RESUMO
YgfB-mediated ß-lactam resistance was recently identified in multi drug resistant Pseudomonas aeruginosa. We show that YgfB upregulates expression of the ß-lactamase AmpC by repressing the function of the regulator of the programmed cell death pathway AlpA. In response to DNA damage, the antiterminator AlpA induces expression of the alpBCDE autolysis genes and of the peptidoglycan amidase AmpDh3. YgfB interacts with AlpA and represses the ampDh3 expression. Thus, YgfB indirectly prevents AmpDh3 from reducing the levels of cell wall-derived 1,6-anhydro-N-acetylmuramyl-peptides, required to induce the transcriptional activator AmpR in promoting the ampC expression and ß-lactam resistance. Ciprofloxacin-mediated DNA damage induces AlpA-dependent production of AmpDh3 as previously shown, which should reduce ß-lactam resistance. YgfB, however, counteracts the ß-lactam enhancing activity of ciprofloxacin by repressing ampDh3 expression and lowering the benefits of this drug combination. Altogether, YgfB represents an additional player in the complex regulatory network of AmpC regulation.
Assuntos
Pseudomonas aeruginosa , Resistência beta-Lactâmica , Pseudomonas aeruginosa/genética , Resistência beta-Lactâmica/genética , Ciprofloxacina/farmacologia , beta-Lactamas/farmacologiaRESUMO
Polarity is a fundamental property of most cell types. The Par protein complex is a major driving force in generating asymmetrically localized protein networks and consists of atypical protein kinase C (aPKC), Par3, and Par6. Dysfunction of this complex causes developmental abnormalities and diseases such as cancer. We identified a PDZ domain-binding motif in Par6 that was essential for its interaction with Par3 in vitro and for Par3-mediated membrane localization of Par6 in cultured cells. In fly embryos, we observed that the PDZ domain-binding motif was functionally redundant with the PDZ domain in targeting Par6 to the cortex of epithelial cells. Our structural analyses by x-ray crystallography and NMR spectroscopy showed that both the PDZ1 and PDZ3 domains but not the PDZ2 domain in Par3 engaged in a canonical interaction with the PDZ domain-binding motif in Par6. Par3 thus has the potential to recruit two Par6 proteins simultaneously, which may facilitate the assembly of polarity protein networks through multivalent PDZ domain interactions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Polaridade Celular , Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Domínios PDZ , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Cristalografia por Raios X , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Moleculares , Ligação ProteicaRESUMO
In epithelia, cells adhere to each other in a dynamic fashion, allowing the cells to change their shape and move along each other during morphogenesis. The regulation of adhesion occurs at the belt-shaped adherens junction, the zonula adherens (ZA). Formation of the ZA depends on components of the Par-atypical PKC (Par-aPKC) complex of polarity regulators. We have identified the Lin11, Isl-1, Mec-3 (LIM) protein Smallish (Smash), the orthologue of vertebrate LMO7, as a binding partner of Bazooka/Par-3 (Baz), a core component of the Par-aPKC complex. Smash also binds to Canoe/Afadin and the tyrosine kinase Src42A and localizes to the ZA in a planar polarized fashion. Animals lacking Smash show loss of planar cell polarity (PCP) in the embryonic epidermis and reduced cell bond tension, leading to severe defects during embryonic morphogenesis of epithelial tissues and organs. Overexpression of Smash causes apical constriction of epithelial cells. We propose that Smash is a key regulator of morphogenesis coordinating PCP and actomyosin contractility at the ZA.
Assuntos
Junções Aderentes/metabolismo , Proteínas de Drosophila/metabolismo , Epiderme/embriologia , Células Epiteliais/metabolismo , Morfogênese/fisiologia , Junções Aderentes/genética , Animais , Proteínas de Drosophila/genética , Drosophila melanogasterRESUMO
Methyl NMR spectroscopy is a powerful tool for studying protein structure, dynamics, and interactions. Yet difficulties with resonance assignment and the low abundance of methyl groups can preclude detailed NMR studies, particularly the determination of continuous interaction surfaces. Here we present a straightforward strategy that overcomes these problems. We systematically substituted solvent-exposed residues with reporter methionines in the expected binding site and performed chemical shift perturbation (CSP) experiments using methyl-TROSY spectra. We demonstrate the utility of this approach for the interaction between the HECT domain of the Rsp5p ubiquitin ligase and its cognate E2, Ubc4. Using these mutants, we could instantaneously assign all newly arising reporter methyl signals, determine the Ubc4 interaction surface on a per-residue basis, and investigate the importance of each individual mutation for ligand binding. Our data show that methionine scanning significantly extends the applicability, information content, and spatial resolution of methyl CSP experiments.
