Germ-line <i>TP53</i> coding SNVs found in the cataract case-control panel (VAF >20%).

<p>Germ-line <i>TP53</i> coding SNVs found in the cataract case-control panel (VAF >20%).</p

Molecular modeling of the effects of mutations presented in on βγ-crystallin structures

Normal structures are shown in red and the overlaid mutant structures are shown in blue (truncations) and blue and white (substitutions). : The normal βB2-crystallin structure is shown in red, and the Q155X mutation is predicted to remove the COOH-terminal 51 amino acids shown in the blue overlay. : The normal γD-crystallin structure is shown in red and the R140X mutation is predicted to remove the COOH-terminal 34 amino acids shown in the blue overlay. : The normal γC-crystallin structure in the vicinity of the R168R mutation is shown in white with positive charges shown in blue and negative charges in red. The overlaid R168W mutant is shown in red, demonstrating the substitution of the polar arginine residue by the apolar tryptophan and consequent displacement of D107. : The normal γS-crystallin structure in the vicinity of the fragments of 3D structures of mutant proteins (γC-crystallin) and S39C (γS-crystallin) mutations are shown in red. Hydrogen-bonding patterns of the mutant γC- and γS-crystallin structures are shown by dashed lines (green).<p><b>Copyright information:</b></p><p>Taken from "Crystallin gene mutations in Indian families with inherited pediatric cataract"</p><p></p><p>Molecular Vision 2008;14():1157-1170.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2435160.</p><p></p

Pedigree, electropherogram, and restriction fragment length polymorphism of ADCC families with a mutation in the α-crystallin gene

In the pedigree, the square symbol represents males while the circle symbol represents females. A circle with a slash denotes a deceased individual, and a blackened symbol denotes an affected individual. The dot on the upper right corner of the symbol means a sample was available from that individual, and the arrow denotes the proband. , , show mutation analyses of . : Family CCW46 shows a heterozygous c.104 C>T resulting in a novel ApaLI restriction site (mutant allele−191 and 63 bp, wild type−254 bp) : Family CCW36 shows a c.130 C>T change that results in the loss of the MspI restriction site (mutant allele-254 bp, wild type-116, 90, and 48 bp) : Family CCW55 shows the gain of a novel HpyCH4V site cosegregating with the affected individual heterozygous for c.230 C>T transition (mutant allele-254, 191, and 63 bp and wild type-254 bp). : Family CCW22 has a mutation in , c.557G>A. This mutation results in the loss of a HpyF10VI site (mutant allele-258 and 118 bp, wild type-258, 77, and 41 bp). M denotes 100 bp DNA ladder, and C denotes unrelated control.<p><b>Copyright information:</b></p><p>Taken from "Crystallin gene mutations in Indian families with inherited pediatric cataract"</p><p></p><p>Molecular Vision 2008;14():1157-1170.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2435160.</p><p></p

Functional Validation of Hydrophobic Adaptation to Physiological Temperature in the Small Heat Shock Protein αA-crystallin

<div><p>Small heat shock proteins (sHsps) maintain cellular homeostasis by preventing stress and disease-induced protein aggregation. While it is known that hydrophobicity impacts the ability of sHsps to bind aggregation-prone denaturing proteins, the complex quaternary structure of globular sHsps has made understanding the significance of specific changes in hydrophobicity difficult. Here we used recombinant protein of the lenticular sHsp α A-crystallin from six teleost fishes environmentally adapted to temperatures ranging from -2°C to 40°C to identify correlations between physiological temperature, protein stability and chaperone-like activity. Using sequence and structural modeling analysis we identified specific amino acid differences between the warm adapted zebrafish and cold adapted Antarctic toothfish that could contribute to these correlations and validated the functional consequences of three specific hydrophobicity-altering amino acid substitutions in αA-crystallin. Site directed mutagenesis of three residues in the zebrafish (V62T, C143S, T147V) confirmed that each impacts either protein stability or chaperone-like activity or both, with the V62T substitution having the greatest impact. Our results indicate a role for changing hydrophobicity in the thermal adaptation of α A-crystallin and suggest ways to produce sHsp variants with altered chaperone-like activity. These data also demonstrate that a comparative approach can provide new information about sHsp function and evolution.</p> </div

Multiple sequence alignment of αA-crystallin amino acid sequences from six bony fish species and human.

<p>Dots indicate residues identical to the zebrafish sequence. Arrows indicate residues that have undergone site-directed mutational analysis in the literature (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034438#pone.0034438.s001" target="_blank">Table S1</a>). Downward facing arrows are sites that have been modified to decrease chaperone activity while upward facing orange arrows are sites that have been modified to increase chaperone-like activity. Purple shaded boxes are the center (midpoint of the window (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034438#pone-0034438-g003" target="_blank">Fig. 3D</a>)) of the regions in which hydrophobicity has undergone positive natural selection as identified by TreeSAAP analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034438#pone-0034438-g004" target="_blank">Fig. 4D</a>). Asterisks indicate three variants (V62T, C143S, T147V) examined in this study. These variants alter hydrophobicity at each residue and were predicted to influence chaperone-like activity based on homology modeling using bacterial sHsp crystal structures. Species shown are (with accession numbers): <i>Cyprinodon variegatus</i> (Cyp; HQ111072), <i>Danio rerio</i> (Dan; NP_694482), <i>Dissostichus mawsoni</i> (Dis; ABA61342), <i>Homo sapiens</i> (Hom; CAG28619), <i>Notothenia angustata</i> (Not; HQ111073), <i>Oncorhynchus kisutch</i> (Onc; HQ111071), <i>Pimephales notatus</i> (Pim; HQ111070).</p

Structure of αA-crystallin domain, location of three modified sites and the effect of modification in position 62 are shown.

<p>(A) A superposition of monomeric molecules of a recent zebrafish crystal structure (PDB id: 3N3E) and the protein obtained using homology modeling with a bovine αA-crystallin structure (PDB id:3L1E) in this work are represented by magenta and white, respectively. Corresponding amino acid residues in zebrafish positions 62, 143 and 147 are shown by ball-and-sticks. (B) The accessible surface of the zebrafish homo-dimer (PDB id: 3N3E) with the non-polar surface shown in green. Non-polar surface of V62 is shown in yellow. This area will become polar when valine is replaced with a threonine residue.</p

Interaction between rs1063192 and rs7916697.

<p>Logistic regression modeling showed that the joint effects between rs1063192 and rs7916697 were interactive (<i>P</i>-interaction = 2.80E−5).The joint odds ratios were estimated for combinations of protective genotypes of rs1063192 (in the dominant model) or rs7916697 (in the dominant model) compared with the combination of both risk genotypes (homozygous major alleles for both markers).</p

Physiological temperatures of species used in this study.

<p>Physiological range and average temperature are indicated for each species. Genus is noted on the ordinate with common names provided to the right of each picture.</p

Association results of rs1063192 and rs7916697 in different models.

∧<p>One sample did not amplify when this marker was tested.</p>*<p>risk allele, odds ratios are calculated for the minor (protective) allele and genotypes.</p>1<p>Chi-Squared <i>P</i>-value. The Bonferroni corrected significance level was 0.0021 ( = 0.05/24, for 18 allelic tests for individual SNPs and 3 model specific tests for each of 2 SNPs).</p>2<p>Odds ratio.</p>3<p>no individuals were homozygous for the risk allele.</p

Allelic tests of SNPs.

*<p>Minor allele frequencies.</p>†<p>Chi-Squared <i>P</i>-value. The Bonferroni corrected significance level was 0.0021 ( = 0.05/24, for 18 allelic tests for individual SNPs and 3 model specific tests for each of 2 SNPs).</p>‡<p>Hardy–Weinberg equilibrium.</p>∧<p>For markers out of HWE association was also tested using the Cochran-Armitage trend test with no association being shown (P = 0.0573 for rs7037117 and P = 0.5561 for rs7961953).</p