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 have shown previously that oxidized mRNA causes a reduction of translation fidelity. In additional study we revealed 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 led to the formation of cross-linking products, e.g. between oxidized RNA and cyt c. The formation of this cross-linking product facilitated 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, led not only to impair RNA normal functions, but it might 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. Our results suggest the involvement of a translation-dependent mRNA oxidation regulatory mechanism, consistent with the notion of mRNA surveilliance mechanisms, e. g. No-go mediated mRNA decay pathway, may participate to cope with damaged mRNA. To investigate whether the level of oxidized mRNA is correlated with ribosome, we subjected the cell lysates of Neuro2 cells to sucrose density gradient centrifugation to separate mRNA into three fractions, polysome, oligo- to mono-some, and free fractions. The RNA was isolated in each fraction, and analyzed their oxidation levels. The results revealed that under normal culture conditions, the mRNA in free fraction exhibited the highest levels, while mRNA in oligosome/monosome fraction yielded the lowest level (i.e. in -actin, polysome: 0.22 %, oligosome/monosome: 0.01 %, free: 0.93 %). It is believed that substantial amount of oxidized mRNA was likely eliminated by degradation pathway. When the cell lysates after treated with 0.2 mM H2O2 for 30 min were analyzed, the results showed the oxidized mRNA levels were elevated in the three fractions to a different levels (i.e. in -actin, polysome: 0.96 %, oligosome/monosome: 0.59 %, free: 1.33 %). The oxidation levels were induced in the oligosome/monosome fraction, with a 3-fold increase in the total amount of mRNA in the fraction, likely derived from the polysome mRNA due to partial dissociation of polysome. Note the oxidation levels in polysome fraction was also increased substantially (4 folds), even though the total mRNA in the fraction was reduced (50 % reduction). It has been suggested that a part of the polysome associated oxidized mRNA was accumulated in the polysome instead of dissociating under acute oxidative stress. However, it remained to be shown whether the oxidized mRNA in the polysome are actively translated or associated with multiple ribosomes stalled. Together these observations showed distinctly the oxidized mRNA are associated to ribosome, implying that oxidized mRNA is metabolized or degraded with translation machinery. (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 its total cellular proteins. OmpB is known to involve in cell adhesion, attachment, and invasion. We and others showed that methylation of lysyl residues in rickettsial OmpB correlated with bacterial virulence. Methylation profiles analysis using LC-MS/MS methods revealed a high correlation in methylation sites between those detected in native proteins purified from bacteria and those attained from in vitro methylation, except the methylation level was found significantly higher in the native proteins relative to those methylated using purified methtyltransferases with overexpressed OmpB fragments as substrates. In addition, the natively purified OmpB from the avirulent strain Madrid E of Rickettsia prowazekii does not contain any trimethyllysine. An observation consistent with the report that the gene encoding the trimethyltransferase, RP027-028, in Madrid E has been interrupted by a frameshift mutation to generate the inactive trimethyltransferase, the RP027 and RP028 fragments. However, OmpB from the highly virulent Rickettsia prowazekii strains Breinl, and RP22 each contains clusters of trimethyllysines located at a relatively close proximity. Thus, the state and type of OmpB methylation is correlated with rickettsial pathogenicity. To this end, 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 Rickettsia prowazekii, both the apo-form and in complex with its cofactors, S-adenosylmethionine or S-adenosylhomocysteine, and for PKMT2 from Rickettsia typhi. The structure of PKMT1 in complex with S-adenosylhomocysteine is solved to a resolution of 1.9 . Both enzymes are dimeric, with each monomer containing an S-adenosylmethionine binding domain with a seven-strand 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.
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