Ketogenic Diet for KARS-Related Mitochondrial Dysfunction and Progressive Leukodystrophy.

KARS encodes lysyl-tRNA synthetase, which is essential for protein translation. KARS mutations sometimes cause impairment of cytoplasmic and mitochondrial protein synthesis, and sometimes lead to progressive leukodystrophies with mitochondrial signature and psychomotor regression, and follow a rapid regressive course to premature death. There has been no disease-modifying therapy beyond supportive treatment. We present a 5-year-old male patient with an asymmetrical leukodystrophy who showed overt evidence of mitochondrial dysfunction, including elevation of lactate on brain MR spectroscopy and low oxygen consumption rate in fibroblasts. We diagnosed this patient's condition as KARS-related leukodystrophy with cerebral calcification, congenital deafness, and evidence of mitochondrial dysfunction. We employed a ketogenic diet as well as multiple vitamin supplementation with the intention to alleviate mitochondrial dysfunction. The patient showed alleviation of his psychomotor regression and even partial restoration of his abilities within 4 months. This is an early report of a potential disease-modifying therapy for KARS-related progressive leukodystrophy without appreciable adverse effects.

Specific Stereoisomeric Conformations Determine the Drug Potency of Cladosporin Scaffold against Malarial Parasite.

The dependence of drug potency on diastereomeric configurations is a key facet. Using a novel general divergent synthetic route for a three-chiral center antimalarial natural product cladosporin, we built its complete library of stereoisomers (cladologs) and assessed their inhibitory potential using parasite-, enzyme-, and structure-based assays. We show that potency is manifest via tetrahyropyran ring conformations that are housed in the ribose binding pocket of parasite lysyl tRNA synthetase (KRS). Strikingly, drug potency between top and worst enantiomers varied 500-fold, and structures of KRS-cladolog complexes reveal that alterations at C3 and C10 are detrimental to drug potency whereas changes at C3 are sensed by rotameric flipping of glutamate 332. Given that scores of antimalarial and anti-infective drugs contain chiral centers, this work provides a new foundation for focusing on inhibitor stereochemistry as a facet of antimicrobial drug development.

Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin.

With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited.

Solid-phase combinatorial synthesis of a lysyl-tRNA synthetase (LysRS) inhibitory library.

The solid-phase combinatorial synthesis of a new library with potential inhibitory activity against the cytoplasmic lysyl-tRNA synthetase (LysRS) isoform of Trypanosoma brucei is described. The library has been specifically designed to mimic the lysyl adenylate complex. The design was carried out by dividing the complex into four modular parts. Proline derivatives (cis-gamma-amino-L-proline or trans-gamma-hydroxy-L-proline) were chosen as central scaffolds. After primary screening, three compounds of the library caused in vitro inhibition of the tRNA aminoacylation reaction in the low micromolar range.

HIV-1 nucleocapsid protein zinc finger structures induce tRNA(Lys,3) structural changes but are not critical for primer/template annealing.

Retroviral reverse transcriptases use host cellular tRNAs as primers to initiate reverse transcription. In the case of human immunodeficiency virus type 1 (HIV-1), the 3' 18 nucleotides of human tRNA(Lys,3) are annealed to a complementary sequence on the RNA genome known as the primer binding site (PBS). The HIV-1 nucleocapsid protein (NC) facilitates this annealing. To understand the structural changes that are induced upon NC binding to the tRNA alone, we employed a chemical probing method using the lanthanide metal terbium. At low concentrations of NC, the strong terbium cleavage observed in the core region of the tRNA is significantly attenuated. Thus, NC binding first results in disruption of the tRNA's metal binding pockets, including those that stabilize the D-TPsiC tertiary interaction. When NC concentrations approach the amount needed for complete primer/template annealing, NC further destabilizes the tRNA acceptor-TPsiC stem minihelix, as evidenced by increased terbium cleavage in this domain. A mutant form of NC (SSHS NC), which lacks the zinc finger structures, is able to anneal tRNA(Lys,3) efficiently to the PBS, and to destabilize the tRNA tertiary core, albeit less effectively than wild-type NC. This mutant form of NC does not affect cleavage significantly in the helical regions, even when bound at high concentrations. These results, as well as experiments conducted in the presence of polyLys, suggest that in the absence of the zinc finger structures, NC acts as a polycation, neutralizing the highly negative phosphodiester backbone. The presence of an effective multivalent cationic peptide is sufficient for efficient tRNA primer annealing to the PBS.

A single point mutation of hamster aminoacyl-tRNA synthetase causes apoptosis by deprivation of cognate amino acid residue.

BACKGROUND: We have isolated a series of temperature-sensitive mutants for cell-proliferation from the BHK21 cell line derived from the golden hamster (Nishimoto & Basilico 1978; Nishimoto et al. 1982). Using these mutants as a recipient of DNA-mediated gene transfer, we have been cloning human genes which complement these ts mutants. RESULTS: Cultures of tsBN269 cells, a temperature-sensitive mutant of the BHK21 cell line, underwent apoptosis at 39.5 degrees C, a nonpermissive temperature. The gene complementing the tsBN269 cells was cloned and found to encode lysyl-tRNA synthetase. Indeed, tsBN269 cells were found to have a single cytosine to a thymine point mutation at the first nucleotide of codon 542 in hamster lysyl-tRNA synthetases. Due to this mutation, the activity of lysyl-tRNA synthetase was reduced--even at 33.5 degrees C, a permissive temperature. Consistent with these findings, while supplementation with lysine permitted tsBN269 cells to grow at a nonpermissive temperature, the deprivation of lysine caused apoptosis in tsBN269 cells, even at 33.5 degrees C. Cycloheximide inhibited the apoptosis caused by lysine starvation at 33.5 degrees C, but not at 39.5 degrees C. We also found that another hamster temperature-sensitive mutant, tsBN250, which is defective in histidyl-tRNA synthetase, entered apoptosis with the deprivation of histidine. CONCLUSION: Our data suggested that the defect in aminoacyl-tRNA synthetase turned on the cascade of apoptosis that was already present in the cells.

Elevated serine catabolism is associated with the heat shock response in Escherichia coli.

The biochemical events associated with the heat shock response are not well understood in any organism, nor have the signals that initiate the induction of heat shock protein synthesis been identified. In this work, we demonstrate that the rate of serine catabolism of Escherichia coli cells grown in glucose minimal medium supplemented with serine is elevated three- to sevenfold when the growth temperature is shifted from 37 to 44 degrees C. Elevations in growth temperature and mutations or treatments that lead to elevated basal rates of serine catabolism at 37 degrees C result in the excretion into the culture medium of acetate derived from exogenous serine. Increases in the basal level of serine catabolism at 37 degrees C do not per se induce a heat shock response but are associated with abnormalities in the pattern of induction of heat shock polypeptides following a temperature shift. We postulate that the events responsible for or resulting from the elevation in serine catabolism associated with a shift-up in temperature modulate the induction of 3 of the 17 heat shock polypeptides identified in E. coli. These observations suggest that heat shock diverts serine away from the production of glycine and C1 units, which are required for initiation of protein synthesis and for nucleotide biosynthesis, and towards acetyl coenzyme A and acetate.

Thialysine- and selenalysine-resistance in a E. coli mutant.

A thialysine-resistant mutant of E. coli strain KL16 also shows a lower sensitivity to selenalysine, the lysine analog containing selenium. No difference between the mutant and the parental strain has been shown regarding the affinities of the transport systems and the lysyl-tRNA synthetase for selenalysine, thialysine and lysine as well as the inhibitory effects of these three aminoacids on the activity of the lysine biosynthetic pathway. A marked difference between the two strains has been evidenced in the AK III repression: in the mutant the repression by selenalysine, thialysine and lysine is much lower than in the parental strain.

Biochemical characterization of a thialysine-resistant clone of CHO cells.

The intracellular transport and the activation of lysine, thialysine and selenalysine have been investigated in a thialysine-resistant CHO cell mutant strain in comparison with the parental strain. The cationic amino acid transport system responsible for the transport of these 3 amino acids shows no differences between the 2 strains as regards its affinity for each of these amino acids. On the other hand the Vmax of the transport system in the mutant is about double that in the parental strain. The lysyl-tRNA synthetase, assayed both as ATP = PPi exchange reaction and lysyl-tRNA synthesis, shows a lower affinity for thialysine and selenalysine than for lysine in both strains; in the mutant, however, the difference is even greater. Thus the thialysine resistance of the mutant is mainly due to the properties of its lysyl-tRNA synthetase, which shows a greater difference of the affinities for lysine and thialysine with respect to the parental strain.

Selenalysine and protein synthesis.

Selenalysine is a lysine analog having the gamma-methylene group substituted by a selenium atom. It has been demonstrated that selenalysine is activated and transferred to tRNAlys by either Escherichia coli or rat liver aminoacyl-tRNA synthetases, and inhibits lysine incorporation into polypeptides in protein-synthesizing systems from E. coli, rat liver or rabbit reticulocytes. All tests were performed in comparison with thialysine, a lysine analog having the gamma-methylene group substituted by a sulfur atom. In all the reactions studied, both thialysine and selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine act as competitive inhibitors of lysine. With respect to thialysine, selenalysine shows a slightly lower activity as lysine inhibitor.