Enhanced C-terminal truncation of alphaA- and alphaB-crystallins in diabetic lenses.

PURPOSE: To investigate the influence of diabetes on the cleavage of C-terminal amino acid residues of alphaA- and alphaB-crystallins in human and rat lenses. METHODS: The human lenses were diabetic or age-matched control lenses from donors 57, 59, 69, and 72 years of age. Lenses were also obtained from streptozotocin-induced diabetic rats. Individual lens crystallins in water-soluble fractions were separated by gel-permeation chromatography. The high (alphaH)- and low (alphaL)-molecular-weight fractions were analyzed by electrospray ionization mass spectrometry. RESULTS: A typical mass spectrum of alphaA-crystallin from human lenses showed intact unmodified alphaA-crystallin, truncated alphaA(1-172), and monophosphorylated alphaA-crystallin. Diabetic lenses showed nearly twofold higher levels of alphaA(1-172) than did the control lenses. Also, the alphaH fraction consistently showed significantly higher levels of alphaA(1-172) than the alphaL fraction. Human alphaB-crystallin showed no evidence of C-terminal truncation. Rat alphaA-crystallin had five C-terminal-truncated components, most of which showed substantial increases in diabetes. Truncated alphaA(1-162) appeared only in the diabetic rat lenses, suggesting specific activation of m-calpain in diabetes. alphaB-crystallin had only one C-terminal-truncated component, alphaB(1-170), which also showed increased levels in diabetes. CONCLUSIONS: These data suggest that diabetic stress causes either enzymatic or nonenzymatic cleavage of peptide bonds between specific C-terminal amino acid residues. Such truncated alpha-crystallins appear to contribute to an increased level of the alphaH fraction generally present in diabetic lenses. Loss of alphaA-crystallin chaperone activity seems to be related to truncation of the C-terminal amino acid residues.

The 5HT1a receptor agonist 8-Oh DPAT induces protection from lipofuscin accumulation and oxidative stress in the retinal pigment epithelium.

Age-related macular degeneration (AMD), a major cause of blindness in the elderly, is associated with oxidative stress, lipofuscin accumulation and retinal degeneration. The aim of this study was to determine if a 5-HT(1A) receptor agonist can reduce lipofuscin accumulation, reduce oxidative damage and prevent retinal cell loss both in vitro and in vivo. Autophagy-derived and photoreceptor outer segment (POS)-derived lipofuscin formation was assessed using FACS analysis and confocal microscopy in cultured retinal pigment epithelial (RPE) cells in the presence or absence of the 5-HT(1A) receptor agonist, 8-OH DPAT. 8-OH DPAT treatment resulted in a dose-dependent reduction in both autophagy- and POS-derived lipofuscin compared to control. Reduction in autophagy-induced lipofuscin was sustained for 4 weeks following removal of the drug. The ability of 8-OH DPAT to reduce oxidative damage following exposure to 200 µM H(2)O(2) was assessed. 8-OH DPAT reduced superoxide generation and increased mitochondrial superoxide dismutase (MnSOD) levels and the ratio of reduced glutathione to the oxidized form of glutathione in H(2)O(2)-treated cells compared to controls and protected against H(2)O(2)-initiated lipid peroxidation, nitrotyrosine levels and mitochondrial damage. SOD2 knockdown mice, which have an AMD-like phenotype, received daily subcutaneous injections of either saline, 0.5 or 5.0 mg/kg 8-OH DPAT and were evaluated at monthly intervals. Systemic administration of 8-OH DPAT improved the electroretinogram response in SOD2 knockdown eyes of mice compared to knockdown eyes receiving vehicle control. There was a significant increase in the ONL thickness in mice treated with 8-OH DPAT at 4 months past the time of MnSOD knockdown compared to untreated controls together with a 60% reduction in RPE lipofuscin. The data indicate that 5-HT(1A) agonists can reduce lipofuscin accumulation and protect the retina from oxidative damage and mitochondrial dysfunction. 5-HT(1A) receptor agonists may have potential as therapeutic agents in the treatment of retinal degenerative disease.

Dietary rice protein isolate attenuates atherosclerosis in apoE-deficient mice by upregulating antioxidant enzymes.

Rice-based diets may have been reported to protect against the development of atherosclerosis; however, the underlying mechanism(s) for this protection remains unknown. In this report, the mechanism(s) contributing to the atheroprotective effects of rice-based diet was addressed using the apolipoprotein E knockout (apoE-/-) mice fed rice protein isolate (RPI) or casein (CAS). Reduced atherosclerotic lesions were observed in aortic sinus and enface analyses of the descending aorta in RPI-fed apoE-/- mice compared with CAS-fed mice. Plasma total- and HDL-cholesterol levels were not different amongst the two groups, suggesting alternative mechanism(s) could have contributed to the atheroprotective effect of rice-based diets. Plasma oxLDL and anti-oxLDL IgG levels were significantly decreased in RPI-fed compared to CAS-fed animals. Plasma and aortic tissue GSH levels and GSH:GSSG ratio were higher in RPI-fed mice compared to CAS-fed group. Interestingly, RPI feeding increased mRNA and protein expression of superoxide dismutase, and mRNA expression of catalase, glutathione peroxidase and glutathione reductase, key antioxidant enzymes implicated inhibiting oxidative stress leading to atherosclerosis. In conclusion, these findings suggest that the reduction in atherosclerotic lesions observed in mice fed the rice-based diet is mediated in part by inhibiting oxidative stress and subsequent oxLDL generation that could result in reduced foam cell formation, an early event during atherogenesis.

Dietary homocysteine promotes atherosclerosis in apoE-deficient mice by inducing scavenger receptors expression.

Elevated plasma homocysteine (Hcy) levels have been recognized as an independent risk factor for atherosclerosis leading to cardiovascular diseases. However, the mechanisms contributing to atherosclerosis have not been delineated. Since, scavenger receptors mediated uptake of oxidized-LDL (oxLDL) by macrophages resulting in foam cell formation is an early event in atherosclerosis, we hypothesized that atherogenic effects of Hcy may be mediated via regulating expression of scavenger receptor(s). We have tested this hypothesis using apoE-/- female mice fed normal rodent chow (NC) diet or NC supplemented with Hcy in drinking water (9 g/L). Hcy-fed mice showed increased fatty streak lesions in aortic sinus/root compared to NC group without alterations in plasma lipid profiles. Similar findings were observed in the enface analysis of the descending aorta. To determine the molecular mechanisms underlying Hcy-mediated progression of fatty streak lesions, expression of scavenger receptors such as CD36 and lectin-like oxidized LDL binding protein-1 (LOX-1) in the aortic lesions were analyzed. Interestingly, Hcy-fed mice had increased immuno-positive staining for CD36 and LOX-1 in the atherosclerotic lesions compared to NC-fed mice. In vitro analyses showed neither Hcy nor HcyLDL directly affect the expression of CD36 and LOX-1 on mouse macrophages. However, Hcy supplementation in apoE-/- mice resulted in elevated oxLDL levels in plasma. Since oxLDL has been shown to upregulate the expression of CD36 and LOX-1, these findings suggest that Hcy may exert its atherogenic effect in part by elevating the levels of oxLDL. Interestingly, interaction of monocytes with Hcy-activated endothelial cells resulted in upregulation of CD36 expression on monocytes, suggesting a possible mechanism by which Hcy may upregulate CD36 expression at the lesion site. Further, these findings suggest a novel mechanism by which Hcy may promote atherogenesis.

Diet regulates the development of gut-associated lymphoid tissue in neonatal piglets.

BACKGROUND: Controversy exists concerning diet-induced changes to gut epithelia and immunocytes that occur during weaning. Furthermore, studies on dietary effects on the development of the neonatal immune system, especially gut-associated lymphoid tissue (GALT), are lacking. OBJECTIVE: The purpose of this study was to investigate growth and development, intestinal morphology, and GALT immune maturation in sow-reared littermates in comparison with early-weaned piglets fed a casein-based liquid diet. METHOD: Piglets were breast fed by the sow or were weaned at 48 h to a casein-based diet (formula) that provided the amount of nutrient requirements recommended by the National Research Council. RESULTS: Gross physical appearance and visual inspection of the gastrointestinal tract and other organs at necropsy revealed normal organogenesis in both cohorts. On postnatal day 21, body weight, liver and kidney weight relative to body weight, small intestine length, and weight-to-length ratio were greater in formula-fed piglets as compared with sow-reared piglets (p<0.05). The CD21+ B lymphocyte component of GALT and spleen was reduced in the formula-fed piglets. This was associated with lower circulating IgG and IgM levels in the formula-fed as compared with the breast-fed neonatal piglets (p<0.001). CONCLUSIONS: Feeding a casein-based formula to newborn piglets may compromise the development of GALT and systemic immune system. Further, the neonatal piglet model may be used to identify the effects of dietary factors on the development of the neonatal immune system.

Musashi regulates the temporal order of mRNA translation during Xenopus oocyte maturation.

A strict temporal order of maternal mRNA translation is essential for meiotic cell cycle progression in oocytes of the frog Xenopus laevis. The molecular mechanisms controlling the ordered pattern of mRNA translational activation have not been elucidated. We report a novel role for the neural stem cell regulatory protein, Musashi, in controlling the translational activation of the mRNA encoding the Mos proto-oncogene during meiotic cell cycle progression. We demonstrate that Musashi interacts specifically with the polyadenylation response element in the 3' untranslated region of the Mos mRNA and that this interaction is necessary for early Mos mRNA translational activation. A dominant inhibitory form of Musashi blocks maternal mRNA cytoplasmic polyadenylation and meiotic cell cycle progression. Our data suggest that Musashi is a target of the initiating progesterone signaling pathway and reveal that late cytoplasmic polyadenylation element-directed mRNA translation requires early, Musashi-dependent mRNA translation. These findings indicate that Musashi function is necessary to establish the temporal order of maternal mRNA translation during Xenopus meiotic cell cycle progression.

Influence of the C-terminal residues on oligomerization of alpha A-crystallin.

Earlier studies have shown that the chaperone activity of alpha-crystallin is significantly affected in diabetic rat and human lenses. Subsequently, mass spectrometric analysis showed diabetic lenses having high levels of the alphaA-crystallins in which different numbers of C-terminal residues were deleted. The present study was aimed to show whether cleavage of these residues influences protein structure, oligomerization, and chaperone function. For generation of various mutants, a stop codon was introduced at the positions of interest, proteins were expressed in BL21(DE3)pLys S E. coli, and the truncated alphaA-crystallins were purified by size-exclusion chromatography. The molecular masses, as determined by molecular sieve HPLC, of mutants with deletions of 1, 5, and 10 C-terminal residues (group-1) were 519-602 kDa, and those of mutants with deletions of 11, 16, and 22 C-terminal residues (group-2) were 148-152 kDa, as compared to 607 kDa for alphaA-wild type. On the basis of circular dichroism measurements, the alpha helix content was 2-fold higher and the tertiary structure was significantly altered in the group-2 mutants. Chaperoning abilities, as determined by the ADH assay and the betaL-crystallin heat denaturation assay, of the group-1 mutants, with the exception of alphaA(1-163), were slightly improved or unchanged, that of alphaA(1-163) was moderately affected, and those of the group-2 mutants were severely affected. Most strikingly, cleavage of 11 C-terminal residues including Arg-163 showed a substantial decrease in oligomeric size and chaperone function and significant changes in protein structure whereas cleavage of 10 residues had either a small effect or no effect at all. This points to an important role for the C-terminal extension, Arg-163 in particular, and no significant role for the C-terminal flexible tail in the oligomer assembly of alphaA-crystallin.

alpha-Crystallin chaperone function in diabetic rat and human lenses.

This study focussed on the effect of diabetes on the chaperone function of alpha-crystallin. The authors relied on diabetic rats with a wide range of plasma glucose levels and non-diabetic control rats to establish a possible relationship between severity of diabetes and alpha-crystallin chaperone activity. In addition, 52-56 and 63-69 year-old diabetic and non-diabetic human lenses were used to show whether diabetes affects alpha-crystallin chaperone activity in human lenses. Correlation between plasma glucose levels and loss of chaperone activity of the alphaL-crystallin fraction in diabetic rats indicated good correlation. The glycemic threshold, reported before for cataract development in diabetic rats, seems to be valid for the chaperone activity loss as well. Analysis of the human lens alphaL-crystallin showed lower chaperone activity in all the diabetic lenses than in the age-matched control lenses. In the 63-69 age group, the loss in chaperone activity due to diabetes was significantly larger than in the 52-56 age group suggesting a dominant effect of duration of diabetes.

Cleavage of in vitro and in vivo formed lens protein cross-links by a novel cross-link breaker.

The purpose of this study was to investigate the effect of N-phenacyl-4,5-dimethylthiazolium bromide (DMPTB), an advanced glycation end product (AGE) cross-link breaker, on lens protein cross-links formed in vitro and in vivo. DMPTB was synthesized and its structure confirmed by its NMR spectrum. To show whether DMPTB can inhibit AGE cross-linking, recombinant human alphaA-crystallin was glycated with glucose-6-phosphate (G6P) in the presence and absence of DMPTB. Reversal of the already formed cross-links was studied by treating pre-glycated alphaA-crystallin with DMPTB. The ability of DMPTB to cleave in vivo formed cross-links was ascertained by treating water-insoluble protein fractions from diabetic human lenses with this compound. Glycation of alphaA-crystallin with G6P showed several high molecular weight (HMW) protein bands on the SDS-PAGE gel; DMPTB inhibited the formation of these HMW proteins. Molecular sieve HPLC confirmed the inhibition of formation of larger aggregates not separated by SDS-PAGE. Treatment of pre-glycated alphaA-crystallin with DMPTB gave evidence for the degradation of the already formed cross-linked HMW aggregates. Both molecular sieve HPLC and reverse-phase HPLC of the water-insoluble protein fractions from two diabetic human lenses showed that DMPTB could degrade a major portion of the cross-linked HMW aggregates to lower molecular weight proteins. This suggests that the cross-linked proteins in human lenses are formed predominantly by the advanced glycation process and cross-link breakers like DMPTB may have application for the intervention of protein cross-linking in the eye lens.

A positive charge preservation at position 116 of alpha A-crystallin is critical for its structural and functional integrity.

An autosomal dominant congenital cataract associated with a missense mutation, Arg-116 to Cys (R116C), in the coding sequence of human alphaA-crystallin has been reported. Subsequent study of this mutant, generated by site-directed mutagenesis, showed significant changes in secondary and tertiary structures, partial loss of chaperone activity, and substantially increased oligomeric size. The study presented here aims to show whether these changes are due to the loss of a positive charge at this position or due to the presence of an extra Cys. To show this, Arg-116 in alphaA-crystallin was mutated to Lys (R116K), Cys (R116C), Gly (R116G), and Asp (R116D) and expressed in Escherichia coli cells. The wild-type (alphaA-wt) and mutant proteins were purified by size exclusion chromatography and characterized by measurements of circular dichroism, intrinsic tryptophan fluorescence, and TNS fluorescence and by determination of molecular masses and chaperone function which was assessed as the ability to suppress target protein aggregation or enhance target protein refolding. Mutation of Arg-116 to a Cys or Gly showed very similar changes in structure, oligomerization, and chaperone function which suggest that the presence of this Cys per se is not the cause of the changes. The R116K mutant, on the other hand, had nearly the same structure, oligomeric size, and chaperone function as alphaA-wt, whereas the mutant with an acidic amino acid in this position, R116D, showed drastic changes in protein structure. Thus, a positive charge must be preserved at this position for the structural and functional integrity of alphaA-crystallin.

Cloning and expression of the trehalose-phosphate phosphatase of Mycobacterium tuberculosis: comparison to the enzyme from Mycobacterium smegmatis.

Two open reading frames in the Mycobacterium tuberculosis genome, Rv3372 and Rv2006, have about 25% sequence identity at the amino acid level to the trehalose-phosphate phosphatase (TPP) purified from Mycobacterium smegmatis. However, the protein produced from the cloned Rv3372 gene has a molecular weight of about 45kDa whereas the trehalose-P phosphatase purified from M. smegmatis has a molecular weight of about 27kDa. We expressed the Rv3372 protein in Escherichia coli and show here that it is a trehalose-P phosphatase with very similar properties to the M. smegmatis TPP, i.e., complete specificity for trehalose-phosphate as the substrate, an almost absolute requirement for Mg(2+), and a pH optimum of 7-7.5. On the other hand, in contrast to the M. smegmatis enzyme, the Rv3372 protein was much less stable to heat and much less sensitive to inhibition by diumycin and moenomycin. In fact, both of these antibiotics stimulate enzyme activity at low concentrations and only inhibit the activity at higher antibiotic concentrations. Antibody prepared against the 27kDa TPP does not cross react with the 45kDa TPP nor does antibody against the 45kDa TPP cross react with the 27kDa TPP. Nevertheless, studies of secondary structure by circular dichroism indicate that the two enzymes are quite similar in structure. The product of the other gene, Rv2006, is a 159kDa protein with no detectable phosphatase activity. Thus, its function is currently unknown.

Purification, cloning, expression, and properties of mycobacterial trehalose-phosphate phosphatase.

The trehalose-phosphate phosphatase (TPP) was purified from the cytosol of Mycobacterium smegmatis to near homogeneity using a variety of conventional steps to achieve a purification of about 1600-fold with a yield of active enzyme of about 1%. Based on gel filtration, the active enzyme had a molecular weight of about 27,000, and the most purified fraction also gave a major band on SDS-PAGE corresponding to a molecular weight of about 27,000. A number of peptides from the 27-kDa protein were sequenced and these sequences showed considerable homology to the trehalose-P phosphatase (otsB) of Escherichia coli. Based on these peptides, the M. smegmatis gene for TPP was cloned and expressed in E. coli. The recombinant protein was synthesized with a (His)(6) tag at the amino terminus. Most of the TPP activity in the crude E. coli sonicate was initially found in the membrane fraction, but it became solubilized in the presence of 0.2% Sarkosyl. The solubilized protein was purified to apparent homogeneity on a metal ion column and this fraction had high phosphatase activity that was completely specific for trehalose-P. The purified enzyme, either isolated from M. smegmatis, or expressed in E. coli, rapidly dephosphorylated trehalose-6-P, but had essentially no activity on any other sugar phosphates, or on p-nitrophenyl phosphate. The K(m) for trehalose-6-P was about 1.6 mm, and the pH optimum was about 7.5. The native enzyme showed an almost absolute requirement for Mg(2+) and was not very active with Mn(2+), whereas both of these cations were equally effective with the recombinant TPP. The enzyme activity was inhibited by the antibiotics, diumycin and moenomycin, but not by a number of other antibiotics or trehalose analogs. TPP activity was strongly inhibited by the detergents, Sarkosyl and deoxycholate, even at 0.025%, but it was not inhibited by Nonidet P-40, Triton X-100, or octyl glucoside, even at concentrations up to 0.3%. The purified enzyme was stable to heating at 60 degrees C for up to 6 min, but was slowly inactivated at 70 degrees C. Circular dichroism studies on recombinant TPP indicate that the secondary structure of this protein has considerable beta-pleated sheet and is very compact. TPP may play a key role in the biosynthesis of trehalose compounds, such as trehalose mycolates, and therefore may represent an excellent target site for chemotherapy against tuberculosis and other mycobacterial diseases.