Proteomic analysis of the oxidation of cysteine residues in human age-related nuclear cataract lenses

Loss of protein thiols is a key feature associated with the onset of age-related nuclear cataract (ARNC), however, little is known about the specific sites of oxidation of the crystallins. We investigated cysteine residues in ARNC lenses and compared them with age-matched normal lenses. Proteomic analysis of tryptic digests revealed ten cysteine residues in older normal lenses that showed no significant oxidation compared to foetal counterparts (Cys 170 in betaA1/3-crystallin, Cys 32 in betaA4-crystallin, Cys 79 in betaB1-crystallin, Cys 22, Cys 78/79, C153 in gammaC-crystallin and Cys 22, Cys 24 and Cys 26 in gammaS-crystallin). Although these thiols were not oxidised in normal lenses past the 6th decade, they were present largely as disulphides in the ARNC lenses. By contrast, two cysteine residues, Cys 41 in gammaC-crystallin and Cys 18 in gammaD-crystallin, were not oxidised, even in advanced ARNC lenses. These cysteines are buried deep within the protein and any unfolding associated with cataract must be insufficient to expose them to the oxidative environment present in the centre of advanced ARNC lenses. The vast majority of the loss of protein thiol observed in such lenses is due to disulphide bond formation.     

One-electron reduction potentials of dietary carotenoid radical cations in aqueous micellar environments

The one-electron reduction potentials of the radical cations of five dietary carotenoids (β-carotene, canthaxanthin, zeaxanthin, astaxanthin and lycopene) in aqueous micellar environments have been obtained from a pulse radiolysis study of electron transfer between the carotenoids and tryptophan radical cations as a function of pH, and lie in the range of 980–1060 mV. These values are consistent with our observation that the carotenoid radical cations oxidise tyrosine and cysteine. The decays of the carotenoid radical cations in the absence of added reactants suggest a distribution of exponential lifetimes. The radicals persist for up to about 1 s, depending on the medium.     

Identification of glutathionyl-3-hydroxykynurenine glucoside as a novel fluorophore associated with aging of the human lens.

A novel fluorophore was isolated from human lenses using high performance liquid chromatography (HPLC). The new fluorophore was well separated from 3-hydroxykynurenine glucoside (3-OHKG) and its deaminated isoform, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside, which are known UV filter compounds. The new compound exhibited UV absorbance maxima at 260 and 365 nm, was fluorescent (Ex(360 nm)/Em(500 nm)), and increased in concentration with age. Further analysis of the purified compound by microbore HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mass of 676 Da. This mass corresponds to that of an adduct of GSH with a deaminated form of 3-OHKG. This adduct was synthesized using 3-OHKG and GSH as starting materials. The synthetic glutathionyl-3-hydroxykynurenine glucoside (GSH-3-OHKG) adduct had the same HPLC elution time, thin-layer chromatography R(F) value, UV absorbance maxima, fluorescence characteristics, and mass spectrum as the lens-derived fluorophore. Furthermore, the (1)H and (13)C NMR spectra of the synthetic adduct were entirely consistent with the proposed structure of GSH-3-OHKG. These data indicate that GSH-3-OHKG is present as a novel fluorophore in aged human lenses. The GSH-3-OHKG adduct was found to be less reactive with beta-glucosidase compared with 3-OHKG, and this could be due to a folded conformation of the adduct that was suggested by molecular modeling.     

Calcium-induced cleavage and breakdown of spectrin in the rat lens

Incubation of intact rat lenses under conditions that stimulated a net influx of calcium resulted in a pronounced loss of transparency and a major decrease in the levels of spectrin. The progressive loss of this cytoskeletal component coincided with the appearance of polypeptides of approximately 150 kDa which showed immunoreactivity with an antibody raised to spectrin. These bands disappeared on further incubation. It is, therefore, suggested that a calcium-activated protease is present in the lens which is capable of degrading spectrin by the initial removal of approximately 90 kDa fragments. This process calcium-induced proteolysis may be the basis for the cytoskeletal reorganisation observed during the differentiation of lens fibre cells and may be involved in cataract development.     

The bacterial N‐end rule pathway: expect the unexpected

The N‐end rule pathway is a highly conserved process that operates in many different organisms. It relates the metabolic stability of a protein to its N‐terminal amino acid. Consequently, amino acids are described as either ‘stabilizing’ or ‘destabilizing’. Destabilizing residues are organized into three hierarchical levels: primary, secondary, and in eukaryotes – tertiary. Secondary and tertiary destabilizing residues act as signals for the post‐translational modification of the target protein, ultimately resulting in the attachment of a primary destabilizing residue to the N‐terminus of the protein. Regardless of their origin, proteins containing N‐terminal primary destabilizing residues are recognized by a key component of the pathway. In prokaryotes, the recognition component is a specialized adaptor protein, known as ClpS, which delivers target proteins directly to the ClpAP protease for degradation. In contrast, eukaryotes use a family of E3 ligases, known as UBRs, to recognize and ubiquitylate their substrates resulting in their turnover by the 26S proteasome. While the physiological role of the N‐end rule pathway is largely understood in eukaryotes, progress on the bacterial pathway has been slow. However, new interest in this area of research has invigorated several recent advances, unlocking some of the secrets of this unique proteolytic pathway in prokaryotes.     

Flux Analysis of Glucose Metabolism in Rhizopus oryzae for the Purpose of Increasing Lactate Yields

A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found.     

Disturbances prevent stem size-density distributions in natural forests from following scaling relationships

Enquist and Niklas propose that trees in natural forests have invariant size-density distributions (SDDs) that scale as a −2 power of stem diameter, although early studies described such distributions using negative exponential functions. Using New Zealand and ‘global’ data sets, we demonstrate that neither type of function accurately describes the SDD over the entire diameter range. Instead, scaling functions provide the best fit to smaller stems, while negative exponential functions provide the best fit to larger stems. We argue that these patterns are consistent with competition shaping the small-stem phase and exogenous disturbance shaping the large-stem phase. Mortality rates, estimated from repeat measurements on 1546 New Zealand plots, fell precipitously with stem size until 18 cm but remained constant after that, consistent with our arguments. Even in the small-stem phase, where SDDs were best described by scaling functions, the scaling exponents were not invariantly −2, but differed significantly from this value in both the ‘global’ and New Zealand data sets, and varied through time in the New Zealand data set.