Characterization and management of ERK inhibitor associated dermatologic adverse events: analysis from a nonrandomized trial of ulixertinib for advanced cancers.

Background Ulixertinib is the first-in-class ERK1/2 kinase inhibitor with encouraging clinical activity in BRAF- and NRAS-mutant cancers. Dermatologic adverse events (dAEs) are common with ulixertinib, so management guidelines like those established for epidermal growth factor receptor inhibitor (EGFRi)-associated dAEs are needed. Patients and Methods This was an open-label, multicenter, phase I dose escalation and expansion trial of ulixertinib evaluating data from 135 patients with advanced malignancies enrolled between March 2013 and July 2017. Histopathological features, management, and dAEs in 34 patients are also reported. Twice daily oral ulixertinib was administered at 10 to 900 mg in the dose escalation cohort (n = 27) and at 600 mg in 21-day cycles in the expansion cohort (n = 108). Results The incidence of ulixertinib-induced dAEs and combined rash were 79% (107/135) and 76% (102/135). The most common dAEs included acneiform rash (45/135, 33%), maculopapular rash (36/135, 27%), and pruritus (34/135, 25%). Grade 3 dAEs were observed in 19% (25/135) of patients; no grade 4 or 5 dAEs were seen. The presence of at least 1 dAE was associated with stable disease (SD) or partial response (PR) (OR = 3.64, 95% CI 1.52-8.72; P = .003). Acneiform rash was associated with a PR (OR = 10.19, 95% CI 2.67-38.91; P < .001). Conclusion The clinical spectrum of ulixertinib-induced dAEs was similar to EGFR and MEK inhibitors; dAEs may serve as a surrogate marker of tumor response. We propose treatment algorithms for common ERK inhibitor-induced dAEs to maintain patients' quality of life and dose intensity for maximal clinical benefit. Clinical Trial Registration: NCT01781429.

Characterization of on-target adverse events caused by TRK inhibitor therapy.

BACKGROUND: The tropomyosin receptor kinase (TRK) pathway controls appetite, balance, and pain sensitivity. While these functions are reflected in the on-target adverse events (AEs) observed with TRK inhibition, these AEs remain under-recognized, and pain upon drug withdrawal has not previously been reported. As TRK inhibitors are approved by multiple regulatory agencies for TRK or ROS1 fusion-positive cancers, characterizing these AEs and corresponding management strategies is crucial. PATIENTS AND METHODS: Patients with advanced or unresectable solid tumors treated with a TRK inhibitor were retrospectively identified in a search of clinical databases. Among these patients, the frequency, severity, duration, and management outcomes of AEs including weight gain, dizziness or ataxia, and withdrawal pain were characterized. RESULTS: Ninety-six patients with 15 unique cancer histologies treated with a TRK inhibitor were identified. Weight gain was observed in 53% [95% confidence interval (CI), 43%-62%] of patients and increased with time on TRK inhibition. Pharmacologic intervention, most commonly with glucagon-like peptide 1 analogs or metformin, appeared to result in stabilization or loss of weight. Dizziness, with or without ataxia, was observed in 41% (95% CI, 31%-51%) of patients with a median time to onset of 2 weeks (range, 3 days to 16 months). TRK inhibitor dose reduction was the most effective intervention for dizziness. Pain upon temporary or permanent TRK inhibitor discontinuation was observed in 35% (95% CI, 24%-46%) of patients; this was more common with longer TRK inhibitor use. TRK inhibitor reinitiation was the most effective intervention for withdrawal pain. CONCLUSIONS: TRK inhibition-related AEs including weight gain, dizziness, and withdrawal pain occur in a substantial proportion of patients receiving TRK inhibitors. This safety profile is unique relative to other anticancer therapies and warrants careful monitoring. These on-target toxicities are manageable with pharmacologic intervention and dose modification.

Phase III randomized study of second line ADI-PEG 20 plus best supportive care versus placebo plus best supportive care in patients with advanced hepatocellular carcinoma.

Background: Arginine depletion is a putative target in hepatocellular carcinoma (HCC). HCC often lacks argininosuccinate synthetase, a citrulline to arginine-repleting enzyme. ADI-PEG 20 is a cloned arginine degrading enzyme-arginine deiminase-conjugated with polyethylene glycol. The goal of this study was to evaluate this agent as a potential novel therapeutic for HCC after first line systemic therapy. Methods and patients: Patients with histologically proven advanced HCC and Child-Pugh up to B7 with prior systemic therapy, were randomized 2 : 1 to ADI-PEG 20 18 mg/m2 versus placebo intramuscular injection weekly. The primary end point was overall survival (OS), with 93% power to detect a 4-5.6 months increase in median OS (one-sided α = 0.025). Secondary end points included progression-free survival, safety, and arginine correlatives. Results: A total of 635 patients were enrolled: median age 61, 82% male, 60% Asian, 52% hepatitis B, 26% hepatitis C, 76% stage IV, 91% Child-Pugh A, 70% progressed on sorafenib and 16% were intolerant. Median OS was 7.8 months for ADI-PEG 20 versus 7.4 for placebo (P = 0.88, HR = 1.02) and median progression-free survival 2.6 months versus 2.6 (P = 0.07, HR = 1.17). Grade 3 fatigue and decreased appetite occurred in <5% of patients. Two patients on ADI-PEG 20 had ≥grade 3 anaphylactic reaction. Death rate within 30 days of end of treatment was 15.2% on ADI-PEG 20 versus 10.4% on placebo, none related to therapy. Post hoc analyses of arginine assessment at 4, 8, 12 and 16 weeks, demonstrated a trend of improved OS for those with more prolonged arginine depletion. Conclusion: ADI-PEG 20 monotherapy did not demonstrate an OS benefit in second line setting for HCC. It was well tolerated. Strategies to enhance prolonged arginine depletion and synergize the effect of ADI-PEG 20 are underway. Clinical Trial number: www.clinicaltrials.gov (NCT 01287585).

Investigation of gammaE-crystallin target protein binding to bovine lens alpha-crystallin by small-angle neutron scattering.

alpha-Crystallin, one of the main constituent proteins in the crystalline lens, is an important molecular chaperone both within and outside the lens. Presently, the structural relationship between alpha-crystallin and its target proteins during chaperone action is poorly understood. It has been hypothesised that target proteins bind within a central cavity. Small-angle neutron-scattering (SANS) experiments in conjunction with isotopic substitution were undertaken to investigate the interaction of a target lens protein (gammaE-crystallin) with alpha-crystallin (alpha(H)) and to measure the radius of gyration (Rg) of the proteins and their binary complexes in solution under thermal stress. The size of the alpha(H) in D(2)O incubated at 65 degrees C increased from 69+/-3 to 81+/-5 A over 40 min, in good agreement with previously published small-angle X-ray scattering (SAXS) and SANS measurements. Deuterated gammaE-crystallin in H(2)O buffer (gammaE(D)/H(2)O) and hydrogenous gammaE-crystallin in D(2)O buffer (gammaE(H)/D(2)O) free in solution were of insufficient size and/or too dilute to provide any measurable scattering over the angular range used, which was selected primarily to investigate gammaE:alpha(H) complexes. The evolution of the aggregation size/shape as an indicator of alpha(H) chaperone action was monitored by recording the neutron scattering in different H:D solvent contrasts under thermally stressed conditions (65 degrees C) for binary mixtures of alpha(H), gammaE(H), and gammaE(D). It was found that Rg(alpha(H):gammaE(D)/D(2)O)>Rg(alpha(H):gammaE(H)/D(2)O)>Rg(alpha(H)/D(2)O) and that Rg(alpha(H):gammaE(H)/D(2)O) approximately Rg(alpha(H)/D(2)O). The relative sizes observed for the complexes weighted by the respective scattering powers of the various components imply that gammaE-crystallin binds in a central cavity of the alpha-crystallin oligomer, during chaperone action.

Effects of N-acetylcysteine and glutathione ethyl ester drops on streptozotocin-induced diabetic cataract in rats.

PURPOSE: To evaluate the effect of N-acetylcysteine (NAC) and glutathione ethyl ester (GSH-EE) eye drops on the progression of diabetic cataract formation induced by streptozotocin (STZ). METHODS: One hundred and thirty Sprague-Dawley (SD) rats were selected, and diabetes was induced by streptozotocin (65 mg/kg bodyweight) in a single intraperitoneal injection. The control group (group I) received only vehicle. Then, 78 rats with random blood glucose above 14 mmol/l were divided into four groups (group II-V). The drug-treated rats received NAC and GSH-EE eye drops five days before STZ injection. Group I and V animals received sodium phosphate buffer drops (pH 7.4), and those in groups II, III, and IV received 0.01% NAC, 0.05% NAC, and 0.1% GSH-EE drops, respectively. Lens transparency was monitored with a slit lamp biomicroscope and classified into six stages. At the end of four weeks, eight weeks, and 13 weeks, animals were killed and components involved in the pathogenesis of diabetic cataract including thiols (from glutathione and protein), glutathione reductase (GR), catalase (CAT), and glycated proteins were investigated in the lens extracts. Blood glucose, urine glucose, and bodyweight were also determined. RESULTS: The progression in lens opacity induced by diabetes showed a biphasic pattern in which an initial slow increase in the first seven weeks after STZ injection was followed by a rapid increase in the next six weeks. The progression of lens opacity in the treated groups (group II-IV) was slower than that of the untreated group (group V) in the earlier period and especially in the fourth week. There were statistically significant differences between the treated groups and the untreated group (p<0.05). However, these differences became insignificant after the sixth week, and the progression of lens opacification in all diabetic groups became aggravated. The content of thiol (from glutathione and protein), glutathione reductase (GR), and catalase (CAT) were lower in the lens extracts of the diabetic rats four weeks, eight weeks, and 13 weeks after the STZ injection while the levels of thiol and CAT activity were both higher in the treated groups (group II-IV) than in the untreated group (group V) at every stage. However, there was no statistically significant difference (p>0.05). Moreover, the diabetes resulted in an increased level of glycated proteins in both the treated groups and the untreated group, but there was no statistically significant difference between all the diabetic groups (p>0.05). CONCLUSIONS: NAC and GSH-EE can slightly inhibit the progression of the diabetic cataract at the earlier stage. They may maintain lens transparency and function by serving as a precursor for glutathione biosynthesis and by protecting sulfhydryl groups from oxidation.

Effect of carnosine, aminoguanidine, and aspirin drops on the prevention of cataracts in diabetic rats.

PURPOSE: To investigate the effect of carnosine (CA), aminoguanidine (AG), and aspirin (ASA) drops, all inhibitors of glycation, on the development of diabetic cataract in rat. METHODS: Rats were made diabetic with streptozotocin, and based on the level of plasma glucose, they were assigned as non-diabetic rats (<14 mmol/l plasma glucose) and diabetic rats (>14 mmol/l plasma glucose). Animals in the treated groups received CA, AG, and ASA as drops to the left eyes starting from the day of streptozotocin injection. Progression of lens opacification was recorded using the slit lamp at regular time intervals. All the rats were killed after the week 13, and the levels of advanced glycation end products (AGE), glutathione reductase (GR), catalase (CAT), and glutathione (GSH) were determined. RESULTS: Lens opacification progressed in a biphasic manner in the diabetic rats, an initial slow increase during the first eight weeks of diabetes followed by a steep increase in the next five weeks. Carnosine treatment delayed the progression of cataracts in diabetic rats, and the delay was statistically significant on the fourth week of diabetes (p<0.05, when compared with untreated moderately diabetic rats). A decrease in the antioxidant enzymes of CAT and the level of GSH was found in the lens of the untreated diabetic rats at 13 weeks after injection. Some protection was provided in the treated eyes. The level of glycation in the untreated diabetic rats was significantly higher than that in the normal rats (p<0.001). After treatment with CA, AG, and ASA, those diabetic rats had a lower level of glycated lens protein compared to the untreated diabetic rats (p<0.001). CONCLUSIONS: These results thus suggest that the effect of CA, AG, and ASA is indeed inhibition of the formation of AGEs. However, the effect of CA, AG, and ASA is overwhelmed by the excessive accumulation of AGEs in the severely diabetic rats. CA compared with AG and ASA treatment can delay the progression of lens opacification in the diabetic rats only during the earlier stages. It also protects against the inactivation of enzymes.

Pinpointing the sites of hydroxylysine glycosides in peptide alpha 1-CB7 of bovine corneal collagen, and their possible role in determining fibril diameter and thus transparency.

Two cyanogen bromide fragments (alpha 1-CB7 and alpha 1-CB8) of bovine corneal stromal collagen have been isolated and characterized. These added to those characterized in our previous work account for 95% of the amino acid sequence of the alpha 1(1)-chain. The hydroxylysine glycoside content of each fragment was determined and in this way the general distribution of glycoside over the entire molecule was deduced accounting for all the galactosylhydroxylysine and most of the glucosylgalactosylhydroxylysine of this heavily glycosylated type I collagen. The characterization of fragments alpha 1-CB7 and alpha 1-CB8 has enabled us to resolve the controversy over the relative mobilities of these fragments on SDS gels. Fragment alpha 1-CB7 of bovine corneal collagen was digested by trypsin and by staphylococcal proteinase V8. The resultant peptides were isolated by gel and ion-exchange chromatography and identified in relation to the known amino acid sequence of type I collagen. The hydroxylysine glycosides were determined in the relevant peptides providing a complete account of their distribution along this part of the collagen molecule. Most of the glycoside was found in the gap region of collagen especially near the edges of the axial holes where it could act as a peg to facilitate fibre formation. In addition, some glycoside was found in the overlap region where, being unable to fit into axial holes, it might impede the growth of the fibre and, with other glycoside of the overlap region, might be responsible for the narrow fibres of corneal collagen that are essential for corneal transparency. This glycoside, with that previously found in the peptide alpha 1-CB3 is the only hydroxylysine glycoside identified in the overlap region of a type I collagen.

Inactivation of glyceraldehyde 3-phosphate dehydrogenase by sugars, prednisolone-21-hemisuccinate, cyanate and other small molecules.

Diabetes, diarrhoea, renal failure and glucocorticoid therapy have all been identified as independent risk factors for cataract. Increased post-translational modification of proteins, leading to inactivation of enzymes and induction of conformational changes within proteins could result in lens opacification and cataract. Aspirin has been associated with many beneficial effects, including protection against cataract, in-vivo. alpha-Crystallin has been shown to act as a molecular chaperone in-vitro. This lenticular protein prevented the thermal aggregation of other lens proteins in-vitro and has sequence and functional homology with the small heat shock proteins. Glyceraldehyde 3-phosphate dehydrogenase (GAP-DH) is constitutively expressed in tissues and is susceptible to chemical modification in-vivo. In-vitro incubations of GAP-DH with sugars, cyanate and prednisolone-21-hemisuccinate, all led to significant loss of enzyme activity with time in two buffer systems. Rapid inactivation occurred when GAP-DH was incubated with fructose 6-phosphate or prednisolone-21-hemisuccinate. Slower inactivation was observed when GAP-DH was incubated with fructose, glucose 6-phosphate or potassium cyanate. Glucose did not inactivate GAP-DH under the conditions of our experiments. Aspirin and ibuprofen were shown to inactivate GAP-DH very rapidly in-vitro. Bovine lenticular alpha-crystallin conferred no protection against GAP-DH inactivation. This is the first occasion that alpha-crystallin has been demonstrated to be unable to protect against inactivation in our chemical enzyme inactivation system. This may have implications for the susceptibility of lenticular GAP-DH to post-translational inactivation.

Identification of the site of glycation of gamma-II-crystallin by (14C)-fructose.

Cataract formation in diabetes may be via non-enzymic glycosylation (glycation) of lens proteins due to increased concentrations of sugars present in the lenses of diabetic patients. The objective of this project was to identify the site(s) of glycation of bovine gamma-II-crystallin by [14C]fructose. gamma-II-crystallin was isolated from soluble lens nucleus proteins by gel chromatography, followed by ion-exchange chromatography and was then glycated by incubation with [14C]fructose. Radioactively labelled gamma-II-crystallin was cleaved with trypsin. Affinity chromatography of the tryptic peptides gave a single main peak containing the majority of the radioactivity. This indicated that fructose had reacted at a single site on the protein. Amino acid analysis of this peptide showed it to contain only lysine and a trace amount of glycine. By relating the results of the amino acid analysis to the amino acid sequence of gamma-II-crystallin, it was concluded that the labelled peptide corresponded to the N-terminal dipeptide. The site of glycation of bovine gamma-II-crystallin by fructose was thereby identified as the alpha-NH2 group of the N-terminal glycine.

The effects of ageing on the chaperone-like function of rabbit alpha-crystallin, comparing three methods of assay.

The lens has a high protein content necessary for focusing light on to the retina. Alpha-Crystallin accounts for approximately 40% of the protein and has been shown to act in a chaperone-like manner. Here we show the effects of ageing on the chaperone-like properties of alpha-crystallin from rabbit lens. Three assays were used to determine chaperone ability. Non-enzymatic glycosylation inactivation of malate dehydrogenase is protected by alpha-crystallin. Thermal aggregation of beta-low crystallin and malate dehydrogenase are both prevented by alpha-crystallin. Three ages of rabbit lens were used. Alpha-Crystallin from the soluble fraction of the cortex and nucleus were investigated as well as alpha-high and alpha-low fractions resolved by size-exclusion chromatography. All three methods complemented each other. There was no age-dependent loss in chaperone-like behaviour for both alpha fractions in the cortex. There was an early decrease with age of the nuclear alpha-low fraction. Nuclear alpha-high shows no age-related decrease but its chaperoning ability is greatly compromised. Post-translational modifications which occur during ageing may be responsible for the effect of alpha-crystallin chaperone-like ability in the lens nucleus.

Inactivation and loss of antigenicity of esterase by sugars and a steroid.

Glycation, the non-enzymic reaction of sugars with proteins, has an important role in the complications of diabetes. It has been studied mostly in structural proteins but more recently has been shown to inactivate enzymes. Previous evidence from our laboratory indicated that glycation-induced inactivation and loss of antigenicity of catalase and superoxide dismutase are simultaneous. Esterase, which decreases activity in the lens in senile cataract and diabetes, was measured by a spectrophotometric assay using p-nitrophenyl acetate as the substrate. Here we investigated the inactivation of carboxylesterase (EC 3.1.1.1) by sugars of different glycating power and prednisolone-21-hemisuccinate while simultaneously monitoring the loss of antigenicity. Antigenicity was assessed by immunoprecipitation and by dot-blotting the glycated and non-glycated fractions of enzymes separated by affinity chromatography. Ribose and fructose inactivated more rapidly than glucose and glucose 6-phosphate. The esterase was progressively inactivated by prednisolone-21-hemisuccinate at a lower concentration. Activity and antigenicity were lost simultaneously. The glycated enzyme had entirely lost its antigenicity. These results further support the idea that inactivation of enzyme and loss of antigenicity are simultaneous.

The reaction of malondialdehyde with lens proteins and the protective effect of aspirin.

Malondialdehyde, a product of lipid peroxidation and a by-product of thromboxane synthesis increases in human cataract. Malondialdehyde bound to soluble lens proteins over 4 h of incubation. Pre-incubation of lens proteins with aspirin offered protection against reaction with MDA. Gel chromatography was used to monitor aggregation of the modified protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the reaction with malondialdehyde led to non-disulphide covalent cross-linking of gamma-crystallin, which was decreased by incubation with aspirin. Malondialdehyde has two carbonyl groups which could react with primary amino groups, forming Schiff-base conjugates and covalently cross-link proteins. The modification and cross-linking could initiate the cataractogenic process.

The reaction of methylglyoxal with human and bovine lens proteins.

Methylglyoxal is an endogenous metabolite that increases in diabetes and has been implicated in some of its long-term complications such as retinopathy, neuropathy and cataract. We investigated the reaction of methylglyoxal with isolated human and bovine lens crystallins (alpha, beta H, beta L and gamma). After 7 days incubation at 37 degrees C and pH 6.9, the reaction of methylglyoxal with lens proteins yielded stable adducts that exhibited fluorescent properties. SDS-polyacrylamide gel electrophoresis was used to monitor aggregation and crosslinking of the modified protein and autoradiography showed that [14C]methylglyoxal was incorporated into all the protein bands. Bovine gamma-crystallin was the most reactive towards methylglyoxal. Reaction of methylglyoxal with bovine gamma II-crystallin, which is found mainly in the lens nucleus, could alter the change surface network of the molecule, resulting in aggregation, increased light scattering and hence cataract. Modification of gamma II-crystallin by methylglyoxal produced an overall loss of positive charge and an increase in molecular weight and non-disulfide covalent crosslinking. Amino acid analysis of the modified gamma II-crystallin showed a loss of 47% of arginine residues.

alpha-crystallin protects glucose 6-phosphate dehydrogenase against inactivation by malondialdehyde.

The present work investigates the effect of malondialdehyde (MDA) binding on the enzymic activity and on some structural properties of glucose 6-phosphate dehydrogenase (G6PD). We studied whether alpha-crystallin could protect the enzyme against MDA damage, and if so, by what mechanism. We also studied whether alpha-crystallin could renature G6PD denatured by MDA. alpha-Crystallin was prepared from bovine lenses by gel chromatography. MDA was freshly prepared and incubated with G6PD with or without alpha-crystallin. The results show that MDA reacted with G6PD non-enzymically causing inactivation at concentrations lower than those used previously on structural proteins. The modified enzyme became fluorescent. alpha-Crystallin, acting as a molecular chaperone, specifically protected the enzyme against inactivation by MDA. The enzyme was not reactivated by alpha-crystallin, but it was stabilised and protected against further denaturation. Complex formation between alpha-crystallin and the modified enzyme was demonstrated by immunoprecipitation. G6PD was very susceptible to MDA and we have shown for the first time that alpha-crystallin is able to protect the enzyme against this damage.

Changes in proteins of the human lens in development and aging.

A number of proteins have been isolated from the human lens at different stages of development, from before birth to old age. These proteins have been characterized and compared with each other and with corresponding proteins from bovine lens. Many similarities were found between human and bovine crystallins, but alpha-crystallin isolated from old human lenses using DEAE-cellulose, unlike bovine alpha-crystallin similarly isolated, is not found as large soluble aggregates. The amide contents of various lens protein fractions were determined. No extensive changes were found during adult life, but there was evidence that significant deamidation of alpha-crystallin had occurred before birth and possibly during infancy. The results are related to the unique development and aging of the lens.

Binding of glucose, galactose and pyridoxal phosphate to lens crystallins.

Glycation of proteins plays an important role in diabetic complications. Both glucose and galactose were shown to bind progressively to lens crystallins with decreased binding in the presence of increasing concentrations of pyridoxal 5-phosphate (PLP). In longer term incubations (10 mM sugar for 21 days) glucose produced no significant yellowing of the protein, that is no detectable advanced glycation products, but pyridoxal phosphate (15 mM) caused an increased absorbance at 325 nm. This increase was greater in the presence of glucose. It appears that PLP becomes firmly attached to the protein and that this binding is enhanced in the presence of glucose or galactose. Changes produced by sodium borohydride indicate that the PLP is attached to protein amino groups as a Schiff base. Incubation of lens crystallins with PLP also led to increased fluorescence which was greater when sugar was present. However, borohydride experiments indicated that glucose and galactose may decrease the formation of non-reducible adducts of PLP. The decreased glycation in the presence of PLP supports the notion that it might be useful in prevention of diabetic complications, but the reaction of PLP itself with protein is less encouraging.

Glycation-induced inactivation of malate dehydrogenase protection by aspirin and a lens molecular chaperone, alpha-crystallin.

Non-enzymic glycosylation (glycation) of structural proteins has been widely studied as a possible mechanism in the long-term complications of diabetes. Here we show that glycation inactivates malate dehydrogenase. Aspirin affords some protection against the glycation, but alpha-crystallin, a lens protein which appears to act as a molecular chaperone in other systems, is much more effective. For example, 5 mM glucose completely inactivates malate dehydrogenase in four days, and 5 micrograms alpha-crystallin/ml provides complete protection against this inactivation. Fructose, a superior glycating agent, inactivates the enzyme in 24 hours but even so the same low concentration of alpha-crystallin is able to protect 80% of the activity. Other proteins provide no protection at the same concentration. The inactivation of malate dehydrogenase and other enzymes by glycation could play a role in diabetic complications, and molecular chaperones like alpha-crystallin could serve to protect them.

A spectroscopic study of glycated bovine alpha-crystallin: investigation of flexibility of the C-terminal extension, chaperone activity and evidence for diglycation.

The effect of glycating the C-terminal extensions of alpha-crystallin on their flexibility was investigated. In the course of the study the reaction sites were identified and double glycation of single lysine residues was found. Alpha-crystallin was incubated until approximately one mole of the sugar had reacted per subunit of the crystallin. The reaction sites were investigated by mass spectrometry and H NMR spectroscopy, and were found to be principally in the short and flexible C-terminal extensions. The chaperone ability of alpha-crystallin was unaffected by this limited glycation. There was little effect on the flexibility of the C-terminal extensions. This result supports the view that the flexibility of the C-terminal extensions of alpha-crystallin is important for chaperone activity. As alpha-crystallin consists of a mixture of unmodified and phosphorylated subunits, a detailed investigation was undertaken of the reaction of galactose with peptides comprising the C-terminal extensions of alphaA- and alphaB-crystallin. The alphaA peptide was incubated with galactose until 0.79 mole of sugar was bound per mole of peptide and the alphaB peptide reacted until 2.2 moles of galactose had been incorporated. The purified glycated peptides were examined by NMR and mass spectrometry to identify glycation site(s), and the effect of glycation on the conformation of the peptides. For both peptides, it was found that extensive glycation of the constituent lysine residues occurred. The addition of two galactose molecules to some lysine residues of the peptides was also noted. This diglycation was confirmed in control experiments with N-acetyl-lysine.

Alpha-crystallin as a molecular chaperone.

The role of alpha-crystallin as a molecular chaperone may explain how the lens stays transparent for so long. Alpha-crystallin prevents the aggregation of other lens crystallins and proteins that have become unfolded by "trapping" the protein in a high molecular weight complex. It also protects enzyme activities. The substrate protein may interact while in a molten globule state. Alpha-crystallin predominantly binds to proteins very early in the denaturation pathways. The amphiphilic nature of alpha-crystallin, a polar C-terminal-region and a hydrophobic N-terminal-region are all essential for chaperone function. The flexible C-terminal extension maintains solubility and can bind to opposing charged residues of unfolding proteins. Hydrophobic regions in the N-terminal region then hold the unfolded protein. Specific areas important for chaperone binding and function have been identified throughout the N-terminal-region, connecting peptide and C-terminal extension. After a substantial amount of chemical data and models, cryo-EM images of alpha-crystallin have confirmed a variable 3D surface with a hollow interior. Alpha-crystallin taken from the lens nucleus shows an age-dependent decrease in chaperone function. High molecular weight aggregates and alpha-crystallin found within the nucleus from clear and cataract lenses have reduced chaperone function. Post-translational modifications, known to occur during ageing, such as glycation, carbamylation, oxidation, phosphorylation and truncation cause a decrease in chaperone function. Alpha-crystallin is expressed outside the lens. AlphaB-crystallin can be induced by heat shock in many tissues where it is translocated from cytoplasm to nucleus. Increased expression of alphaB-crystallin has been seen in many pathological states. Conformational disorders, including cataract may have a common aetiology and potentially a common therapy.

Amino acid sequence of bovine gamma E (IVa) lens crystallin.

When electrospray ionization mass spectrometry (ESMS) was used to analyze purified bovine gamma E (gamma IVa)-crystallin, it yielded a relative molecular mass (M(r)) of 20.955 +/- 5. This mass is significantly different from that calculated from the published sequence (M(r) 20.894) (White HE et al., 1989, J Mol Biol 207:217-235). Further, ES-MS analysis of the protein after it had been reduced and carboxymethylated indicated the presence of five cysteine residues, whereas the published sequence contains six (Kilby GW et al., 1995, Eur Mass Spectrom 1:203-208). The entire protein sequence of gamma E crystallin has therefore been studied via a combination of ES-MS, ES-MS/MS, and Edman amino acid sequencing. The corrected sequence gives an M(r) of 20.955.3, which matches that obtained by ES-MS analysis of the purified native protein. The corrected sequence is also in agreement with a recent cDNA sequence obtained for a bovine gamma-crystallin by R. Hay (pers. comm.).