In this program, we focused on the following projects: (i) RNA oxidation. Oxidative damage to RNA has received relatively little attention despite the fact that growing evidence indicates that mRNA oxidation is correlated with a number of age-related neurodegenerative diseases, including Alzheimer's disease and the finding reveals that mRNA oxidation occurs early in motor neuron deterioration in Amyotrophic lateral sclerosis. We showed previously that oxidized mRNA causes a reduction of translation fidelity.In additional study we showed that in vitro RNA oxidation catalyzed by cytochrome c (cyt c)/H2O2 or by the Fe(II)/ascorbate/H2O2 system yielded different covalently modified RNA derivatives. Guanosine in RNA was the predominant ribonucleoside oxidized in cytochrome c-mediated oxidation, while Fe(II)/ascorbate system oxidized all ribonucleoside with no obvious preference. GC/MS and LC/MS analyses showed that the guanine base was not only oxidized but it also depurinated to form an abasic sugar moiety. The aldehyde moieties on the abasic site formed Schiff base with the amino groups in the proteins and lead to the formation of cross-linking products, e.g. between oxidized RNA and cyt c. The formation of this cross-linking product facilitates the release of cyt c from cardiolipin-containing liposomes which may represent the release of cyt c from the mitochondria to the cytosol. Thus, the oxidative modification of RNA, including cross-linking, leads not only to impair RNA normal functions, but it may also gain a protective signal to facilitate cellular apoptosis in response to oxidative stress. To investigate the molecular basis of this observation led us to carry out microarray analysis of oxidized mRNA species in Neuro2a cells. The data indicate that mRNA oxidation was selective either with or without hydrogen peroxide treatment. Furthermore, we found that the oxidation levels were not co-related with the mRNA half-life, suggesting that mRNA oxidation is regulated. To investigate the regulatory mechanism, the cells were treated with hydrogen peroxide in the presence of cycloheximide or puromycin. The results indicate that the addition of translation inhibitors led to an increase in the levels of oxidized mRNA. In addition, when cells were treated with hydrogen peroxide for 20 min then transferred to fresh medium, the elevated mRNA oxidation was found reduced to 50% within 1h. Addition of cycloheximide in the medium blocked the reduction of oxidized mRNA. These results suggest the involvement of a translation-dependent mRNA oxidation regulatory mechanism, an observation consistents with the notion that mRNA surveilliance mechanisms, e. g. No-go mediated mRNA decay pathway, may participate to cope with damaged mRNA. (ii) Rickettsiae are obligatory intracellular infectious Gram-negative bacteria that responsible for major rickettsiosis, which include epidemic typhus, spotted fever, and scrub typhus, without the availability of vaccine or early detection method. The outer membrane of Rickettsia is largely composed of an outer membrane protein called OmpB that accounts for up to 15 % of total cellular proteins. OmpB is a virulence factor that is involved in cell adhesion, attachment, and invasion. It has been shown to be methylated at lysine residues and methylation appears to associate with rickettsial pathogenicity. Our previous studies revealed that OmpB proteins from virulent rickettsial strains contain multiple trimethylated lysyl residues while avirulent strains contain predominantly monomethylated lysyl residues. Thus, knowledge on the enzyme(s) that catalyzes OmpB methylation and on the nature of the methylated OmpB could provide new insight on OmpB-methylation and its role on virulent effect. Through bioinformatics analysis of genomic DNA sequences of Rickettsia, Dr. Yangs lab has revealed five potential sequences of putative protein lysine methylatransferases. Syntthesis and expression of these genes, follow with purification and characterization of the gene products revealed the presence two distinct types of protein lysine methyltransferases. They are the PKMT1 and PKMT2, which catalyzes predominantly monomethylation and trimethylation, respectively. Among known protein lysine methyltransferases, rickettsial PKMT1 and PKMT2 are unique in that their substrates appears to be limited to OmpB and both methyltransferases are capable of methylating multiple lysyl residues with broad sequence specificity. To better understand the mechanism by which PKMT1 and PKMT2 differentially catalyze OmpB methylation, we carry out crystal structural analysis for PKMT1 from R. prowazekii, both the apo-form and in complex with its cofactors AdoMet and AdoHcy, and for PKMT2 from R. typhi. Our data reveal that both enzymes are dimeric, with each monomer containing an AdoMet binding domain with a core Rossmann fold, a dimerization domain, a middle domain, a C-terminal domain, and a centrally located open cavity. Based on the crystal structures, residues involved in catalysis, cofactor binding and substrate interactions were examined using site-directed mutagenesis followed by steady-state kinetic analysis to ascertain their catalytic functions in solution. Together, our data provide new structural and mechanistic insights on how rickettsial methyltransferases catalyze OmpB methylation.
Showing the most recent 10 out of 15 publications
