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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.

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 [alpha]- and [beta]-crystallins. Here, we investigated the effects of the deamidation of [alpha]A- and [alpha]B-crystallins on their interaction with [beta]A3-crystallin using surface plasmon resonance (SPR) and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer (FLIM-FRET) methods. SPR analysis confirmed adherence of WT [alpha]A- and WT [alpha]B-crystallins and their deamidated mutants with [beta]A3-crystallin. The deamidated mutants of [alpha]A-crystallin ([alpha]A N101D and [alpha]A N123D) displayed lower adherence propensity for [beta]A3-crystallin relative to the binding affinity shown by WT [alpha]A-crystallin. Among [alpha]B-crystallin mutants, [alpha]B N78D displayed higher adherence propensity whereas [alpha]B N146D mutant showed slightly lower binding affinity for [beta]A3-crystallin relative to that shown by WT [alpha]B-crystallin. Under the in vivo condition (FLIM-FRET), both [alpha]A-deamidated mutants ([alpha]A N101D and [alpha]A N123D) exhibited strong interaction with [beta]A3-crystallin (32±4% and 36±4% FRET efficiencies, respectively) compared to WT [alpha]A-crystallin (18±4%). Similarly, the [alpha]B N78D and [alpha]B N146D mutants showed strong interaction (36±4% and 22±4% FRET efficiencies, respectively) with [beta]A3-crystallin compared to 18±4% FRET efficiency of WT [alpha]B-crystallin. Further, FLIM-FRET analysis of the C-terminal domain (CTE), N-terminal domain (NTD), and core domain (CD) of [alpha]A- and [alpha]B-crystallins with [beta]A3-crystallin suggested that interaction sites most likely reside in the [alpha]A CTE and [alpha]B NTD regions, respectively, as these domains showed the highest FRET efficiencies. Overall, results suggest that similar to WT [alpha]A- and WT[alpha]B-crystallins, the deamidated mutants showed strong interactionfor [beta]A3-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 [alpha]-crystallin may facilitate greater interaction and the formation of large oligomers with other crystallins, and this may contribute to the cataractogenic mechanism.

Protein glycosylation has been described as the most abundant and complex post-translational modification occurring in nature. Recent studies have enhanced our view of how this modification occurs in bacteria highlighting the role of protein glycosylation in various processes such as biofilm formation, virulence and host-microbe interactions. We recently showed that the collagen- and laminin-binding adhesin Cnm of the dental pathogen Streptococcus mutans is post-translationally modified by the PgfS glycosyltransferase. Following this initial identification of Cnm as a glycoprotein, we have now identified additional genes ( pgfM1 , pgfE and pgfM2 ) that are also involved in the posttranslational modification of Cnm. Similar to the previously characterized Δ pgfS strain, inactivation of pgfM1 , pgfE or pgfM2 directly impacts Cnm by altering its migration pattern, proteolytic stability and function. In addition, we identified the wall-associated protein A (WapA) as an additional substrate of Pgf-dependent modification. We conclude that the pgS-pgfM1-pgfE-pgfM2 operon encodes for a protein machinery that can modify, likely through the addition of glycans, both core and non-core gene products in S . mutans .

Mitochondria link the energy -- releasing activities of electron transport and proton pumping with the energy conserving process of oxidative phosphorylation to form ATP. A declined mitochondrial performance has been generally observed during aging. In the present investigation, the activities of tricarboxylic acid cycle enzymes such as isocitrate, alpha-ketoglutarate, succinate and malate dehydrogenases and electron transport complexes I-IV were measured in mitochondria isolated from brain regions like cortex, striatum and hippocampus of young and aged rats before and after L-Carnitine supplementation. All the three brain regions of aged rats showed decreased activities of isocitrate, alpha-ketoglutarate and succinate dehydrogenases, complexes I and IV when compared to control young rats. Striatum seems to be the most susceptible region when compared to hippocampus and cortex. L-Carnitine supplementation to aged rats reversed the activities of these enzymes to near normal whereas treatment to young rats did not show any significant alterations. These results confirm that L-Carnitine can alleviate the age-associated decline in the metabolic efficiency of mitochondria in all three brain regions under investigation.