction and leads to aggregate formation. Various studies have established interaction among the α- and β-crystallins. Here, we investigated the effects of the deamidation of αA- and αB-crystallins on their interaction with βA3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT αA- and WT αB-crystallins and their deamidated mutants with βA3-crystallin. The deamidated mutants of αA-crystallin (αA N101D and αA N123D) displayed lower adherence propensity for βA3-crystallin relative to the binding affinity shown by WT αA-crystallin. Among αB-crystallin mutants, αB N78D displayed higher adherence propensity whereas αB N146D mutant showed slightly lower binding affinity for βA3-crystallin relative to that shown by WT αB-crystallin. Under the in vivo condition (FLIM-FRET), both αA-deamidated mutants (αA N101D and αA N123D) exhibited strong interaction with βA3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT αA-crystallin (18±4%). Similarly, the αB N78D and αB N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with βA3-crystallin compared to 18±4% FRET efficiency of WT αB-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of αA- and αB-crystallins with βA3-crystallin suggested that interaction sites most likely reside in the αA CTE and αB NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT αA- and WTαB-crystallins, the deamidated mutants showed strong interactionfor βA3-crystallin. Variable in vitro and in vivo interactions are most likely due to the mutant's large size oligomers, reduced hydrophobicity, and altered structures. Together, the results suggest that deamidation of α-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.

The CRYAAN101D transgenic mouse model expressing deamidated αA-crystallin (deamidation at N101 position to D) develops cortical cataract at the age of 7 to 9 months. The present study was carried out to explore the molecular mechanism that leads to the development of cortical opacity in CRYAAN101D lenses. RNA sequence analysis was carried out on 2- and 4-month-old αA-N101D and wild type (WT) lenses. To understand the biologic relevance and function of significantly altered genes, Ingenuity Pathway Analysis (IPA) was done. To elucidate terminal differentiation defects, immunohistochemical, and Western blot analyses were carried out. RNA sequence and IPA data suggested that the genes belonging to gene expression, cellular assembly and organization, and cell cycle and apoptosis networks were altered in N101D lenses. In addition, the tight junction signaling and Rho A signaling were among the top three canonical pathways that were affected in N101D mutant. Immunohistochemical analysis i...

Gamma glutamyl transpeptidase from Bacillus pumilus KS12 (GGTBP) was cloned,
expressed in pET-28-E.coli expression system as a heterodimeric enzyme with
molecular weights of 45 and 20 kDa for large and small subunit respectively. It was
purified by nickel affinity chromatography with hydrolytic and transpeptidase activity
of 1.82 U/mg and 4.35 U/mg respectively. Sequence analysis revealed that GGTBP
was most closely related to Bacillus licheniformis GGT and had all the catalytic
residues and nucleophiles for autoprocessing recognized from E.coli. It was optimally
active at pH 8 and 60C. It exhibited pH stability from pH 6-9 and high
thermostability with t1/2 of 15 min at 70C. It had Km, Vmax of 0.045 mM, 4.35
μmol/mg/min respectively. Decoupling of autoprocessing by co-expressing large and
small subunit in pET-Duet1-E.coli expression system yielded active enzyme with
transpeptidase activity of 5.31 U/mg. Though N-terminal truncations of rGGTBP upto
95 aa did not affect autoprocessing of GGT however activity was lost with truncation
beyond 63 aa.

A feather degrading strain of Bacillus licheniformis ER-15 was isolated which also degraded a-keratin of hooves. A detailed analysis revealed that a novel monomeric g-glutamyl transpeptidase (GGT30), a proteolytic product of heterodimeric 67kDa g-glutamyl transpeptidase (GGT67), assists subtilisin during its action on a keratin. An equimolar combination of
subtilisin and GGT30 was designated as KerN and was used as ungual enhancer for topical application. KerN was effective in releasing proteins from nail plate surface and 300mg of enzyme could release 41 mg protein/mg of nail after 24 h treatment. Scanning electron micrograph (SEM) revealed loosening of nail matrix confirming the action of KerN on nail keratin. Drug permeation studies revealed permeation of clotrimazole through both enzymatically pretreated nail plates and also through nail plates in presence of KerN. Nearly 58% drug could be retained by nail plates after 24 h of 300mg/mL KerN which further enhanced up to 97% by prolonging the enzyme application. The enzyme was found to be stable in presence of drug even after 72 h. Thus, KerN can be used as an additive in formulation of topical drug for onchomycosis.

Keratinase (ker BL) from Bacillus licheniformis ER-15 was cloned into vector pEZZ18 for extracellular expression in Escherichia coli HB101. Recombinant keratinase was secreted with high specific activity (75 units/mg) under non-inducible conditions after 36 h at 37
C and 300 rpm in a shake flask. Protein was concentrated and, subsequently, purified by ion-exchange chromatography
using Q-sepharose with 95.8% yield. The recombinant keratinase was a serine protease and most active in the pH range of 8-12 and at 60
C. The enzyme was stable over a wide pH range of 4-12 for 3 h. ker BL degraded bovine serum albumin, casein, azocasein, gelatin, and feather. E. coli HB101 harboring pEZZ18 ker BL2 degraded chicken feather completely within 24 h at 37
C.

Monomeric 30 kDa c-glutamyl transpeptidase (GGT30) was purified from culture broth of Bacillus licheniformis ER-15 along with a heterodimeric 67 kDa GGT (GGT67). In presence of subtilisin, GGT30 had improved catalytic efficiency (Vmax/Km) of 59 min-1, altered pH and temperature optima of pH 11 and 70C.and had salt-tolerant glutaminase activity. Glutaminase activity was retained even in protease-inhibited condition in presence of 2 mM PMSF. GGT30 and subtilisin complexation was also confirmed by relative electrophoretic mobility and fluorescence quenching experiment.

A novel dimeric 58 kDa keratinase is reported from Bacillus licheniformis ER-15. The bacterium produced 244 U/ml keratinase in 48 h which was increased by eight fold (1962 U/ml) after medium optimization by one-variable-at-a-time and response surface methodology. Enzyme was concentrated by ultrafiltration
followed by acetone precipitation and purified by gel filtration chromatography. It had subunit of 30 and 28 kDa and pI of 8.4. Enzyme was maximally active at pH 11 and 70 C. It hydrolyzed various complex proteins viz. haemoglobin, feather, hooves, fibrin and meat protein. It was a thiol activated serine protease and 6.25-fold enhancement in activity was observed in presence of 5 mM mercaptoethanol.Nearly 1200 U keratinase degraded 1.5 g feather in 12 h at pH 8, 50 C in redox free environment. This
enzyme also dehaired buffalo hide within 16 h in presence of 3% Ca (OH)2.