| dc.description.abstract | Cycloheximide (CHI) is a eukaryotic protein synthesis inhibitor whose action has been implicated in all three steps of translation-initiation, elongation, and termination. It has been shown that CHI binds to the 60S ribosomal subunit and blocks the release of deacylated tRNA from ribosomes. Microsporum canis, a human pathogenic fungus, can be enriched by culturing in the presence of CHI. In vitro studies on protein synthesis in this organism have shown that resistance is expressed at the level of ribosomes. A related but distinct pattern of resistance to CHI has been reported in Tetrahymena thermophila, where the presence of CHI in the medium inhibits protein synthesis and growth; upon prolonged incubation, the cells become “adapted” to CHI and resume protein synthesis and growth. Enhanced phosphorylation of ribosomal proteins has been reported to play a critical role in CHI “adaptation.”
In this thesis, attempts have been made to study native ribosomal particles, ribosomal proteins and phosphoproteins, and methylation of ribosomal RNA from cells grown in the presence and absence of CHI, in order to elucidate the mechanism of CHI resistance in M. canis.
A brief account of the physicochemical properties of eukaryotic ribosomes, the role of ribosomal protein phosphorylation in protein biosynthesis, the process of ribosome biogenesis, and mechanisms of resistance to CHI is presented in Chapter I.
Ribosome Characterization (Chapter II)
The physicochemical characteristics of M. canis ribosomal subunits and monosomes were determined and compared with those from other eukaryotes. The sedimentation coefficients of derived monosomes and ribosomal subunits are 82S (80S), 58S (60S), and 38S (40S), respectively. Their buoyant densities are 1.585, 1.610, and 1.550 g/ml.
The ribosomal RNAs of M. canis show sedimentation coefficients of 25S, 18S, 5.8S, and 5S, with molecular weights of approximately 1.82 × 10 , 0.68 × 10 , 52,000, and 39,000, respectively. The molecular weights of the ribosomal particles were calculated from the aggregate masses of rRNA and proteins. The native 80S and 60S preparations have buoyant densities of 1.585 and 1.540 g/ml, respectively. The native 40S preparation contains four species differing in densities-1.535, 1.450, 1.430, and 1.395 g/ml. The native ribosomal particles, irrespective of whether they are from control cells or cells grown in the presence of CHI, do not differ in their buoyant densities.
CHI–Ribosome Interactions by NMR (Chapter III)
Nuclear magnetic resonance (NMR) spectroscopy was used to study CHI–ribosome interactions. The two methyl resonances of CHI exhibit line broadening upon interaction with ribosomes from Saccharomyces cerevisiae in a concentration dependent manner, indicating that the drug is in fast exchange with ribosomes. Tetracycline does not compete with CHI for binding site(s) on ribosomes. A comparison of CHI induced line broadening by ribosomes from S. cerevisiae and M. canis revealed that less CHI is bound to M. canis ribosomes, consistent with ribosomal resistance.
Ribosomal Proteins and Phosphoproteins (Chapter IV)
Ribosomal proteins from M. canis were analysed by 2 D gel electrophoresis (basic acidic). Twenty eight and thirty seven proteins were identified in the 40S and 60S subunits, numbered S2–S20 and L3–L39, respectively. The molecular weights (Mr) were in the ranges 32,500–7,600 (40S) and 48,000–11,000 (60S). Separation on gradient gels revealed 18, 25, and 30 bands in 40S, 60S, and 80S particles, respectively. Comparison of patterns in 1 D and 2 D gels helped identify three additional proteins in the 40S and two in the 60S subunits.
Analysis of ribosomal phosphoproteins by 2 D gels (basic acidic) showed a single major phosphoprotein (S6) of Mr ~32,000 in the small subunit. By 1 D gradient gels, three phosphoproteins (Mr 32,000; 23,500; 18,000) were observed in the small subunit and two (Mr 41,000; 17,000) in the large subunit. Since S6 is the only phosphoprotein detected in basic–acidic 2 D gels, the other four are considered acidic in nature.
Mycelia grown in the presence of CHI did not show changes in the overall pattern of proteins or phosphoproteins. However, a marginal increase in ^32P incorporation into different phosphoproteins was observed in CHI grown cells. Phosphoserine was predominant, with trace phosphothreonine, in both control and CHI treated cells.
rRNA Methylation (Chapter V)
rRNAs isolated from cells grown in the presence and absence of CHI were analysed. Separation of nucleotides from total rRNA after RNase T digestion showed an identical pattern except for 7 methylguanosine (m G) (note: original “mJA” appears to be a typographical rendering; context indicates m G or a specific methylated base), which was present only in control cells. Analyses of 5S, 5.8S, and 18S rRNA from control and CHI grown cells did not show differences in base or sugar methylations.
The 25S rRNA from control cells contained 37–38 ribose methylations, of which 31–32 were monomethylations. It also contained 4–5 moles of m C and 36–37 pseudouridine residues. A similar analysis of 25S rRNA from CHI grown cells did not reveal significant differences except for m G: about 1–2 residues per 25S rRNA present in controls were absent in CHI grown cells.
Proposed Mechanism
Based on the present studies and in light of available literature, it is proposed that methylated bases (notably m G; see note above) are present in or near the peptidyl transferase center of eukaryotic ribosomes. The absence of this methylated base in M. canis cells grown in the presence of CHI might underlie their “adaptation” to the drug, by altering ribosomal sensitivity while preserving translational function. | |
