Functional sequences in human alphaB crystallin

Abstract

Background: Human alphaB crystallin (HspB5) contains the alpha crystallin core domain, a series of antiparallel beta-strands organized into the characteristic beta sandwich of small heat shock proteins (sHsps). The full 3-dimensional structure for alpha crystallin has not been determined and the mechanism for the biological activity remains elusive because sHsps participate in multiple interactions with a broad range of target proteins that favor self-assembly of polydisperse fibrils and complexes. We selected human alphaB crystallin to study interactive sequences because it is involved in many human condensation, amyloid, and aggregation diseases and it is very sensitive to the destabilization of unfolding proteins. Sophisticated methods are being used to analyze and complete the structure of alphaB crystallin with the expectation of understanding sHsp function. This review considers the identification of interactive sites on the surface of the alphaB crystallin, which may be the key to understanding the multifunctional activity of human alphaB crystallin.

Scope of review: This review summarizes the research on the identification of the bioactive interactive sequences responsible for the function of human alphaB crystallin, an sHsp with chaperone-like activity.

Major conclusions: The multifunctional activity of human alphaB crystallin results from the interactive peptide sequences exposed on the surface of the molecule. The multiple, non-covalent, interactive sequences can account for the selectivity and sensitivity of alphaB crystallin to the initiation of protein unfolding.

General significance: Human alphaB crystallin may be an important part of an endogenous protective mechanism in aging cells and tissues. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Keywords: Amyloid; Cataract; Crystallin; Human; Lens; Pin array.

FIGURE 1:. Interactive Sequences in AlphaB Crystallin Identified Using a Pin Array.

The full length sequence of human alphaB crystallin (top row) is compared with the 19 residue sequence of the mini-alpha A crystallin, MAC, (second row) discovered by Sharma. Pin array studies identified a number of interactive peptide sequences in human alphaB Crystallin listed as Ghosh frag 1–12. Ghosh frag 2 is the sequence in the C-terminal domain of alphaB crystallin that corresponds to MAC in alpha A crystallin. Ghosh frag 1 is an interactive peptide sequence in the N-terminal domain of alphaB crystallin. Ghosh frags 3–8 are four amino acid bioactive sequences based on the alphaB crystallin sequence corresponding to MAC. Ghosh frag 9 is a second bioactive sequence identified in the alpha crystallin core domain and Ghosh frags 10–12 are four amino acid bioactive sequences based on Ghosh frag 9. The functional activity of Ghosh peptide fragments are compared in figure 4.

FIGURE 2:. Cellular Opacification (cataract) in Mouse is an Excellent Model for the Study of Protein Aggregation and Amyloid Formation in vivo.

Transmission electron micrographs of transparent (left) and opaque (right) lens cells illustrate differences between cytoplasmic structure in the normal transparent lens and a dense cataract. In the transparent cells, soluble, concentrated proteins are distributed uniformly throughout the cells and fluctuations in the index of refraction are small relative to the wavelength of visible light (390–700nm). The distribution of the cytoplasmic proteins is uniform and homogeneous similar to window glass. In opaque cells, the cytoplasmic proteins self-assemble into condensed, light scattering aggregates separated by spaces filled with cytoplasmic fluid. The fluctuations in the index of refraction are large relative to the wavelength of visible light similar to frosted glass. In this example the condensed protein appears to have a filamentous structure connecting the cell membranes, but various morphologies are observed in opacities, and they can be very subtle [–50]. A lens containing opaque cells like those in figure 2B would appear completely opaque as in the cataract in figure 3. It is well known, but not widely recognized, that only 3 percent of the cytoplasmic proteins need to self- assemble into HMW for complete cellular opacity. Regulation of the interactions between cytoplasmic proteins is a function of alphaB crystallin that is thought to assist in stabilization of transparent cytoplasm both during the differentiation of transparency and during aging when post-translational modification and proteolysis can favor aggregation and disordered cytoplasmic structure. (bar = one micron)

FIGURE 3:. In Vivo Transparency and Opacity in Cells of a Mouse Lens Cataract.

In a transparent lens, protection against self-assembly of HMW aggregates can be evaluated easily in vivo. In a photograph of a normal live mouse eye at approximately 45 days of age, the lens is transparent (3A). Lens cell transparency allows the observation of changes in light scattering from individual cells using quantitative spectroscopic methods. Large HMW aggregates are responsible for the opacity (3B) observed in the lenses of numerous animal models for cataract, and dynamic light scattering can be used to measure the progressive transition from soluble protein monomers to multimeric light scattering complexes at the earliest stages of opacification. The protective activity of modulators of the self-assembly of proteins can be evaluated in vivo using these methods. A number of small molecular weight compounds and biomolecules have protective effects when administered during the early stages of aggregation in vivo [51, 52]. Pantethine (MW = 555 Daltons) is among the most effective molecules that protect against protein aggregation and opacity (3C) [53, 54]. Pantethine can enhance the natural protective action of alphaB crystallin.

FIGURE 4:. The Interactive Sequences in Human AlphaB Crystallin Enhanced or Inhibited the Self-Assembly of Amyloid Proteins.

The relative activity for each bioactive peptide identified in human alphaB crystallin was assessed using a Thioflavin T fluorescence assay for fibrillation of amyloid proteins. All experiments were conducted using 1:1 molar ratio of peptide to amyloid target protein. Target proteins were the amyloid protein Amyloid beta 1–42 (Abeta) associated with Alzheimer’s disease (column 2) or alpha synuclein (column 3) associated with Parkinson’s disease. In the Thioflavin T assay, relative fluorescence was measured and “1.0” is normal fibrillation in the absence of bioactive peptide, and “0.0” is complete protection against fibrillation observed with full size alphaB crystallin. Peptides STSLSPFYLRPPSFLRAP and HGKHEERQDE were the most effective inhibitors of Abeta fibril formation. While HGKH had no effect, HEER and RQDE enhanced fibril formation of Abeta. The protective activity of the peptides was different with alpha synuclein where STSLSPFYLRPPSFLRAP enhanced fibril formation and Abeta, where there was complete inhibition of fibril formation. In contrast, the sequence DRFSVNLDVKHFS and the four residue peptides, RFSV, FSVN, VNLD, LDVK, KHFS and HGKH were effective inhibitors of alpha synuclein fibrillation and less effective with Abeta. The studies determined that human alphaB crystallin contains a number of inhibitory peptide sequences that can modulate amyloid formation in vitro. This is an excellent example of how a peptide sequence that is an effective inhibitor of fibril formation of one amyloid protein can be ineffective against fibril formation of another amyloid protein.

FIGURE 5:. Model for the interactive sequences in a ribbon model of the small heat shock protein, human alphaB crystallin.

Possibly the most surprising result of the study of the interactive sequences in human alphaB crystallin was the identification of multiple interactive sequences. The sequences form a diverse network of potential protective sites for selective interaction with several categories of target proteins. The biological, biophysical and pharmacological advantages of combining a number of multifunctional interactive sites on a single intracellular protective molecule, instead of numerous soluble cytoplasmic peptides, as small as four amino acids, each with protective activity remains to be determined.