Genetic analysis of interactions with eukaryotic rRNA identify the mitoribosome as target in aminoglycoside ototoxicity

Abstract

Aminoglycoside ototoxicity has been related to a surprisingly large number of cellular structures and metabolic pathways. The finding that patients with mutations in mitochondrial rRNA are hypersusceptible to aminoglycoside-induced hearing loss has indicated a possible role for mitochondrial protein synthesis. To study the molecular interaction of aminoglycosides with eukaryotic ribosomes, we made use of the observation that the drug binding site is a distinct domain defined by the small subunit rRNA, and investigated drug susceptibility of bacterial hybrid ribosomes carrying various alleles of the eukaryotic decoding site. Compared to hybrid ribosomes with the A site of human cytosolic ribosomes, susceptibility of mitochondrial hybrid ribosomes to various aminoglycosides correlated with the relative cochleotoxicity of these drugs. Sequence alterations that correspond to the mitochondrial deafness mutations A1555G and C1494T increased drug-binding and rendered the ribosomal decoding site hypersusceptible to aminoglycoside-induced mistranslation and inhibition of protein synthesis. Our results provide experimental support for aminoglycoside-induced dysfunction of the mitochondrial ribosome. We propose a pathogenic mechanism in which interference of aminoglycosides with mitochondrial protein synthesis exacerbates the drugs' cochlear toxicity, playing a key role in sporadic dosedependent and genetically inherited, aminoglycoside-induced deafness.decoding ͉ mitochondria ͉ ribosomes ͉ toxicity ͉ translation

Dissociation of antibacterial activity and aminoglycoside ototoxicity in the 4-monosubstituted 2-deoxystreptamine apramycin

“…In the hybrid ribosomes a central 34-nucleotide part of the bacterial 16S rRNA helix 44 is replaced with various homologs resulting in rRNA-decoding A sites corresponding to those in human cytosolic and mitochondrial (wild-type and A1555G-mutant) ribosomes (except when it is referred to as mtDNA position 1555, the Escherichia coli rRNA numbering is used throughout the paper). These hybrid ribosomes have been used previously to study the ribosomal specificity of aminoglycoside antibiotics (29,35). Dose-response curves of aminoglycoside-induced inhibition of luciferase synthesis were analyzed to define the IC 50 values of individual aminoglycosides ( Table 1).…”

“…The origin of ROS generation remains unresolved, however, and both nonenzymatic and enzymatic mechanisms have been suggested (27). Recent studies imply mitochondrial protein synthesis as a key element in aminoglycoside ototoxicity, because experimental evidence has been presented for both aminoglycoside-induced dysfunction of the mitochondrial ribosome, in particular drug-mediated misreading, and A1555G-linked mitochondrial hypersusceptibility to aminoglycosides (28,29). Defective mitochondrial function reportedly elicits the formation of ROS (30,31), and hence the antimitoribosomal activity of aminoglycosides may provoke ROS generation and/or impaired resistance to oxidative stress.…”

Structure-Activity Relationships among the Kanamycin Aminoglycosides: Role of Ring I Hydroxyl and Amino Groups

“…The single rRNA allelic Mycobacterium smegmatis ⌬rrnB strain (SZ380) was obtained by unmarked deletion mutagenesis (21) and used for all genetic constructions; mutant strains 1408G, 1491A, and 1491C have been described previously (25). Residues 1408 and 1491 are 16S rRNA residues critical for aminoglycoside binding (24,32,33) and main determinants of acquired drug resistance (34,36,37); their phylogenetic polymorphism provides the basis for the compounds' selective mode of action (6,20,22 Table S1 in the supplemental material) were kindly provided by P. Courvalin, Institut Pasteur, Paris, France.…”

Molecular Basis for the Selectivity of Antituberculosis Compounds Capreomycin and Viomycin

“…Mitochondrial mistranslation has been identified as an important mechanism contributing to aminoglycoside ototoxicity (14,16). In addition, aminoglycoside ototoxicity occurs in a genetically inherited form, with drug hypersusceptibility linked to alterations A1555G or C1494U in mitochondrial rRNA (29,39).…”

“…However, the structural determinants of selectivity, i.e., the ability to differentiate between prokaryotic and eukaryotic ribosomes, are notably different. While for the clinically used 6ЈNH 2 4,6-aminoglycosides selectivity to a large extent rests upon 16S rRNA residue 1408 (14,16), selectivity of tuberactinomycin antibiotics involves mainly 16S rRNA residue 1491. We suggest that the structures available together with the determinants of selectivity identified will enable the creation of new compounds by targeted drug design.…”