Structural and functional studies on drosophila ADH

The enzyme alcohol dehydrogenase of the fruit-fly Drosophila (DADH) catalyzes the same reaction as the mammalian alcohol dehydrogenases, transforming alcohols into aldehydes through the reduction of nicotinamideadenine-dinucleotide. Despite this identical chemical behaviour the enzyme's structure belongs to a different class of proteins, called short-chain dehydrogenases, which have a totally different three-dimensional architecture to the mammalian alcohol dehydrogenases. The study of the structure-fuction relationships of DADH is of interest because, while we still lack a crystal structure for the protein, large amounts of biochemicafevolutionary and genetical data have accumulated which require structural information on the enzyme for its proper interpretation.The aim of this project was two-fold: to set up a suitable system for the undertaking of protein engineering studies on DADH and to start such studies by producing and analyzing a first set of site-directed mutants. The first part of the project involved: a) creating suitable genetic vectors for the introduction of mutations, their sequencing and the expression of the mutated enzymes in yeast; b) the development of a purification method to obtain pure enzyme solutions from the expressing yeast culture; c) the development of biochemical and biophysical assays for the evaluation of the mutation's effects and, d) the construction of a three-dimensional model for the enzyme that could offer structural explanations to such effects.The second part consisted of the actual introduction of five different mutations in the enzyme's sequence, and their further evaluation using the system set up in the first place

More than an adaptor molecule: The emerging role of tRNA in cell signaling and disease

This FEBS Letters ‘FOCUS ON’ series of short reviews on tRNA captures the essence of the Barcelona BioMed Conference on Gene Translation: Fidelity and Quality Control, which was held at the Institut d’Estudis Catalans in Barcelona on December 2–4, 2013. This meeting was powered by the dramatic resurgence of interest in tRNA biochemistry following the realization that tRNA is much more than a simple adaptor of the genetic code

A-to-I editing on tRNAs: Biochemical, biological and evolutionary implications

AbstractInosine on transfer RNAs (tRNAs) are post-transcriptionally formed by a deamination mechanism of adenosines at positions 34, 37 and 57 of certain tRNAs. Despite its ubiquitous nature, the biological role of inosine in tRNAs remains poorly understood. Recent developments in the study of nucleotide modifications are beginning to indicate that the dynamics of such modifications are used in the control of specific genetic programs. Likewise, the essentiality of inosine-modified tRNAs in genome evolution and animal biology is becoming apparent. Here we review our current understanding on the role of inosine in tRNAs, the enzymes that catalyze the modification and the evolutionary link between such enzymes and other deaminases

Malaria parasite tyrosyl-tRNA synthetase secretion triggers pro-inflammatory responses

Malaria infection triggers pro-inflammatory responses in humans that are detrimental to host health. Parasite-induced enhancement in cytokine levels correlate with malaria-associated pathologies. Here we show that parasite tyrosyl-tRNA synthetase (PfTyrRS), a housekeeping protein translation enzyme, induces pro-inflammatory responses from host immune cells. PfTyrRS exits from the parasite cytoplasm into the infected red blood cell (iRBC) cytoplasm, from where it is released into the extracellular medium on iRBC lysis. Using its ELR peptide motif, PfTyrRS specifically binds to and internalizes into host macrophages, leading to enhanced secretion of the pro-inflammatory cytokines TNF-α ± and IL-6. PfTyrRS-macrophage interaction also augments expression of adherence-linked host endothelial receptors ICAM-1 and VCAM-1. Our description of PfTyrRS as a parasite-secreted protein that triggers pro-inflammatory host responses, along with its atomic resolution crystal structure in complex with tyrosyl-adenylate, provides a novel platform for targeting PfTyrRS in anti-parasitic strategies

Analogs of natural aminoacyl-tRNA synthetase inhibitors clear malaria in vivo

Malaria remains a major global health problem. Emerging resistance to existing antimalarial drugs drives the search for new antimalarials, and protein translation is a promising pathway to target. Here we explore the potential of the aminoacyl-tRNA synthetase (ARS) family as a source of antimalarial drug targets. First, a battery of known and novel ARS inhibitors was tested against Plasmodium falciparum cultures, and their activities were compared. Borrelidin, a natural inhibitor of threonyl-tRNA synthetase (ThrRS), stands out for its potent antimalarial effect. However, it also inhibits human ThrRS and is highly toxic to human cells. To circumvent this problem, we tested a library of bioengineered and semisynthetic borrelidin analogs for their antimalarial activity and toxicity. We found that some analogs effectively lose their toxicity against human cells while retaining a potent antiparasitic activity both in vitro and in vivo and cleared malaria from Plasmodium yoelii-infected mice, resulting in 100% mice survival rates. Our work identifies borrelidin analogs as potent, selective, and unexplored scaffolds that efficiently clear malaria both in vitro and in vivo.Human Frontier Science Program (Strasbourg, France) (Postdoctoral Fellowship LT000307/2013

Elucidation of tRNA-dependent editing by a class II tRNA synthetase and significance for cell viability

Editing of misactivated amino acids by class I tRNA synthetases is encoded by a specialized internal domain specific to class I enzymes. In contrast, little is known about editing activities of the structurally distinct class II enzymes. Here we show that the class II alanyl-tRNA synthetase (AlaRS) has a specialized internal domain that appears weakly related to an appended domain of threonyl-tRNA synthetase (ThrRS), but is unrelated to that found in class I enzymes. Editing of misactivated glycine or serine was shown to require a tRNA cofactor. Specific mutations in the aforementioned domain disrupt editing and lead to production of mischarged tRNA. This class-specific editing domain was found to be essential for cell growth, in the presence of elevated concentrations of glycine or serine. In contrast to ThrRS, where the editing domain is not found in all three kingdoms of living organisms, it was incorporated early into AlaRSs and is present throughout evolution. Thus, tRNA-dependent editing by AlaRS may have been critical for making the genetic code sufficiently accurate to generate the tree of life