RESUMO
Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Fúngicas/metabolismo , Mitocôndrias , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Peroxiredoxins (Prxs) are cysteine-based peroxidases that play a central role in keeping the H2O2 at physiological levels. Eukaryotic cells express different Prxs isoforms, which differ in their subcellular locations and substrate specificities. Mitochondrial Prxs are synthesized in the cytosol as precursor proteins containing N-terminal cleavable presequences that act as mitochondrial targeting signals. Due to the fact that presequence controls the import of the vast majority of mitochondrial matrix proteins, the mitochondrial Prxs were initially predicted to be localized exclusively in the matrix. However, recent studies showed that mitochondrial Prxs are also targeted to the intermembrane space by mechanisms that remain poorly understood. While in yeast the IMP complex can translocate Prx1 to the intermembrane space, the maturation of yeast Prx1 and mammalian Prdx3 and Prdx5 in the matrix has been associated with sequential cleavages of the presequence by MPP and Oct1/MIP proteases. In this review, we describe the state of the art of the molecular mechanisms that control the mitochondrial import and maturation of Prxs of yeast and human cells. Once mitochondria are considered the major intracellular source of H2O2, understanding the mitochondrial Prx biogenesis pathways is essential to increase our knowledge about the H2O2-dependent cellular signaling, which is relevant to the pathophysiology of some human diseases.
RESUMO
Multidrug resistance (MDR) is a serious threat to public health, making the development of new antimicrobials an urgent necessity. Pyocins are protein antibiotics produced by Pseudomonas aeruginosa strains to kill closely related cells during intraspecific competition. Here, we report an in-depth biochemical, microbicidal, and structural characterization of a new S-type pyocin, named S8. Initially, we described the domain organization and secondary structure of S8. Subsequently, we observed that a recombinant S8 composed of the killing subunit in complex with the immunity (ImS8) protein killed the strain PAO1. Furthermore, mutation of a highly conserved glutamic acid to alanine (Glu100Ala) completely inhibited this antimicrobial activity. The integrity of the H-N-H motif is probably essential in the killing activity of S8, as Glu100 is a highly conserved residue of this motif. Next, we observed that S8 is a metal-dependent endonuclease, as EDTA treatment abolished its ability to cleave supercoiled pUC18 plasmid. Supplementation of apo S8 with Ni2+ strongly induced this DNase activity, whereas Mn2+ and Mg2+ exhibited moderate effects and Zn2+ was inhibitory. Additionally, S8 bound Zn2+ with a higher affinity than Ni2+ and the Glu100Ala mutation decreased the affinity of S8 for these metals, as shown by isothermal titration calorimetry (ITC). Finally, we describe the crystal structure of the Glu100Ala S8 DNase-ImS8 complex at 1.38 Å, which gave us new insights into the endonuclease activity of S8. Our results reinforce the possibility of using pyocin S8 as an alternative therapy for infections caused by MDR strains, while leaving commensal human microbiota intact.IMPORTANCE Pyocins are proteins produced by Pseudomonas aeruginosa strains that participate in intraspecific competition and host-pathogen interactions. They were first described in the 1950s and since then have gained attention as possible new antibiotics. However, there is still only scarce information about the molecular mechanisms by which these molecules induce cell death. Here, we show that the metal-dependent endonuclease activity of pyocin S8 is involved with its antimicrobial action against strain PAO1. We also describe that this killing activity is dependent on a conserved Glu residue within the H-N-H motif. The potency and selectivity of pyocin S8 toward a narrow spectrum of P. aeruginosa strains make this protein an attractive antimicrobial alternative for combatting MDR strains, while leaving commensal human microbiota intact.
Assuntos
Antibacterianos/química , Desoxirribonuclease I/química , Pseudomonas aeruginosa/metabolismo , Piocinas/química , Motivos de Aminoácidos , Ácido Glutâmico/química , Relação Estrutura-AtividadeRESUMO
Yeast Prx1 is a mitochondrial 1-Cys peroxiredoxin that catalyzes the reduction of endogenously generated H2O2 Prx1 is synthesized on cytosolic ribosomes as a preprotein with a cleavable N-terminal presequence that is the mitochondrial targeting signal, but the mechanisms underlying Prx1 distribution to distinct mitochondrial subcompartments are unknown. Here, we provide direct evidence of the following dual mitochondrial localization of Prx1: a soluble form in the intermembrane space and a form in the matrix weakly associated with the inner mitochondrial membrane. We show that Prx1 sorting into the intermembrane space likely involves the release of the protein precursor within the lipid bilayer of the inner membrane, followed by cleavage by the inner membrane peptidase. We also found that during its import into the matrix compartment, Prx1 is sequentially cleaved by mitochondrial processing peptidase and then by octapeptidyl aminopeptidase 1 (Oct1). Oct1 cleaved eight amino acid residues from the N-terminal region of Prx1 inside the matrix, without interfering with its peroxidase activity in vitro Remarkably, the processing of peroxiredoxin (Prx) proteins by Oct1 appears to be an evolutionarily conserved process because yeast Oct1 could cleave the human mitochondrial peroxiredoxin Prx3 when expressed in Saccharomyces cerevisiae Altogether, the processing of peroxiredoxins by Imp2 or Oct1 likely represents systems that control the localization of Prxs into distinct compartments and thereby contribute to various mitochondrial redox processes.
Assuntos
Metaloproteases/metabolismo , Mitocôndrias/enzimologia , Peroxidases/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Humanos , Metaloproteases/genética , Mitocôndrias/genética , Peroxidases/genética , Transporte Proteico/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
A novel transposon belonging to the Tn3-like family was identified on the chromosome of a commensal strain of Pseudomonas aeruginosa sequence type 2343 (ET02). Tn6350 is 7,367 bp long and harbors eight open reading frames (ORFs), an ATPase (IS481 family), a transposase (DDE catalytic type), a Tn3 resolvase, three hypothetical proteins, and genes encoding the new pyocin S8 with its immunity protein. We show that pyocin S8 displays activity against carbapenemase-producing P. aeruginosa, including IMP-1, SPM-1, VIM-1, GES-5, and KPC-2 producers.
Assuntos
Elementos de DNA Transponíveis/genética , Pseudomonas aeruginosa/genética , Piocinas/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Humanos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , beta-Lactamases/biossíntese , beta-Lactamases/genéticaRESUMO
This study reports the presence of hospital-associated high-risk lineages of OXA-23-producing ST79 Acinetobacter baumannii and SPM-1-producing ST277 Pseudomonas aeruginosa in urban rivers in Brazil. These findings indicate that urban rivers can act as reservoirs of clinically important multidrug-resistant bacteria, which constitute a potential risk to human and animal health.