Doctor of Philosophy
School of Biological Sciences
Thornell, Erin, The Role of Phosphorylated αB-Crystallin in Age-related Nuclear Cataract, Doctor of Philosophy thesis, School of Biological Sciences, University of Wollongong, 2016. http://ro.uow.edu.au/theses/4904
The transparency of the lens is maintained throughout life, at least in part, by the chaperone activity of the small heat shock protein (sHSP) αB-crystallin (αB). However, modified forms of αB, including phosphorylated and deamidated isoforms, accumulate in insoluble protein fractions from the lens with age. This has led to the suggestion that modification of αB is a precursor to age-related nuclear cataract formation. This thesis investigated whether phosphorylated αB could contribute to cataract formation by facilitating the formation of the nucleocortical barrier, a physical barrier to the diffusion of small molecules into the nucleus of the lens that forms at middle age. To determine how phosphorylation may modulate the chaperone activity of αB, single (S19D; S45D; S59D), double (S19D and S45D) and triple (S19D, S45D and S59D) mutants of αB which mimic phosphosphorylation at three key serine residues (Ser19, Ser45 and Ser59) were used in an in vitro aggregation assay and their activity compared to wild type (WT) αB. Whilst WTαB failed to inhibit the heat-induced aggregation of creatine phosphokinase (CPK), the triple mutant (i.e. αB S19D, S45D and S59D) inhibited the aggregation of CPK by 83.9±1.3%. Thus, it is concluded that the addition of negative charges to αB, similar to those added by phosphorylation, increases its chaperone activity.
Next, protein was isolated from adult human lenses via sucrose density centrifugation and separated using 2-dimensional sodium dodecylsulphate polyacrylamide gel electrophoresis (2D SDS PAGE) to identify changes in αB phosphorylation and the deamidation of cytoskeletal proteins that occurs with age across different regions of the lens. The amount of multiply phosphorylated αB (i.e. phosphorylated at multiple residues) was increased in highdensity protein fractions from the lens (representing membrane-bound protein) compared to low density protein fractions. The amount of multiply phosphorylated αB increased towards the nucleus in the aged lenses. In some high density protein fractions there was also evidence of crosslinked high molecular weight complexes (HMWCs), involving αA-crystallin (αA), αB and cytoskeletal proteins including filensin. This provides evidence for the involvement of α-crystallin, via binding to intermediate filaments, in the formation of a network that supports the fibre cell membrane.