Antiapoptotic Properties of α-Crystallin–Derived Peptide Chaperones and Characterization of Their Uptake Transporters in Human RPE Cells

Abstract

Citation: Sreekumar PG, Chothe P, Sharma KK, et al. Antiapoptotic properties of a-crystallin-derived peptide chaperones and characterization of their uptake transporters in human RPE cells. Invest Ophthalmol Vis Sci. 2013;54:278754: -279854: . DOI:10.1167 PURPOSE. The chaperone proteins, a-crystallins, also possess antiapoptotic properties. The purpose of the present study was to investigate whether 19 to 20-mer a-crystallin-derived mini-chaperone peptides (a-crystallin mini-chaperone) are antiapoptotic, and to identify their putative transporters in human fetal RPE (hfRPE) cells. METHODS.Cell death and caspase-3 activation induced by oxidative stress were quantified in early passage hfRPE cells in the presence of 19 to 20-mer aA-or aB-crystallin-derived or scrambled peptides. Cellular uptake of fluorescein-labeled, a-crystallin-derived mini-peptides and recombinant full-length aB-crystallin was determined in confluent hfRPE. The entry mechanism in hfRPE cells for a-crystallin mini-peptides was investigated. The protective role of polycaprolactone (PCL) nanoparticle encapsulated aB-crystallin mini-chaperone peptides from H 2 O 2 -induced cell death was studied.RESULTS. Primary hfRPE cells exposed to oxidative stress and either aA-or aB-crystallin minichaperones remained viable and showed marked inhibition of both cell death and activation of caspase-3. Uptake of full-length aB-crystallin was minimal while a time-dependent uptake of aB-crystallin-derived peptide was observed. The mini-peptides entered the hfRPE cells via the sodium-coupled oligopeptide transporters 1 and 2 (SOPT1, SOPT2). PCL nanoparticles containing aB-crystallin mini-chaperone were also taken up and protected hfRPE from H 2 O 2 -induced cell death at significantly lower concentrations than free aB-crystallin minichaperone peptide.CONCLUSIONS. aA-and aB-crystallin mini-chaperones offer protection to hfRPE cells and inhibit caspase-3 activation. The oligopeptide transporters SOPT1 and SOPT2 mediate the uptake of these peptides in RPE cells. Nanodelivery of aB-crystallin-derived mini-chaperone peptide offers an alternative approach for protection of hfRPE cells from oxidant injury.Keywords: a-crystallin, chaperone peptides, oxidative stress, RPE protection, oligopeptide transporters T he superfamily of small heat shock proteins (sHSPs) has attracted considerable attention in recent years because of its multifunctional cellular properties. The human genome encodes 10 members of the sHSP family, among which aAcrystallin and aB-crystallin are considered important members. 1 Both aA-and aB-crystallins have been studied extensively in the lens for their chaperone and related functions. However, recent studies have identified several novel functions for aA-and aBcrystallins in retina and other tissues in addition to their wellrecognized chaperone function. 2 Both a-crystallins are expressed in RPE cells and in the retina; higher expression of aBcrystallin was found in the RPE while aA-crystallin was found mostly in photoreceptors and astroglial and Mül...

Protein polymer nanoparticles engineered as chaperones protect against apoptosis in human retinal pigment epithelial cells

“…Protein chaperones protect RPE cells against apoptosis by interfering with caspase activation and/or mitochondrial processes that lead to cell death [26]. Having demonstrated that crySI and cryS96 both have chaperone activity, their potential anti-apoptotic properties were evaluated on RPE cells co-incubated with 200 μM H 2 O 2 for 24 hours.…”

“…We have previously shown that exogenous recombinant αB crystallin and the mini-αB crystallin peptide (cry) can be taken up by RPE cells [26], which may play a role in the suppression of apoptosis. To determine if exogenous ELP fusions to the mini-peptide would also have access to the cytosol, confocal microscopy was used to track the internalization of rhodamine labeled material.…”

Therapeutic potential of α-crystallin

“…A recent study showed that the intraperitoneal injection of MACs or of their acetyl derivatives inhibited selenite-induced cataracts in rats, which was accompanied by the inhibition of protein aggregation and lens epithelial cell apoptosis [97]. A MAC derived from αB-crystallin has been shown to inhibit oxidative stress-mediated apoptosis in cultured retinal pigmented epithelial cells [98]; this study also showed that MAC entry into cells was mediated by sodium-coupled oligopeptide transporters. The inhibition of amyloid fibrillogenesis and toxicity of the αA-crystallin MAC [99] could be further exploited for therapy against Alzheimer’s disease.…”

“…Because this peptide is expected to be refractory to proteases as well, it might be retained longer in the system and thus might better protect cells. The discovery of oligopeptide transporters for α-crystallin [98] is another exciting area. If such transportation can be improved and engineered for selected delivery to tissues, the functionality of the MACs could not only be improved but could also be directed toward specific tissues.…”

Extracellular α-crystallin protects astrocytes from cell death through activation of MAPK, PI3K/Akt signaling pathway and blockade of ROS release from mitochondria

“…Recently published data shed light on possible mechanisms of uptake of αB-crystallin/HSPB5. A study observed the time-dependent uptake of αB-crystallin-derived mini-peptides via the sodiumcoupled oligopeptide transporters 1 and 2 (Sreekumar et al, 2013). The αB-crystallin protein uptake may involve the process of molecular diffusion, as characterized for γD crystallin (Danysh et al, 2010) in lens capsule.…”