Promotion of Protein Solubility and Reduction in Stiffness in Human Lenses by Aggrelyte-1: Implications for Reversing Presbyopia.

With aging, human lenses lose the ability to focus on nearby objects due to decreases in accommodative ability, a condition known as presbyopia. An increase in stiffness or decrease in lens elasticity due to protein aggregation and insolubilization are the primary reasons for presbyopia. In this study, we tested aggrelyte-1 (S,N-diacetyl glutathione diethyl ester) for its ability to promote protein solubility and decrease the stiffness of lenses through its dual property of lysine acetylation and disulfide reduction. Treatment of water-insoluble proteins from aged human lenses (58-75 years) with aggrelyte-1 significantly increased the solubility of those proteins. A control compound that did not contain the S-acetyl group (aggrelyte-1C) was substantially less efficient in solubilizing water-insoluble proteins. Aggrelyte-1-treated solubilized protein had significant amounts of acetyllysine, as measured by Western blotting and LC-MS/MS. Aggrelytes increased the protein-free thiol content in the solubilized protein. Aged mouse (7 months) and human (44-66 years) lenses treated with aggrelyte-1 showed reduced stiffness accompanied by higher free thiol and acetyllysine levels compared with those treated with aggrelyte-1C or untreated controls. Our results suggested that aggrelyte-1 reduced lens stiffness through acetylation followed by disulfide reduction. This proof-of-concept study paves the way for developing aggrelyte-1 and related compounds to reverse presbyopia.

Transforming growth factor-ß2-mediated mesenchymal transition in lens epithelial cells is repressed in the absence of RAGE.

Transforming growth factor-ß2 (TGFß2)-mediated epithelial to mesenchymal transition (EMT) in lens epithelial cells (LECs) has been implicated in fibrosis associated with secondary cataracts. In this study, we investigated whether the receptor for advanced glycation end products (RAGE) plays a role in TGFß2-mediated EMT in LECs. Unlike in the LECs from wild-type mice, TGFß2 failed to elicit an EMT response in LECs from RAGE knockout mice. The lack of RAGE also diminished TGFß2-mediated Smad signaling. In addition, treatment with TGFß2 increased IL-6 levels in LECs from wild-type mice but not in those from RAGE knockout mice. Treatment of human LECs with the RAGE inhibitor FPS-ZM1 reduced TGFß2-mediated Smad signaling and the EMT response. Unlike that in wild-type lenses, the removal of fiber cell tissue in RAGE knockout lenses did not result in elevated levels of α-smooth muscle actin (α-SMA), fibronectin (FN), and integrin ß1 in capsule-adherent LECs. Taken together, these results suggest that TGFß2 signaling is intricately linked to RAGE. Targeting RAGE could be explored as a therapeutic strategy against secondary cataracts.

Glycation-mediated inter-protein cross-linking is promoted by chaperone-client complexes of α-crystallin: Implications for lens aging and presbyopia.

Lens proteins become increasingly cross-linked through nondisulfide linkages during aging and cataract formation. One mechanism that has been implicated in this cross-linking is glycation through formation of advanced glycation end products (AGEs). Here, we found an age-associated increase in stiffness in human lenses that was directly correlated with levels of protein-cross-linking AGEs. α-Crystallin in the lens binds to other proteins and prevents their denaturation and aggregation through its chaperone-like activity. Using a FRET-based assay, we examined the stability of the αA-crystallin-γD-crystallin complex for up to 12 days and observed that this complex is stable in PBS and upon incubation with human lens-epithelial cell lysate or lens homogenate. Addition of 2 mm ATP to the lysate or homogenate did not decrease the stability of the complex. We also generated complexes of human αA-crystallin or αB-crystallin with alcohol dehydrogenase or citrate synthase by applying thermal stress. Upon glycation under physiological conditions, the chaperone-client complexes underwent greater extents of cross-linking than did uncomplexed protein mixtures. LC-MS/MS analyses revealed that the levels of cross-linking AGEs were significantly higher in the glycated chaperone-client complexes than in glycated but uncomplexed protein mixtures. Mouse lenses subjected to thermal stress followed by glycation lost resilience more extensively than lenses subjected to thermal stress or glycation alone, and this loss was accompanied by higher protein cross-linking and higher cross-linking AGE levels. These results uncover a protein cross-linking mechanism in the lens and suggest that AGE-mediated cross-linking of α-crystallin-client complexes could contribute to lens aging and presbyopia.

Advanced glycation end products in human diabetic lens capsules.

Advanced glycation end products (AGEs) accumulate with age in human lens capsules. AGEs in lens capsules potentiate the transforming growth factor beta-2-mediated mesenchymal transition of lens epithelial cells, which suggests that they play a role in posterior capsule opacification after cataract surgery. We measured AGEs by liquid chromatography-mass spectrometry in capsulorhexis specimens obtained during cataract surgery from nondiabetic and diabetic patients with and without established retinopathy. Our data showed that the levels of most AGEs (12 out of 13 measured) were unaltered in diabetic patients and diabetic patients with retinopathy compared to nondiabetic patients. There was one exception: glucosepane, which was significantly higher in diabetic patients, both with (6.85 pmol/µmol OH-proline) and without retinopathy (8.32 pmol/µmol OH-proline), than in nondiabetic patients (4.01 pmol/µmol OH-proline). Our study provides an explanation for the similar incidence of posterior capsule opacification between nondiabetic and diabetic cataract patients observed in several studies.

Glycation-mediated protein crosslinking and stiffening in mouse lenses are inhibited by carboxitin in vitro.

Proteins in the eye lens have negligible turnover and therefore progressively accumulate chemical modifications during aging. Carbonyls and oxidative stresses, which are intricately linked to one another, predominantly drive such modifications. Oxidative stress leads to the loss of glutathione (GSH) and ascorbate degradation; this in turn leads to the formation of highly reactive dicarbonyl compounds that react with proteins to form advanced glycation end products (AGEs). The formation of AGEs leads to the crosslinking and aggregation of proteins contributing to lens aging and cataract formation. To inhibit AGE formation, we developed a disulfide compound linking GSH diester and mercaptoethylguanidine, and we named it carboxitin. Bovine lens organ cultured with carboxitin showed higher levels of GSH and mercaptoethylguanidine in the lens nucleus. Carboxitin inhibited erythrulose-mediated mouse lens protein crosslinking, AGE formation and the formation of 3-deoxythreosone, a major ascorbate-derived AGE precursor in the human lens. Carboxitin inhibited the glycation-mediated increase in stiffness in organ-cultured mouse lenses measured using compressive mechanical strain. Delivery of carboxitin into the lens increases GSH levels, traps dicarbonyl compounds and inhibits AGE formation. These properties of carboxitin could be exploited to develop a therapy against the formation of AGEs and the increase in stiffness that causes presbyopia in aging lenses.

Transient elevation of temperature promotes cross-linking of α-crystallin-client proteins through formation of advanced glycation endproducts: A potential role in presbyopia and cataracts.

The chaperone activity of α-crystallin is important for maintaining the transparency of the human lens. αB-crystallin (αBC) is a long-lived protein in the lens that accumulates chemical modifications during aging. The formation of advanced glycation end products (AGEs) through glycation is one such modification. αBC is a small heat shock protein that exhibits chaperone activity. We have previously shown that αBC-client protein complexes can undergo AGE-mediated interprotein cross-linking. Here, we demonstrate that short-term (1 h) exposure to elevated temperatures and methylglyoxal (MGO) during the chaperoning of client proteins by αBC promotes AGE-mediated interprotein cross-linking. Liquid chromatography/mass spectrometry (LC-MS/MS) analyses revealed the rapid formation of AGEs by MGO. Interestingly, we found that despite protein cross-linking, the chaperone activity of αBC increased during the transient elevation of temperature in the presence of MGO. Together, these results imply that transient and subtle elevation of temperature in the lens of the eye can promote protein cross-linking through AGEs, and if this phenomenon recurs over a period of many years, it could lead to early onset of presbyopia and age-related cataracts.

Kynurenic Acid Protects Against Ischemia/Reperfusion-Induced Retinal Ganglion Cell Death in Mice.

BACKGROUND: Glaucoma is an optic neuropathy and involves the progressive degeneration of retinal ganglion cells (RGCs), which leads to blindness in patients. We investigated the role of the neuroprotective kynurenic acid (KYNA) in RGC death against retinal ischemia/reperfusion (I/R) injury. METHODS: We injected KYNA intravenously or intravitreally to mice. We generated a knockout mouse strain of kynurenine 3-monooxygenase (KMO), an enzyme in the kynurenine pathway that produces neurotoxic 3-hydroxykynurenine. To test the effect of mild hyperglycemia on RGC protection, we used streptozotocin (STZ) induced diabetic mice. Retinal I/R injury was induced by increasing intraocular pressure for 60 min followed by reperfusion and RGC numbers were counted in the retinal flat mounts. RESULTS: Intravenous or intravitreal administration of KYNA protected RGCs against I/R injury. The I/R injury caused a greater loss of RGCs in wild type than in KMO knockout mice. KMO knockout mice had mildly higher levels of fasting blood glucose than wild type mice. Diabetic mice showed significantly lower loss of RGCs when compared with non-diabetic mice subjected to I/R injury. CONCLUSION: Together, our study suggests that the absence of KMO protects RGCs against I/R injury, through mechanisms that likely involve higher levels of KYNA and glucose.

Phosphatidic acid binding proteins display differential binding as a function of membrane curvature stress and chemical properties.

Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins.

Identification and functional characterization of the Arabidopsis†Snf1-related protein kinase SnRK2.4 phosphatidic acid-binding domain.

Phosphatidic acid (PA) is an important signalling lipid involved in various stress-induced signalling cascades. Two SnRK2 protein kinases (SnRK2.4 and SnRK2.10), previously identified as PA-binding proteins, are shown here to prefer binding to PA over other anionic phospholipids and to associate with cellular membranes in response to salt stress in Arabidopsis roots. A 42 amino acid sequence was identified as the primary PA-binding domain (PABD) of SnRK2.4. Unlike the full-length SnRK2.4, neither the PABD-YFP fusion protein nor the SnRK2.10 re-localized into punctate structures upon salt stress treatment, showing that additional domains of the SnRK2.4 protein are required for its re-localization during salt stress. Within the PABD, five basic amino acids, conserved in class 1 SnRK2s, were found to be necessary for PA binding. Remarkably, plants overexpressing the PABD, but not a non-PA-binding mutant version, showed a severe reduction in root growth. Together, this study biochemically characterizes the PA-SnRK2.4 interaction and shows that functionality of the SnRK2.4 PABD affects root development.

Thiol-Mediated Enhancement of Nε-Acetyllysine Formation in Lens Proteins.

Lysine acetylation (AcK) is a prominent post-translational modification in eye lens crystallins. We have observed that AcK formation is preferred in some lysine residues over others in crystallins. In this study, we have investigated the role of thiols in such AcK formation. Upon incubation with acetyl-CoA (AcCoA), αA-Crystallin, which contains two cysteine residues, showed significantly higher levels of AcK than αB-Crystallin, which lacks cysteine residues. Incubation with thiol-rich γS-Crystallin resulted in higher AcK formation in αB-Crystallin from AcCoA. External free thiol (glutathione and N-acetyl cysteine) increased the AcK content in AcCoA-incubated αB-Crystallin. Reductive alkylation of cysteine residues significantly decreased (p < 0.001) the AcCoA-mediated AcK formation in αA-Crystallin. Introduction of cysteine residues within ∼5 Å of lysine residues (K92C, E99C, and V169C) in αB-Crystallin followed by incubation with AcCoA resulted in a 3.5-, 1.3- and 1.3-fold increase in the AcK levels when compared to wild-type αB-Crystallin, respectively. Together, these results suggested that AcK formation in α-Crystallin is promoted by the proximal cysteine residues and protein-free thiols through an S → N acetyl transfer mechanism.

Lens capsule advanced glycation end products induce senescence in epithelial cells: Implications for secondary cataracts.

Posterior capsule opacification (PCO) is a common complication after cataract surgery. Residual lens epithelial cells (LECs) on the anterior lens capsule, after cataract surgery, migrate to the posterior lens capsule and undergo transdifferentiation into myofibroblast-like cells. Those cells synthesize excessive amounts of extracellular matrix and contribute to fibrosis during PCO. Cellular senescence, a phenomenon that increases with aging, has been implicated in several fibrotic diseases. Here, we have investigated the prevalence of senescent LECs within the lens posterior capsule and the ability of advanced glycation end products (AGEs) in lens capsules to induce senescence, contributing to PCO. Aged lens capsules from pseudophakic human cadaver eyes showed the presence of senescent LECs. In human capsular bags, LECs showed an age-dependent increase in senescence after 28 days of culture. Human LECs cultured on aged lens capsules for 3 days underwent senescence; this effect was not seen in LECs cultured on young lens capsules. Human LECs cultured on an AGE-modified extracellular matrix (ECM-AGEs) showed an AGE-concentration-dependent increase in the expression of senescence markers and reactive oxygen species (ROS) levels. Treatment with a RAGE antagonist and ROS inhibitor reduced the expression of senescence and fibrotic markers. Additionally, conditioned media from ECM-AGEs-treated cells induced the expression of fibrotic markers in naïve LECs. Together, these suggest that AGEs in the capsule induce senescence of LECs, which triggers the mesenchymal transition of neighboring non-senescent LECs and contributes to PCO.

Topical ocular application of aggrelyte-2A reduces lens stiffness in mice.

Presbyopia is the progressive loss of the ability of the lens to focus on nearby objects due to its increased stiffness. It occurs in the mid-40s and continues to worsen until the mid-60s. The age-associated increase in protein cross-linking in the lens leads to protein aggregation and water insolubility, especially in the nuclear region, contributing to lens stiffness. This study reports the development of aggrelyte-2A (methyl S-acetyl-N-(3,3-dimethylbutanoyl) cysteinate, a derivative of our previously reported aggrelyte-2) for reversing the stiffness of aged lenses. Aggrelyte-2A showed minimal toxicity in cultured mouse lens epithelial cells (up to 2000 µM) and human lens epithelial cells (up to 250 µM). Lenses from aged mice (age: 24-25 months) treated with 1 mM aggrelyte-2A for 24 h, and human lenses (age: 47-67 years) treated with 250 µM aggrelyte-2A for 48 h showed 11-14% reductions in stiffness, accompanied by an increase in acetyllysine in lens proteins, and free-thiols in the lens. Topical application of aggrelyte-2A (40 mM, 5 µl twice daily for 4 weeks) on mouse eyes significantly reduced lens stiffness. The topical application showed no toxicity to the lens, cornea, or retina, as revealed by morphological examination, H&E staining, and optical coherence tomography. These data suggest that aggrelyte-2A could be developed as a presbyopia-reversing therapeutic.

Aggrelyte-2 promotes protein solubility and decreases lens stiffness through lysine acetylation and disulfide reduction: Implications for treating presbyopia.

Aging proteins in the lens become increasingly aggregated and insoluble, contributing to presbyopia. In this study, we investigated the ability of aggrelyte-2 (N,S-diacetyl-L-cysteine methyl ester) to reverse the water insolubility of aged human lens proteins and to decrease stiffness in cultured human and mouse lenses. Water-insoluble proteins (WI) of aged human lenses (65-75 years) were incubated with aggrelyte-2 (500 µM) for 24 or 48 h. A control compound that lacked the S-acetyl group (aggrelyte-2C) was also tested. We observed 19%-30% solubility of WI upon treatment with aggrelyte-2. Aggrelyte-2C also increased protein solubility, but its effect was approximately 1.4-fold lower than that of aggrelyte-2. The protein thiol contents were 1.9- to 4.9-fold higher in the aggrelyte-2- and aggrelyte-2C-treated samples than in the untreated samples. The LC-MS/MS results showed Nε -acetyllysine (AcK) levels of 1.5 to 2.1 nmol/mg protein and 0.6 to 0.9 nmol/mg protein in the aggrelyte-2- and aggrelyte-2C-treated samples. Mouse (C57BL/6J) lenses (incubated for 24 h) and human lenses (incubated for 72 h) with 1.0 mM aggrelyte-2 showed significant decreases in stiffness with simultaneous increases in soluble proteins (human lenses) and protein-AcK levels, and such changes were not observed in aggrelyte-2C-treated lenses. Mass spectrometry of the solubilized protein revealed AcK in all crystallins, but more was observed in α-crystallins. These results suggest that aggrelyte-2 increases protein solubility and decreases lens stiffness through acetylation and disulfide reduction. Aggrelyte-2 might be useful in treating presbyopia in humans.

Properties of membrane-incorporated WALP peptides that are anchored on only one end.

Peptides of the "WALP" family, acetyl-GWW(LA)(n)LWWA-[ethanol]amide, have proven to be opportune models for investigating lipid-peptide interactions. Because the average orientations and motional behavior of the N- and C-terminal Trp (W) residues differ, it is of interest to investigate how the positions of the tryptophans influence the properties of the membrane-incorporated peptides. To address this question, we synthesized acetyl-GGWW(LA)(n)-ethanolamide and acetyl-(AL)(n)WWG-ethanolamide, in which n = 4 or 8, which we designate as "N-anchored" and "C-anchored" peptides, respectively. Selected (2)H or (15)N labels were incorporated for solid-state nuclear magnetic resonance (NMR) spectroscopy. These peptides can be considered "half"-anchored WALP peptides, having only one pair of interfacial Trp residues near either the amino or the carboxyl terminus. The hydrophobic lengths of the (n = 8) peptides are similar to that of WALP23. These longer half-anchored WALP peptides incorporate into lipid bilayers as α-helices, as reflected in their circular dichroism spectra. Solid-state NMR experiments indicate that the longer peptide helices assume defined transmembrane orientations with small non-zero average tilt angles and moderate to high dynamic averaging in bilayer membranes of 1,2-dioleoylphosphatidylcholine, 1,2-dimyristoylphosphatidylcholine, and 1,2-dilauroylphosphatidylcholine. The intrinsically small apparent tilt angles suggest that interactions of aromatic residues with lipid headgroups may play an important role in determining the magnitude of the peptide tilt in the bilayer membrane. The shorter (n = 4) peptides, in stark contrast to the longer peptides, display NMR spectra that are characteristic of greatly reduced motional averaging, probably because of peptide aggregation in the bilayer environment, and CD spectra that are characteristic of ß-structure.

Proline kink angle distributions for GWALP23 in lipid bilayers of different thicknesses.

By using selected (2)H and (15)N labels, we have examined the influence of a central proline residue on the properties of a defined peptide that spans lipid bilayer membranes by solid-state nuclear magnetic resonance (NMR) spectroscopy. For this purpose, GWALP23 (acetyl-GGALW(5)LALALALALALALW(19)LAGA-ethanolamide) is a suitable model peptide that employs, for the purpose of interfacial anchoring, only one tryptophan residue on either end of a central α-helical core sequence. Because of its systematic behavior in lipid bilayer membranes of differing thicknesses [Vostrikov, V. V., et al. (2010) J. Biol. Chem. 285, 31723-31730], we utilize GWALP23 as a well-characterized framework for introducing guest residues within a transmembrane sequence; for example, a central proline yields acetyl-GGALW(5)LALALAP(12)ALALALW(19)LAGA-ethanolamide. We synthesized GWALP23-P12 with specifically placed (2)H and (15)N labels for solid-state NMR spectroscopy and examined the peptide orientation and segmental tilt in oriented DMPC lipid bilayer membranes using combined (2)H GALA and (15)N-(1)H high-resolution separated local field methods. In DMPC bilayer membranes, the peptide segments N-terminal and C-terminal to the proline are both tilted substantially with respect to the bilayer normal, by ~34 ± 5° and 29 ± 5°, respectively. While the tilt increases for both segments when proline is present, the range and extent of the individual segment motions are comparable to or smaller than those of the entire GWALP23 peptide in bilayer membranes. In DMPC, the proline induces a kink of ~30 ± 5°, with an apparent helix unwinding or "swivel" angle of ~70°. In DLPC and DOPC, on the basis of (2)H NMR data only, the kink angle and swivel angle probability distributions overlap those of DMPC, yet the most probable kink angle appears to be somewhat smaller than in DMPC. As has been described for GWALP23 itself, the C-terminal helix ends before Ala(21) in the phospholipids DMPC and DLPC yet remains intact through Ala(21) in DOPC. The dynamics of bilayer-incorporated, membrane-spanning GWALP23 and GWALP23-P12 are less extensive than those observed for WALP family peptides that have more than two interfacial Trp residues.

Small Heat Shock Proteins in Retinal Diseases.

This review summarizes the latest findings on small heat shock proteins (sHsps) in three major retinal diseases: glaucoma, diabetic retinopathy, and age-related macular degeneration. A general description of the structure and major cellular functions of sHsps is provided in the introductory remarks. Their role in specific retinal diseases, highlighting their regulation, role in pathogenesis, and possible use as therapeutics, is discussed.

Cell behavior on surface modified polydimethylsiloxane (PDMS).

Designing complex tissue culture systems requires cell alignment and directed extracellular matrix (ECM) and gene expression. Here, a micro-rough, polydimethylsiloxane (PDMS) surface, that also integrates a micro-pattern of 50 µm wide lines of fibronectin (FN) separated by 60 µm wide lines of bovine serum albumin (BSA), is developed. Human fibroblasts cultured on the rough, patterned substrate have aligned growth and a significant change in morphology when compared to cells on a flat, patterned surface. The rough PDMS topography significantly decreases cell area and induces the upregulation of several ECM related genes by two-fold when compared to cells cultured on flat PDMS. This study describes a simple surface engineering procedure for creating surface architecture for scaffolds to design and control the cell-surface interface.

Liposome-binding assays to assess specificity and affinity of phospholipid-protein interactions.

Protein-lipid interactions play an important role in cellular protein relocation, activation and signal transduction. The liposome-binding assay is a simple and inexpensive method to examine protein-lipid binding in vitro. The phospholipids used for liposome production are dried and hydrated. Subsequent extrusion of the phospholipid mixture ensures the production of large unilamellar vesicles (LUV) filled with raffinose. Those LUVs can be easily separated from the aqueous solution by centrifugation. By incubating a protein of interest with the LUVs and subsequent centrifugation steps, the bound protein fraction can be determined using Western Blot or Coomassie staining. This technique enables analysis of protein-lipid binding affinity and specificity.