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Peptide-based Treatment Strategies for Cataract

Cetinel, Sibel PhD*,†; Unsworth, Larry PhD*,†,‡; Montemagno, Carlo PhD*,†

doi: 10.1097/IJG.0000000000000111
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Cataract produces vision loss due to opacification of the lens. Two possible reasons for cataract formation are insolubility of crystallin proteins and fibril (aggregate) formation. Currently, the only available treatment is surgical replacement of the lens with a synthetic one. An alternative treatment that is both economically and technologically accessible is needed in developing countries where untreated cataract and coincident blindness is high, access to trained surgeons is limited, and the cost of surgery may be prohibitive. We present current progress toward the development of a nonsurgical treatment strategy focusing on promoting protein solubility and/or dissolving fibrillar aggregates.

*Department of Chemical and Materials Engineering, Faculty of Engineering

Ingenuity Laboratory, National Institute of Nanotechnology (NINT), University of Alberta

National Research Council, National Institute for Nanotechnology, Edmonton, AB, Canada

Disclosure: The authors declare no conflict of interest.

Reprints: Carlo Montemagno, PhD, Ingenuity Laboratory, National Institute of Nanotechnology (NINT), University of Alberta, 1-070C, 11421 Saskatchewan Drive, Edmonton, AB, Canada T6G 2M9 (e-mail: montemag@ingenuitylab.ca).

Received August 2, 2014

Accepted August 2, 2014

The lens is a highly organized transparent optical syncytial tissue that focuses light onto the retina. Its transparency arises from fiber cells, which are composed of highly concentrated and short range ordered crystallin proteins.1 Because of the lack of cellular organelles, there is almost no protein turnover in the lens. Therefore, in a healthy eye, the protein organization and structure stability need to be maintained for life.

The major proteins found in fiber cells crystallins are lens-specific structural proteins. The mammalian lens contains 3 types of crystallins—α, β, γ, and various subunits of each. Among them, α-crystallin is the most abundant lens protein and is a member of a small heat-shock protein family acting as a molecular chaperone.2 α-crystallin is composed of 2 subunits, αA and αB. Each subunit with a molecular weight of 20 kDa comes together to form the heterogeneous multimeric α-crystallin complex of ∼300 kDa.3 The chaperone activity of α-crystallin inhibits the aggregation and insolubility of all other crystallins4,5 and, thus maintains the organization of the lens tissue. The other crystallin proteins, β-crystallins and γ-crystallins, are important for lens stability. Each of them contains closely sequence related subunits. The γ-crystallins have 7 subunits (A, B, C, D, E, F, S) with a mass of ∼21 kDa, whereas β-crystallins have 4 acidic (A) and 3 basic (B) subunits with a mass of ∼22 to 28 kDa. They share a similar molecular structure.6 Both β-crystallin and γ-crystallin are homologous β-sheet proteins.4,7,8 Even though they both contain 2 domains, because of the difference in connecting region between domains, γ-crystallins are monomers, whereas β-crystallins are dimers or oligomers. All crystallin proteins are very stable in their native state and in native environmental conditions.

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CATARACT FORMATION

The lens tissue seems to be well maintained due to the durable crystallin proteins and the presence of a high level of chaperone concentration that deals with any misfolding of its structural proteins.9 However, with aging, accumulation of destabilizing factors can result in protein insolubility and aggregation. These phenomena change the refractive index of the lens, which impairs vision, and is the process of cataract formation. These destabilizing conditions could be caused by a variety of events, including changes in cellular conditions as well as the mutations and posttranslational modifications of proteins.7 The most probable pathway for cataract formation is solubility loss of crystallin proteins resulting in fibril (aggregate) formation.

The lens crystallin proteins are covalently damaged with aging through UV radiation, deamination, oxidation, racemization, truncation, and phosphorylation.10,11 Deamination is the most common posttranslational modification observed in cataracts, results from the transformation of glutamine and asparagine residues to glutamic and aspartic acid.12 This modification introduces a negative charge to the protein.13 As deamination decreases the thermodynamic and kinetic stability of the protein, it is believed to cause protein aggregation.14

Oxidation due to UV radiation and oxidative stress is another common damage mechanism in the lens. The most oxidation-susceptible residue is tryptophan, followed by cysteine and methionine.15–17 Tryptophan oxidation generates a less aromatic residue that leads to protein destabilization because these residues are buried in core regions18 of the molecule. Moreover, a recent study showed that during the oxidation of a tryptophan residue next to an aspartic acid, the racemization of that residue is accelerated.19 UV radiation also causes protein truncation, which is associated with backbone cleavage of the molecule, mainly in the interdomain region.20 Truncated crystallin proteins and peptides lose their structural stability, which may lead to the formation of insoluble aggregates. Through the chaperone activity of α-crystallin, aggregation of deaminated and truncated crystallins could be suppressed.21,22 However, α-crystallin truncations result in a loss of chaperone activity,23 which results in the inevitable aggregation of various crystallins.

Beside covalent damages on crystallin proteins, mutations are also responsible for cataract formation, albeit mainly in early-onset cases. Many mutations have been characterized in crystallin proteins, each causing cataract formation in different pathways. Some of the mutations in β-crystallin and γ-crystallin decrease the solubility without affecting the protein structural stability.24,25 Whereas some others lead to destabilization and protein misfolding26 and/or change the chaperone recognition regions.27 In contrast, mutations on α-crystallin results in either the loss of chaperone activity or protein insolubility.28–30

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PROSPECTIVE TREATMENTS

The only available cataract treatment is surgical replacement of the lens with a synthetic one. Finding alternative treatments is crucial particularly for third-world countries where access to trained surgeons is limited and the cost of surgery is prohibitive and cataract formation can directly affect quality of life for whole families.

In recent decades, cataract has beeen considered an aggregation disease like Alzheimer, Huntington, or Parkinson disease. The aggregate formation is initiated with misfolded but soluble proteins in all aggregation diseases but not in cataracts or exfoliation syndrome. Hence, for these diseases, aggregation/plaque formation is believed to be a protection mechanism against toxic monomers and fibril disruption is not considered as a treatment. However, in the case of the 2 eye diseases, cataracts and exfoliation syndrome, protein misfolding (and insolubility) and aggregation is the pathology causing the diseases. Therefore, prevention of formation of aggregates as well as their destruction could be major treatment strategies.

As α-crystallins exhibit the major maintenance responsibility in the lens, increasing their cellular concentration could be a preventive approach. The chaperone functional region of α-crystallin is already shown to be effective for inhibition of amyloid fibrillogenesis.31 Also, α-crystallin delivery to lens tissue has recently been demonstrated as a promising method.32 Beside molecular chaperones, chemical chaperones, due to their lower molecular mass, could be investigated. In contrast, chemical compounds enhancing chaperone activity are considered as drug targets for cataract treatment.33,34

Utilization of β-sheet breakers that stabilize the native conformation or destabilize the misfolded structure could be considered as a fibril dissociation strategy.35 Even though β-sheet breakers have been tested for different aggregation diseases, no study has been undertaken to evaluate their effectiveness on treating cataracts.

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PEPTIDE-BASED TREATMENT STRATEGY

The screening of small molecules that inhibit protein aggregation and cell damage is a promising strategy for identification of drug candidates. The screened molecules are commonly chemical compounds and the screening is mainly applied on whole cells.36 Therefore, the detailed inhibitory mechanism of drug candidates is not generally considered and further improvements using this strategy are limited. A similar but targeted specific strategy, peptide screening, could be achieved by using peptide display libraries. Because of their specific binding abilities, peptides are promising candidates for recognition of crystallin proteins and their aggregates.37 Furthermore, peptides could influence stabilization of protein/aggregate as well as activity depending on the binding region.38 Therefore, crystalline-specific peptides can exhibit potential not only as drug delivery mediators but also aggregation inhibitory molecules.

The specific peptides for insoluble crystallin proteins and aggregates could be selected using either cell or phage display techniques. As recombinant crystallin proteins exhibit similar aggregation profiles with natural lens proteins, peptide selection could be performed against aggregated recombinant proteins as well as cataract lens tissue. Working with aggregates of individual crystallin proteins (α, β, γ) will allow the identification of peptide regions recognizing different crystallin families. Thus, peptides could be identified that stabilize the structure and increase the solubility of β-crystallin and/or γ-crystallin. In addition, it may be possible to develop peptides that enhance chaperone activity of α-crystallin protein.

Once the specific peptides are selected, their effect on aggregation processes could be followed using a variety of in situ techniques, including time-sequenced atomic force microscopy visualization. Peptides can be screened for particular inhibitory properties to be considered as potential therapeutical agents and evaluated on lens tissue and animal models (Fig. 1).

FIGURE 1

FIGURE 1

Peptides that exhibit specific recognition activity, but are not functionally inhibitory, could be used as drug mediators. These peptides could be subsequently tailored through the incorporation of chemical, thermal, or mechanical β-sheet disrupters such as magnetic nanoparticles. Collectively, this strategy may provide a new treatment moiety for cataracts in developing nations and provide a path forward for the clinical treatment of exfoliation syndrome and glaucoma.

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Keywords:

exfoliation syndrome; cataract; affinity peptides; treatment

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