The role of the conserved COOH-terminal triad in alphaA-crystallin aggregation and functionality.

PURPOSE: The sequentially variable COOH-terminal region of small heat shock protein superfamily members usually contains a conserved IXI/V feature where X is typically a proline. When present in solved sHsp crystal structures (e.g. MjHsp16.5 and wheat Hsp16.9), this short sequence forms an isolated beta strand apparently involved in the alignment of dimers into larger oligomers. Because it is a common feature of many sHsp family members, it is possible that this triad has a similar role in alphaA-crystallin. This study was undertaken to determine the contribution of this conserved triad to the quaternary structure and function of alphaA-crystallin. METHODS: A series of site-directed mutants was generated in both wild type alphaA and in an alphaA deletion mutant lacking the NH2-terminal residues 1-50. After overexpression and purification, each protein's oligomer size was characterized by size-exclusion fast protein liquid chromatography (FPLC), thermal transition temperature by non-denaturing composite gel electrophoresis, and chaperone activity by the inhibition of DL-dithiothreitol (DTT)-induced insulin aggregation. RESULTS: Using the alphaA-crystallin NH2-deletion mutant, the hydrophobic triad was changed from IPV to TPT, GPG, IGV, ITV, or GGG. All six D51 mutants associated into tetramers with small amounts of dimer and monomer also present. Chaperone-like activity was reduced but not eliminated in some of these triad mutants with GGG and ITV the most strongly affected. Similar modifications to wild type alphaA-crystallin (IPV to ITV, IGV, or GGG) restored oligomer sizes similar, but not identical to, native alphaA-crystallin, with additional small amounts of tetramer and dimer. Interestingly, equivalent mutants of wild type alphaA-crystallin did not have reduced chaperone-like activity but differed considerably in their thermal transition temperatures. CONCLUSIONS: The conserved COOH-terminal triad does not appear to have a strong effect on the steady-state aggregation of wild type alphaA-crystallin or its 50-residue deletion mutant at 25 degrees C. However, it can exert a considerable effect on chaperone-like activity in the absence of the NH2-terminal 50-residue sequence extension and can influence the thermal transition temperature in its presence. These results suggest that the conserved triad in alphaA-crystallin contributes to the stability of higher order oligomers but is not essential for the formation of tetramers.

NH2-terminal stabilization of small heat shock protein structure: a comparison of two NH2-terminal deletion mutants of alphaA-crystallin.

PURPOSE: To assess the role of the NH2-terminal in alphaA-crystallin folding and chaperone-like activity. METHODS: Two NH2-terminal deletion mutants of alphaA-crystallin were generated by standard mutagenesis methods, one with and one without a leader sequence in place of the first 50 residues. Aggregate size of each before and after thermal stress was assessed by FPLC, and chaperone-like activity was assessed using DTT-induced insulin denaturation. RESULTS: Both mutants assemble primarily into tetramers, and both exhibit similar levels of chaperone-like activity, but are less protective than recombinant alphaA-crystallin. After a cycle of heat stress to 70 degrees C, tetramers of the mutant without the leader sequence dissociate into dimers and monomers and show severely reduced chaperone-like activity. In contrast, the mutant with the leader sequence retains its tetrameric form and its chaperone-like activity. CONCLUSIONS: The NH2-terminal region is an important determinant of alpha-crystallin aggregate size, but is not required for folding of the alpha-crystallin domain, since the aggregate size and chaperone-like activity of the two mutants at room temperature are essentially the same. The leader sequence appears to increase the thermal stability of the alpha-crystallin domain and/or to contribute to the reformation of the active form after cooling, suggesting that the native NH2-terminal also plays a role in alpha-crystallin's resistance to environmental stress.

N-terminal control of small heat shock protein oligomerization: changes in aggregate size and chaperone-like function.

The small heat shock protein superfamily is composed of proteins from throughout the phylogenetic spectrum that are induced upon environmental stress. Their structural stability under stress derives in large part from the central region of the proteins, which forms two beta sheets held together by hydrophobic interactions and appears to be present in all superfamily members. The length, sequence, and amino acid composition of the N- and C-terminals, in contrast, are quite variable. The role of the N-terminal has been hypothesized to control species-specific assembly of subunits into higher level structures. To test this, a set of constructs was designed and expressed: the N-terminal sequences preceding the start of the core regions of alphaA-crystallin and HSP 16.5 from Methanococcus jannaschii were swapped; the N-terminal of each protein was removed, and replaced with a brief N-terminal extension sequence; and two nonsense N-terminal sequences of approximately the same length and hydropathicity as the original replaced the alphaA-crystallin N-terminal. All constructs, plus the original recombinant sequences, could be overexpressed except for the 16.5 N-terminal extension, and all showed chaperone-like activity except for the hybrid with the 16.5 C-terminal. Size and properties of the replacement N-terminal place limits on aggregate size. Additional restrictions are imposed by the structure of the dimer.

The mechanism of presbyopia.

Accommodation in humans refers to the ability of the lens to change shape in order to bring near objects into focus. Accommodative loss begins during childhood, with symptomatic presbyopia, or presbyopia that affects one's day to day activities, striking during midlife. While symptomatic presbyopia has traditionally been treated with reading glasses or contact lenses, a number of surgical interventions and devices are being actively developed in an attempt to restore at least some level of accommodation. This is occurring at a time when the underlying cause of presbyopia remains unknown, and even the mechanism of accommodation is occasionally debated. While Helmholtz' theory regarding the mechanism of accommodation is generally accepted with regard to broad issues, additional details continue to emerge. Age-related changes in anterior segment structures associated with accommodation have been documented, often through in vitro and/or rhesus monkey studies. A review of these findings suggests that presbyopia develops very differently in humans compared to non-human primates. Focusing on non-invasive in vivo human imaging technologies, including Scheimpflug photography and high-resolution magnetic resonance imaging (MRI), the data suggest that the human uveal tract acts as a unit in response to age-related increasing lens thickness and strongly implicates lifelong lens growth as the causal factor in the development of presbyopia.

Structural diversity in the small heat shock protein superfamily: control of aggregation by the N-terminal region.

The small heat shock protein superfamily, extending over all kingdoms, is characterized by a common core domain with variable N- and C-terminal extensions. The relatively hydrophobic N-terminus plays a critical role in promoting and controlling high-order aggregation, accounting for the high degree of structural variability within the superfamily. The effects of N-terminal volume on aggregation were studied using chimeric and truncated proteins. Proteins lacking the N-terminal region did not aggregate above the tetramers, whereas larger N-termini resulted in large aggregates, consistent with the N-termini packing inside the aggregates. Variation in an extended internal loop differentiates typical prokaryotic and plant superfamily members from their animal counterparts; this implies different geometry in the dimeric building block of high-order aggregates.

Aging of the human lens: changes in lens shape upon accommodation and with accommodative loss.

Accommodation in the human eye occurs through controlled changes in crystalline lens shape, thickness, and refractive surface placement relative to the cornea. The changes in lens curvatures, whether surface or internal, have been characterized as a function of accommodation and subject age by use of quantitative analysis of Scheimpflug slit-lamp photographic images. Radii of curvature of the major lens refractive surfaces--the external and nuclear boundaries--decrease linearly with increasing accommodation in all eyes that are capable of accommodation. The rates at which they change with accommodation are age dependent, decreasing steadily toward zero with increased age. For the curves visible in each lens half, arising from boundaries between adjacent zones of discontinuity, radius of curvature and location are linearly related over the entire accommodative range for a given lens and over the age range for the population. The slope of this relationship changes with both accommodation and age, decreasing linearly in both cases. The relationship between these geometric changes and the loss of accommodative amplitude is discussed.