RESUMO
BCL-2-associated X protein (BAX) is a promising therapeutic target for activating or restraining apoptosis in diseases of pathologic cell survival or cell death, respectively. In response to cellular stress, BAX transforms from a quiescent cytosolic monomer into a toxic oligomer that permeabilizes the mitochondria, releasing key apoptogenic factors. The mitochondrial lipid trans-2-hexadecenal (t-2-hex) sensitizes BAX activation by covalent derivatization of cysteine 126 (C126). In this study, we performed a disulfide tethering screen to discover C126-reactive molecules that modulate BAX activity. We identified covalent BAX inhibitor 1 (CBI1) as a compound that selectively derivatizes BAX at C126 and inhibits BAX activation by triggering ligands or point mutagenesis. Biochemical and structural analyses revealed that CBI1 can inhibit BAX by a dual mechanism of action: conformational constraint and competitive blockade of lipidation. These data inform a pharmacologic strategy for suppressing apoptosis in diseases of unwanted cell death by covalent targeting of BAX C126.
Assuntos
Apoptose , Proteína X Associada a bcl-2 , Proteína X Associada a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Humanos , Apoptose/efeitos dos fármacos , Cisteína/química , Cisteína/metabolismo , Animais , Aldeídos/química , Aldeídos/farmacologia , Modelos Moleculares , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismoRESUMO
BCL-2 family proteins converge at the mitochondrial outer membrane to regulate apoptosis and maintain the critical balance between cellular life and death. This physiologic process is essential to organism homeostasis and relies on protein-protein and protein-lipid interactions among BCL-2 family proteins in the mitochondrial lipid environment. Here, we find that trans-2-hexadecenal (t-2-hex), previously implicated in regulating BAX-mediated apoptosis, does so by direct covalent reaction with C126, which is located on the surface of BAX at the junction of its α5/α6 core hydrophobic hairpin. The application of nuclear magnetic resonance spectroscopy, hydrogen-deuterium exchange mass spectrometry, specialized t-2-hex-containing liposomes, and BAX mutational studies in mitochondria and cells reveals structure-function insights into the mechanism by which covalent lipidation at the mitochondria sensitizes direct BAX activation. The functional role of BAX lipidation as a control point of mitochondrial apoptosis could provide a therapeutic strategy for BAX modulation by chemical modification of C126.
Assuntos
Apoptose , Cisteína/metabolismo , Lipídeos/química , Proteína X Associada a bcl-2/metabolismo , Aldeídos , Animais , Humanos , Lipossomos , Espectroscopia de Ressonância Magnética , Membranas Artificiais , Camundongos , Mitocôndrias/metabolismo , Conformação Proteica , Multimerização Proteica , Proteínas Recombinantes/metabolismo , Proteína X Associada a bcl-2/químicaRESUMO
A cytochrome P450 was engineered to selectively incorporate Ir(Me)-deuteroporphyrinâ IX (Ir(Me)-DPIX), in lieu of heme, in bacterial cells. Cofactor selectivity was altered by introducing mutations within the heme-binding pocket to discriminate the deuteroporphyrin macrocycle, in combination with mutations to the P450 axial cysteine to accommodate a pendant methyl group on the Ir(Me) center. This artificial metalloenzyme was investigated for activity in non-native metallocarbenoid-mediated olefin cyclopropanation reactions and showed enhanced activity for aliphatic and electron-deficient olefins when compared to the native heme enzyme. This work provides a general strategy to augment the chemical functionality of heme enzymes in cells with application towards abiotic catalysis.
Assuntos
Biocatálise , Metaloproteínas/síntese química , Engenharia de Proteínas , Bactérias/citologia , Sistema Enzimático do Citocromo P-450/química , Deutério , Heme/química , Irídio/química , Porfirinas/químicaRESUMO
We introduce a strategy that expands the functionality of hemoproteins through orthogonal enzyme/heme pairs. By exploiting the ability of a natural heme transport protein, ChuA, to promiscuously import heme derivatives, we have evolved a cytochrome P450 (P450BM3) that selectively incorporates a nonproteinogenic cofactor, iron deuteroporphyrin IX (Fe-DPIX), even in the presence of endogenous heme. Crystal structures show that selectivity gains are due to mutations that introduce steric clash with the heme vinyl groups while providing a complementary binding surface for the smaller Fe-DPIX cofactor. Furthermore, the evolved orthogonal enzyme/cofactor pair is active in non-natural carbenoid-mediated olefin cyclopropanation. This methodology for the generation of orthogonal enzyme/cofactor pairs promises to expand cofactor diversity in artificial metalloenzymes.