Oxidation of proteins by reactive oxygen species is associated with aging, oxidative stress, and many diseases. Although free and protein-bound methionine residues are particularly sensitive to oxidation to methionine sulfoxide derivatives, these oxidations are readily repaired by the action of methionine sulfoxide reductase (MsrA). To gain a better understanding of the biological roles of MsrA in metabolism, we have created a strain of mouse that lacks the MsrA gene. Compared with the wild type, this mutant: (i) exhibits enhanced sensitivity to oxidative stress (exposure to 100% oxygen); (ii) has a shorter lifespan under both normal and hyperoxic conditions; (iii) develops an atypical (tip-toe) walking pattern after 6 months of age; (iv) accumulates higher tissue levels of oxidized protein (carbonyl derivatives) under oxidative stress; and (v) is less able to up-regulate expression of thioredoxin reductase under oxidative stress. It thus seems that MsrA may play an important role in aging and neurological disorders. P rotein-bound methionine residues are among the most susceptible to oxidation by reactive oxygen species (ROS), resulting in formation of methionine sulfoxide [Met(O)] residues. However, this modification can be repaired by methionine sulfoxide reductase (MsrA), which catalyzes the thioredoxindependent reduction of free and protein-bound Met(O) to methionine, both in vitro (1) and in vivo (2). Bacteria and yeast cells lacking the msrA gene show increased sensitivity to oxidative stress and lower survival rates (3, 4), with yeast showing accumulation of high levels of both free and protein-bound Met(O) (2, 4). In addition, overexpression of the MsrA enzyme in human T cells prolongs their life under conditions of oxidative stress (4). Because methionine residues are particularly susceptible to oxidation by ROS, MsrA could have at least three important functions in cellular metabolism: (i) as an antioxidant enzyme that scavenges ROS by facilitating the cyclic interconversion of methionine͞protein-methionine residues between oxidized and reduced forms (2); (ii) as a repair enzyme by keeping critical methionine residues in their reduced form; and (iii) as a regulator of critical enzyme activity through cyclic interconversion of specific methionine residues between oxidized and reduced forms (5, 6). Escherichia coli and Saccharomyces cerevisiae both contain at least two Msrs. One (MsrA) is able to reduce both free and protein-bound Met(O), and the other can reduce only free Met(O). The MsrA protein is highly expressed in liver, kidney, pigment epithelial cells of the retina, macrophages, cerebellum, and brain neurons (7). These tissues͞ cells are sensitive to oxidative stress damages. Therefore, abolishing the MsrA enzyme could lead to loss of antioxidant defense, resulting in enhanced oxidative damage and a decreased lifespan. To investigate the possible role of MsrA as an antioxidant in mammals and its possible influence on lifespan, we created a strain of mouse lacking the MsrA protein. Materials and ...

The structure of the infectious prion protein (PrPSc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrPSc replicates by converting the non-infectious, cellular prion protein (PrPC) into the misfolded, infectious conformer through an unknown mechanism. PrPSc and its N-terminally truncated variant, PrP 27–30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27–30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 Å. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 Å3, predicted the height of each PrP 27–30 molecule as ~17.7 Å. Together, the data indicate a four-rung β-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding.

Metal-catalyzed oxidation results in loss of function and structural alteration of proteins. The oxidative process affects a variety of side amino acid groups, some of which are converted to carbonyl compounds. Spectrophotometric measurement of these moieties, after their reaction with 2,4-dinitrophenylhydrazine, is a simple, accurate technique that has been widely used to reveal increased levels of protein carbonyls in aging and disease. We have initiated studies aimed at elucidating the chemical nature of protein carbonyls. Methods based on gas chromatography͞mass spectrometry with isotopic dilution were developed for the quantitation of glutamic and aminoadipic semialdehydes after their reduction to hydroxyaminovaleric and hydroxyaminocaproic acids. Analysis of model proteins oxidized in vitro by Cu 2؉ ͞ascorbate revealed that these two compounds constitute the majority of protein carbonyls generated. Glutamic and aminoadipic semialdehydes were also detected in rat liver proteins, where they constitute Ϸ60% of the total protein carbonyl value. Aminoadipic semialdehyde was also measured in protein extracts from HeLa cells, and its level increased as a consequence of oxidative stress to cell cultures. These results indicate that glutamic and aminoadipic semialdehydes are the main carbonyl products of metal-catalyzed oxidation of proteins, and that this reaction is a major route leading to the generation of protein carbonyls in biological samples.

Disrupted-in-schizophrenia 1 (DISC1) and other genes have been identified recently as potential molecular players in chronic psychiatric diseases such as affective disorders and schizophrenia. A molecular mechanism of how these genes may be linked to the majority of sporadic cases of these diseases remains unclear. The chronic nature and irreversibility of clinical symptoms in a subgroup of these diseases prompted us to investigate whether proteins corresponding to candidate genes displayed subtle features of protein aggregation. Here, we show that in postmortem brain samples of a distinct group of patients with phenotypes of affective disorders or schizophrenia, but not healthy controls, significant fractions of DISC1 could be identified as cold Sarkosyl-insoluble protein aggregates. A loss-offunction phenotype could be demonstrated for insoluble DISC1 through abolished binding to a key DISC1 ligand, nuclear distribution element 1 (NDEL1): in human neuroblastoma cells, DISC1 formed expression-dependent, detergent-resistant aggregates that failed to interact with endogenous NDEL1. Recombinant (r) NDEL1 expressed in Escherichia coli selectively bound an octamer of an rDISC1 fragment but not dimers or high molecular weight multimers, suggesting an oligomerization optimum for molecular interactions of DISC1 with NDEL1. For DISC1-related sporadic psychiatric disease, we propose a mechanism whereby impaired cellular control over self-association of DISC1 leads to excessive multimerization and subsequent formation of detergent-resistant aggregates, culminating in loss of ligand binding, here exemplified by NDEL1. We conclude that the absence of oligomer-dependent ligand interactions of DISC1 can be associated with sporadic mental disease of mixed phenotypes.

Recent studies have shown that a sizable fraction of PrPSc present in prion-infected tissues is, contrary to previous conceptions, sensitive to digestion by proteinase K (PK). This finding has important implications in the context of diagnosis of prion disease, as PK has been extensively used in attempts to distinguish between PrPSc and PrPC. Even more importantly, PK-sensitive PrPSc (sPrPSc) might be essential to understand the process of conversion and aggregation of PrPC leading to infectivity. We have isolated a fraction of sPrPSc. This material was obtained by differential centrifugation at an intermediate speed of Syrian hamster PrPSc obtained through a conventional procedure based on ultracentrifugation in the presence of detergents. PK-sensitive PrPSc is completely degraded under standard conditions (50 mug/mL of proteinase K at 37 degrees C for 1 h) and can also be digested with trypsin. Centrifugation in a sucrose gradient showed sPrPSc to correspond to the lower molecular weight fractions of the continuous range of oligomers that constitute PrPSc. PK-sensitive PrPSc has the ability to convert PrPC into protease-resistant PrPSc, as assessed by the protein misfolding cyclic amplification assay (PMCA). Limited proteolysis of sPrPSc using trypsin allows for identification of regions that are particularly susceptible to digestion, i.e., are partially exposed and flexible; we have identified as such the regions around residues K110, R136, R151, K220, and R229. PK-sensitive PrPSc isolates should prove useful for structural studies to help understand fundamental issues of the molecular biology of PrPSc and in the quest to design tests to detect preclinical prion disease.

Metal-catalyzed oxidation may result in structural damage to proteins and has been implicated in aging and disease, including neurological disorders such as Alzheimer's disease and amyotrophic lateral sclerosis. The selective modification of specific amino acid residues with high metal ion affinity leads to subtle structural changes that are not easy to detect but may have dramatic consequences on physical and functional properties of the oxidized protein molecules. PrP contains a histidine-rich octarepeat domain that binds copper. Because copper-binding histidine residues are particularly prone to metal-catalyzed oxidation, we investigated the effect of this reaction on the recombinant prion protein SHaPrP(29 -231). Using Cu 2؉ ͞ascorbate, we oxidized SHaPrP(29 -231) in vitro. Oxidation was demonstrated by liquid chromatography͞mass spectrometry, which showed the appearance of protein species of higher mass, including increases in multiples of 16, characteristic of oxygen incorporation. Digestion studies using Lys C indicate that the 29 -101 region, which includes the histidinecontaining octarepeats, is particularly affected by oxidation. Oxidation was time-and copper concentration-dependent and was evident with copper concentrations as low as 1 M. Concomitant with oxidation, SHaPrP(29 -231) suffered aggregation and precipitation, which was nearly complete after 15 min, when the prion protein was incubated at 37°C with a 6-fold molar excess of Cu 2؉ . These findings indicate that PrP, a copper-binding protein, may be particularly susceptible to metal-catalyzed oxidation and that oxidation triggers an extensive structural transition leading to aggregation.

Prions are unusual protein assemblies that propagate their conformationally-encoded information in absence of nucleic acids. The first prion identified, the scrapie isoform (PrP Sc ) of the cellular prion protein (PrP C ), caused epidemic and epizootic episodes [ 1 ]. Most aggregates of other misfolding-prone proteins are amyloids, often arranged in a Parallel-In-Register-β-Sheet (PIRIBS) [ 2 ] or β-solenoid conformations [ 3 ]. Similar folding models have also been proposed for PrP Sc , although none of these have been confirmed experimentally. Recent cryo-electron microscopy (cryo-EM) and X-ray fiber-diffraction studies provided evidence that PrP Sc is structured as a 4-rung β-solenoid (4RβS) [ 4 , 5 ]. Here, we combined different experimental data and computational techniques to build the first physically-plausible, atomic resolution model of mouse PrP Sc , based on the 4RβS architecture. The stability of this new PrP Sc model, as assessed by Molecular Dynamics (MD) simulations, was found to be comparable to that of the prion forming domain of Het-s, a naturally-occurring β-solenoid. Importantly, the 4RβS arrangement allowed the first simulation of the sequence of events underlying PrP C conversion into PrP Sc . This study provides the most updated, experimentally-driven and physically-coherent model of PrP Sc , together with an unprecedented reconstruction of the mechanism underlying the self-catalytic propagation of prions.

Many organisms have been shown to possess a methionine sulfoxide reductase (MsrA), exhibiting high specificity for reduction the S form of free and proteinbound methionine sulfoxide to methionine. Recently, a different form of the reductase (referred to as MsrB) has been detected in several organisms. We show here that MsrB is a selenoprotein that exhibits high specificity for reduction of the R forms of free and proteinbound methionine sulfoxide. The enzyme was partially purified from mouse liver and a derivative of the mouse MsrB gene, in which the codon specifying selenocystein incorporation was replaced by the cystein codon, was prepared, cloned, and overexpressed in Escherichia coli. The properties of the modified MsrB protein were compared directly with those of MsrA. Also, we have shown that in Staphylococcus aureus there are two MsrA and one nonselenoprotein MsrB, which demonstrates the same substrate stereospecificity as the mouse MsrB.Key Words: methionine sulfoxide; oxidative stress; methionine sulfoxide reductase; free radicals; methionine oxidation; selenoprotein; stereospecificity.Methionine residues of proteins are readily oxidized to methionine sulfoxide (MetO) by most reactive oxygen species However, in contrast to most other oxidative posttranslational modifications, the oxidation of methionine residues is repaired by the action of methionine sulfoxide reductase (MsrA) which catalyzes reduction of MetO to methionine, both in vitro and in vivo (1, 2). In addition to limiting the steady-state level of oxidized methionine, the cyclic oxidation/reduction of protein methionine residues constitutes a mechanism for the scavenging of ROS, and thereby provides increased resistance to oxidative cellular damage and to enhanced survival under conditions of oxidative stress (3-5). It was shown previously that a mouse strain lacking MsrA is more prone to oxidative stress damage, has shorter life span and exhibits a typical "tip toe" walking behavior starting at six months of age (5). Other studies have shown that the level of MsrA declines with age (6), and that over expression of the enzyme in human T cells increases their survival rate under conditions of oxidative stress. Furthermore, in addition to its repair and antioxidant functions, MsrA may play a regulatory role in regulation of various biological functions (7,8).In view of the fact that the ROS-mediated oxidation of protein-bound methionine residues leads to a racemic mixture of the R and S forms of MetO, it was disturbing that MsrA exhibits high specificity toward the S form only (9, 10). Moreover, an enzyme (Fmsr) that catalyzes specifically the reduction of free MetO, is also specific for the S isomer (9). Nevertheless, results of other studies indicated that cells have the ability to convert the R form of MetO to methionine, by an unknown mechanism (9). In an effort to identify the enzyme(s) responsible for the conversion of R-MetO to methionine, we examined the substrate stereospecificity of three different Msrs in Staphylococcus aureus (11), ...