RESUMEN
High-throughput screening has shown that Retro-1 inhibits ricin and Shiga toxins by diminishing their intracellular trafficking via the retrograde route, from early endosomes to the Golgi apparatus. To improve the activity of Retro-1, a structure-activity relationship (SAR) study was undertaken and yielded an analogue with a roughly 70-fold better half-maximal effective concentration (EC50) against Shiga toxin cytotoxicity measured in a cell protein synthesis assay.
Asunto(s)
Benzodiazepinonas/química , Benzodiazepinonas/farmacología , Toxinas Shiga/antagonistas & inhibidores , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Toxinas Shiga/metabolismo , Relación Estructura-ActividadRESUMEN
The bacterial cell wall peptidoglycan contains unusual l- and d-amino acids assembled as branched peptides. Insight into the biosynthesis of the polymer has been hampered by limited access to substrates and to suitable polymerization assays. Here we report the full synthesis of the peptide stem of peptidoglycan precursors from two pathogenic bacteria, Enterococcus faecium and Mycobacterium tuberculosis, and the development of a sensitive post-derivatization assay for their cross-linking by l,d-transpeptidases. Access to series of stem peptides showed that amidation of free carboxyl groups is essential for optimal enzyme activity, in particular the amidation of diaminopimelate (DAP) residues for the cross-linking activity of the l,d-transpeptidase LdtMt2 from M. tuberculosis. Accordingly, construction of a conditional mutant established the essential role of AsnB indicating that this DAP amidotransferase is an attractive target for the development of anti-mycobacterial drugs.
Asunto(s)
Enterococcus faecium/enzimología , Mycobacterium tuberculosis/enzimología , Peptidoglicano/biosíntesis , Peptidil Transferasas/metabolismo , Transaminasas/metabolismo , Pared Celular/metabolismo , Enterococcus faecium/química , Enterococcus faecium/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Peptidil Transferasas/efectos de los fármacos , beta-Lactamas/químicaRESUMEN
Hypomorphic mutations in the X-linked human NEMO gene result in various forms of anhidrotic ectodermal dysplasia with immunodeficiency. NEMO function is mediated by two distal ubiquitin binding domains located in the regulatory C-terminal domain of the protein: the coiled-coil 2-leucine zipper (CC2-LZ) domain and the zinc finger (ZF) domain. Here, we investigated the effect of the D406V mutation found in the NEMO ZF of an ectodermal dysplasia with immunodeficiency patients. This point mutation does not impair the folding of NEMO ZF or mono-ubiquitin binding but is sufficient to alter NEMO function, as NEMO-deficient fibroblasts and Jurkat T lymphocytes reconstituted with full-length D406V NEMO lead to partial and strong defects in NF-κB activation, respectively. To further characterize the ubiquitin binding properties of NEMO ZF, we employed di-ubiquitin (di-Ub) chains composed of several different linkages (Lys-48, Lys-63, and linear (Met-1-linked)). We showed that the pathogenic mutation preferentially impairs the interaction with Lys-63 and Met-1-linked di-Ub, which correlates with its ubiquitin binding defect in vivo. Furthermore, sedimentation velocity and gel filtration showed that NEMO ZF, like other NEMO related-ZFs, binds mono-Ub and di-Ub with distinct stoichiometries, indicating the presence of a new Ub site within the NEMO ZF. Extensive mutagenesis was then performed on NEMO ZF and characterization of mutants allowed the proposal of a structural model of NEMO ZF in interaction with a Lys-63 di-Ub chain.
Asunto(s)
Displasia Ectodérmica/metabolismo , Quinasa I-kappa B/metabolismo , Síndromes de Inmunodeficiencia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mutación Missense , FN-kappa B/metabolismo , Ubiquitina/metabolismo , Sustitución de Aminoácidos , Animales , Displasia Ectodérmica/genética , Humanos , Quinasa I-kappa B/química , Quinasa I-kappa B/genética , Síndromes de Inmunodeficiencia/genética , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Células Jurkat , Ratones , Ratones Mutantes , Modelos Moleculares , FN-kappa B/química , FN-kappa B/genética , Unión Proteica/genética , Estructura Terciaria de Proteína , Ubiquitina/genética , Dedos de ZincRESUMEN
Nuclear factor-κB essential modulator (NEMO), the regulatory subunit of the IκB kinase complex, is a critical component of the NF-κB pathway. Hypomorphic mutations in the X-linked human NEMO gene cause various forms of anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID). All known X-linked EDA-ID-causing mutations impair NEMO protein expression, folding, or both. We describe here 2 EDA-ID-causing missense mutations that affect the same residue in the CC2-LZ domain (D311N and D311G) that do not impair NEMO production or folding. Structural studies based on pull-down experiments showed a defect in noncovalent interaction with K63-linked and linear polyubiquitin chains for these mutant proteins. Functional studies on the patients' cells showed an impairment of the classic NF-κB signaling pathways after activation of 2 NEMO ubiquitin-binding-dependent receptors, the TNF and IL-1ß receptors, and in the CD40-dependent NF-κB pathway. We report the first human NEMO mutations responsible for X-linked EDA-ID found to affect the polyubiquitin binding of NEMO rather than its expression and folding. These experiments demonstrate that the binding of human NEMO to polyubiquitin is essential for NF-κB activation. They also demonstrate that the normal expression and folding of NEMO do not exclude a pathogenic role for NEMO mutations in patients with EDA-ID.
