Herbert Weissbach

Association between Lupus Psychosis and Antiribosomal P Protein Antibodies

In 18 of 20 patients with psychosis secondary to systemic lupus erythematosus (SLE), autoantibodies to ribosomal P proteins were detected by immunoblotting and measured with a new radioimmunoassay using a synthetic peptide as antigen. The frequency of anti-P was not increased in patients with other central nervous system manifestations of SLE (3 of 20, by radioimmunoassay), in patients with transient behavioral abnormalities due to SLE (none of 8), in patients with psychosis who did not have SLE (none of 13), or in normal controls (none of 20). In four of five paired serum samples, anti-P-peptide antibody levels increased 5-fold to 30-fold during the active phase of lupus psychosis. Longitudinal studies of anti-P activity in two patients with psychosis revealed that anti-P levels increased before and during the active phases of psychosis but not during sepsis or other exacerbations of SLE, and that the elevations were selective for anti-P antibodies, as opposed to anti-DNA antibodies. Longitudinal studies of anti-P activity in two patients with anti-P but without psychosis showed less than threefold changes in anti-P levels despite exacerbations of disease. We conclude that anti-P is associated with lupus psychosis and that synthetic peptide antigens may be useful for the detection and measurement of autoantibodies to intracellular proteins.

High-quality life extension by the enzyme peptide methionine sulfoxide reductase

Cumulative oxidative damages to cell constituents are considered to contribute to aging and age-related diseases. The enzyme peptide methionine sulfoxide reductase A (MSRA) catalyzes the repair of oxidized methionine in proteins by reducing methionine sulfoxide back to methionine. However, whether MSRA plays a role in the aging process is poorly understood. Here we report that overexpression of the msrA gene predominantly in the nervous system markedly extends the lifespan of the fruit fly Drosophila. The MSRA transgenic animals are more resistant to paraquat-induced oxidative stress, and the onset of senescence-induced decline in the general activity level and reproductive capacity is delayed markedly. The results suggest that oxidative damage is an important determinant of lifespan, and MSRA may be important in increasing the lifespan in other organisms including humans.

Peptide methionine sulfoxide reductase from Escherichia coli and Mycobacterium tuberculosis protects bacteria against oxidative damage from reactive nitrogen intermediates

Inducible nitric oxide synthase (iNOS) plays an important role in host defense. Macrophages expressing iNOS release the reactive nitrogen intermediates (RNI) nitrite and S-nitrosoglutathione (GSNO), which are bactericidal in vitro at a pH characteristic of the phagosome of activated macrophages. We sought to characterize the active intrabacterial forms of these RNI and their molecular targets. Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) catalyzes the reduction of methionine sulfoxide (Met-O) in proteins to methionine (Met). E. coli lacking MsrA were hypersensitive to killing not only by hydrogen peroxide, but also by nitrite and GSNO. The wild-type phenotype was restored by transformation with plasmids encoding msrA from E. coli or M. tuberculosis, but not by an enzymatically inactive mutant msrA, indicating that Met oxidation was involved in the death of these cells. It seemed paradoxical that nitrite and GSNO kill bacteria by oxidizing Met residues when these RNI cannot themselves oxidize Met. However, under anaerobic conditions, neither nitrite nor GSNO was bactericidal. Nitrite and GSNO can both give rise to NO, which may react with superoxide produced by bacteria during aerobic metabolism, forming peroxynitrite, a known oxidant of Met to Met-O. Thus, the findings are consistent with the hypotheses that nitrite and GSNO kill E. coli by intracellular conversion to peroxynitrite, that intracellular Met residues in proteins constitute a critical target for peroxynitrite, and that MsrA can be essential for the repair of peroxynitrite-mediated intracellular damage.

The mirrored methionine sulfoxide reductases of Neisseria gonorrhoeae pilB.

Methionine sulfoxide reductases (Msr) protect against oxidative damage that can contribute to cell death. The tandem Msr domains (MsrA and MsrB) of the pilB protein from Neisseria gonorrhoeae each reduce different epimeric forms of methionine sulfoxide. The overall fold of the MsrB domain revealed by the 1.85 Å crystal structure shows no resemblance to the previously determined MsrA structures from other organisms. Despite the lack of homology, the active sites show approximate mirror symmetry. In each case, conserved amino acid motifs mediate the stereo-specific recognition and reduction of the substrate. Unlike the MsrA domain, the MsrB domain activates the cysteine or selenocysteine nucleophile through a unique Cys-Arg-Asp/Glu catalytic triad. The collapse of the reaction intermediate most likely results in the formation of a sulfenic or selenenic acid moiety. Regeneration of the active site occurs through a series of thiol-disulfide exchange steps involving another active site Cys residue and thioredoxin. These observations have broad implications for modular catalysis, antibiotic drug design and continuing longevity studies in mammals.

[22] Elongation factor Tu and the aminoacyl-tRNA · EFTu · GTP complex

Publisher Summary The bacterial elongation factors EFTu and EFTs promote the binding of AA-tRNA to ribosomes via an AA-tRNA·EFTu·GTP intermediate. When this ternary complex interacts with ribosomes in the presence of messenger-RNA, the AA-tRNA is transferred to the ribosome and GTP is hydrolyzed with the formation of EFTu·GDP and P 1 . The EFTu·GDP complex dissociates very slowly. The function of EFTs is to catalyze the exchange of the tightly bound GDP with free GTP to form EFTu·GTP that can interact with another molecule of AA-tRNA, thus allowing EFTu to function catalytically in the binding cycle.

Free methionine-(R)-sulfoxide reductase from Escherichia coli reveals a new GAF domain function

The reduction of methionine sulfoxide (MetO) is mediated by methionine sulfoxide reductases (Msr). The MsrA and MsrB families can reduce free MetO and MetO within a peptide or protein context. This process is stereospecific with the S- and R-forms of MetO repaired by MsrA and MsrB, respectively. Cell extracts from an MsrA−B− knockout of Escherichia coli have several remaining Msr activities. This study has identified an enzyme specific for the free form of Met-(R)-O, fRMsr, through proteomic analysis. The recombinant enzyme exhibits the same substrate specificity and is as active as MsrA family members. E. coli fRMsr is, however, 100- to 1,000-fold more active than non-selenocysteine-containing MsrB enzymes for free Met-(R)-O. The crystal structure of E. coli fRMsr was previously determined, but no known function was assigned. Thus, the function of this protein has now been determined. The structural similarity of the E. coli and yeast proteins suggests that most fRMsrs use three cysteine residues for catalysis and the formation of a disulfide bond to enclose a small active site cavity. This latter feature is most likely a key determinant of substrate specificity. Moreover, E. coli fRMsr is the first GAF domain family member to show enzymatic activity. Other GAF domain proteins substitute the Cys residues and others to specifically bind cyclic nucleotides, chromophores, and many other ligands for signal potentiation. Therefore, Met-(R)-O may represent a signaling molecule in response to oxidative stress and nutrients via the TOR pathway in some organisms.

Molecular cloning and functional expression of a human peptide methionine sulfoxide reductase (hMsrA)

Oxidation of methionine residues in proteins to methionine sulfoxide can be reversed by the enzyme peptide methionine sulfoxide reductase (MsrA, EC 1.8.4.6). We cloned the gene encoding a human homologue (hMsrA) of the enzyme, which has an 88% amino acid sequence identity to the bovine version (bMsrA). With dot blot analyses based on RNA from human tissues, expression of hMsrA was found in all tissues tested, with highest mRNA levels in adult kidney and cerebellum, followed by liver, heart ventricles, bone marrow and hippocampus. In fetal tissue, expression was highest in the liver. No expression of hmsrA was detected in leukemia and lymphoma cell lines. To test if hMsrA is functional in cells, we assayed its effect on the inactivation time course of the A‐type potassium channel ShC/B since this channel property strongly depends on the oxidative state of a methionine residue in the N‐terminal part of the polypeptide. Co‐expression of ShC/B and hMsrA in Xenopus oocytes significantly accelerated inactivation, showing that the cloned enzyme is functional in an in vivo assay system. Furthermore, the activity of a purified glutathione‐S‐transferase‐hMsrA fusion protein was demonstrated in vitro by measuring the reduction of [3H]N‐acetyl methionine sulfoxide.

Origin and evolution of the protein-repairing enzymes methionine sulphoxide reductases.

The majority of extant life forms thrive in an O2‐rich environment, which unavoidably induces the production of reactive oxygen species (ROS) during cellular activities. ROS readily oxidize methionine (Met) residues in proteins/peptides to form methionine sulphoxide [Met(O)] that can lead to impaired protein function. Two methionine sulphoxide reductases, MsrA and MsrB, catalyse the reduction of the S and R epimers, respectively, of Met(O) in proteins to Met. The Msr system has two known functions in protecting cells against oxidative damage. The first is to repair proteins that have lost activity due to Met oxidation and the second is to function as part of a scavenger system to remove ROS through the reversible oxidation/reduction of Met residues in proteins. Bacterial, plant and animal cells lacking MsrA are known to be more sensitive to oxidative stress. The Msr system is considered an important cellular defence mechanism to protect against oxidative stress and may be involved in ageing/senescence. MsrA is present in all known eukaryotes and eubacteria and a majority of archaea, reflecting its essential role in cellular life. MsrB is found in all eukaryotes and the majority of eubacteria and archaea but is absent in some eubacteria and archaea, which may imply a less important role of MsrB compared to MsrA. MsrA and MsrB share no sequence or structure homology, and therefore probably emerged as a result of independent evolutionary events. The fact that some archaea lack msr genes raises the question of how these archaea cope with oxidative damage to proteins and consequently of the significance of msr evolution in oxic eukaryotes dealing with oxidative stress. Our best hypothesis is that the presence of ROS‐destroying enzymes such as peroxiredoxins and a lower dissolved O2 concentration in those msr‐lacking organisms grown at high temperatures might account for the successful survival of these organisms under oxidative stress.

Regulation of voltage-dependent K+ channels by methionine oxidation: effect of nitric oxide and vitamin C

Methionine oxidation is known to alter functional properties of a transient A‐type potassium channel expressed in Xenopus oocytes. We show here that nitric oxide (NO) slows down the K+ channel inactivation time course by oxidizing a critical methionine residue in the inactivation ball domain of the channel protein. We also demonstrate that the channel protein is protected from methionine oxidation by the enzyme methionine sulfoxide reductase and the antioxidant vitamin C.

Selenocompounds Can Serve as Oxidoreductants with the Methionine Sulfoxide Reductase Enzymes

In a recent study on the reducing requirement for the methionine sulfoxide reductases (Msr) (Sagher, D., Brunell, D., Hejtmancik, J. F., Kantorow, M., Brot, N. & Weissbach, H. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 8656–8661), we have shown that thioredoxin, although an excellent reducing system for Escherichia coli MsrA and MsrB and bovine MsrA, is not an efficient reducing agent for either human MsrB2 (hMsrB2) or human MsrB3 (hMsrB3). In a search for another reducing agent for hMsrB2 and hMsrB3, it was recently found that thionein, the reduced, metal-free form of metallothionein, could function as a reducing system for hMsrB3, with weaker activity using hMsrB2. In the present study, we provide evidence that some selenium compounds are potent reducing agents for both hMsrB2 and hMsrB3.