In this program, we focused on the following projects: (i) RNA oxidation. Growing evidence indicates that RNA oxidation is correlated with a number of age-related neurodegenerative diseases, including the recent finding showing that mRNA oxidation occurs early in motor neuron deterioration in ALS. We showed previously that oxidized mRNA causes a reduction of translation fidelity despite the fact that the oxidized mRNA shows a similar binding constant to polysomes as that of non-oxidized mRNA. Our recent study 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. To this end, guanosine in RNA was the predominant ribonucleoside oxidized in cytochrome c (cyt 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 generated cross-linking products, e.g. between oxidized RNA and cyt c. Interestingly, the formation of the cross-linking product between oxidized RNA and cyt c 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. It has been reported that certain mRNA was selectively oxidized in AD and ALS. Our attempt to understand the molecular basis of this observation led us to carry out genome-wide study to quantify oxidation levels of individual mRNA in Neuro2a cells. Preliminary results indicate that the abundantly expressed mRNA appears to be more protective from oxidative damage. (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. Methylation of rickettsial OmpB has been implicated in bacteria virulence. 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. Synthesis 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. Kinetic and products analysis revealed that PKMT1 catalyzes primarily monomethylation while PKMT2 functions as trimethyltransferase. The proteins, RP789 from R. prowazekii and RT0776 from R. typhi were found to be PKMT1 while proteins RP027-028 from R. prowazekii and RT0101 from R. Typhi to be PKMT2. Semiquantitative integrated liquid chromatography-tandem mass spectrometer was used to characterize the location, state and level of methylated rOmpB fragments catalyzed by the purified methyltransferases as well as the native OmpB purified from Rickettsia. Our results revealed that methylation on OmpB occurs at multiple sites in both purified native proteins and methyltransferase-catalyzed rOmpB fragments. Furthermore, in vitro trimethylation, proceeds via a processive mechanism, occurs at relatively specific locations in OmpB with consensus motifs, KX(G/A/V/I)N and KT(I/L/F), while monomethylation is pervasive in OmpB. Interestingly, native OmpB from R. Typhi contains mono- and trimethllysine residues at location well correlated with those catalyzed by PKMT1 and PKMT2. We revealed that clusters of highly trimethylated lysine residues are present in OmpB from virulent strains but not in avirulent strain. Furthermore, the number of highly trimethylated lysine clusters is correlated with the virulent of the strains.
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