• Cloning, Expression and Characterization of Bovine aB-crystallin

      Kelley, Patrick; Department of Biochemistry and Molecular Biology (2000-01)
      The lens protein, aB-crystallin, plays central role in slowing the formation of pre-cataractous protein aggregates. By binding to newly exposed, hydrophobic regions of damaged proteins, aB-crystallin creates stable, soluble complexes that are resistant to further unfolding and aggregation. Several regions o f aB-crystallin have been identified as possible sites for this chaperone-like binding, three of which are explored further in the present work using assays for both the physical properties and in vitro function of sitedirected mutants of aB-crystallin. A priority in this study was the generation of a bovine aB-crystallin cDNA clone. One of the regions of putative chaperone binding in aB - crystallin, residues 24-32, which was implicated by deuterium exchange experiments, contains several hydrophobic residues. Since hydrophobic residues are thought to play a central role in chaperone binding, the present work investigates a mutant of one of these residues, F28, which was changed to serine. Two charged residues within this same domain, E30, and E34 were each separately mutated to glutamine in order to assess the role of negatively charged residues in chaperone activity. Another two regions implicated as possible binding sites in aB-crystallin, residues 59-68 and residues 92-108, were previously identified using SAED binding studies. In the present study, one hydrophobic residue from each of these domains was mutated to a less hydrophobic residue: 16IS and L94Q. The results show that at 25°C, a serine as position 28 (F28S) causes moderate alterations in secondary structure, tertiary structure and oligomeric assembly. At 58°C, however, this mutant suffers from a disintegration o f oligomeric structure as well as a loss of chaperone function. The data implicate F28 as a critical residue for maintaining the structural integrity of aB-crystallin. The other mutants, with the exception o f L94Q, behaved similar to wild type aB-crystallin whether assayed at 37 ° or 58 °. The L94Q mutant displayed slightly better chaperone activity than wild type in the high temperature (58°) assay. The present work supports the idea that the chaperone-like behavior of aBcrystallin requires intact oligomeric structure and that the activity may not be associated with a discreet binding site but instead a diverse array of residues spread out over the surface of aB-crystallin.
    • Significance of Small Heat Shock Proteins HSPB1 and HSPB5 in Aggregation and Degradation of Amyloid Proteins

      Ojha, Juhi; Department of Biochemistry and Molecular Biology (2011-06-10)
      Molecular chaperones protect cells from the deleterious effects of protein misfolding and aggregation. Neurotoxicity of amyloid-beta (Ap) aggregates and its deposition in senile plaques are hallmarks of Alzheimer's disease (AD). We observed that the overall content of aB-crystallin, a small heat shock protein molecular chaperone, decreased in AD model mice in an age-dependent manner. We hypothesized that aB-crystallin protects cells against Ap toxicity. To test this, we crossed aB-crystallin/HspB2 deficient (CRYAB'A HSPB2'A) mice with AD model transgenic mice expressing mutant human amyloid precursor protein. Transgenic and non-transgenic mice in chaperone-sufficient or deficient backgrounds were examined for representative behavioral paradigms for locomotion and memory network functions - (i) spatial orientation and locomotion was monitored by open field test; (ii) sequential organization and associative learning was monitored by fear conditioning and (iii) evoked behavioral response was tested by hot plate method. Interestingly, aB-crystallin/HspB2 deficient transgenic mice were severely impaired in locomotion compared to each genetic model separately. Our results highlight a synergistic effect of combining chaperone deficiency in a transgenic mouse model for AD underscoring an important role for chaperones in protein misfolding diseases.