RESUMEN
LACTB is a mammalian active-site serine protein that has evolved from a bacterial penicillin-binding protein. Penicillin-binding proteins are involved in the metabolism of peptidoglycan, the major bacterial cell wall constituent, implying that LACTB has been endowed with novel biochemical properties during eukaryote evolution. Here we demonstrate that LACTB is localized in the mitochondrial intermembrane space, where it is polymerized into stable filaments with a length extending more than a hundred nanometers. We infer that LACTB, through polymerization, promotes intramitochondrial membrane organization and micro-compartmentalization. These findings have implications for our understanding of mitochondrial evolution and function.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Proteínas de Unión a las Penicilinas/metabolismo , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Immunoblotting , Masculino , Espectrometría de Masas , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Datos de Secuencia Molecular , Plásmidos/genética , Polímeros/metabolismo , Ratas , Ratas WistarRESUMEN
BACKGROUND: Bacterial penicillin-binding proteins and beta-lactamases (PBP-betaLs) constitute a large family of serine proteases that perform essential functions in the synthesis and maintenance of peptidoglycan. Intriguingly, genes encoding PBP-betaL homologs occur in many metazoan genomes including humans. The emerging role of LACTB, a mammalian mitochondrial PBP-betaL homolog, in metabolic signaling prompted us to investigate the evolutionary history of metazoan PBP-betaL proteins. RESULTS: Metazoan PBP-betaL homologs including LACTB share unique structural features with bacterial class B low molecular weight penicillin-binding proteins. The amino acid residues necessary for enzymatic activity in bacterial PBP-betaL proteins, including the catalytic serine residue, are conserved in all metazoan homologs. Phylogenetic analysis indicated that metazoan PBP-betaL homologs comprise four alloparalogus protein lineages that derive from alpha-proteobacteria. CONCLUSION: While most components of the peptidoglycan synthesis machinery were dumped by early eukaryotes, a few PBP-betaL proteins were conserved and are found in metazoans including humans. Metazoan PBP-betaL homologs are active-site-serine enzymes that probably have distinct functions in the metabolic circuitry. We hypothesize that PBP-betaL proteins in the early eukaryotic cell enabled the degradation of peptidoglycan from ingested bacteria, thereby maximizing the yield of nutrients and streamlining the cell for effective phagocytotic feeding.
Asunto(s)
Bacterias/genética , Evolución Molecular , Proteínas de Unión a las Penicilinas/metabolismo , beta-Lactamasas/metabolismo , Secuencia de Aminoácidos , Animales , Bacterias/enzimología , Bacterias/metabolismo , Sitios de Unión , Dominio Catalítico , Secuencia Conservada , Humanos , Proteínas de la Membrana/genética , Proteínas Mitocondriales , Peso Molecular , Proteínas Ribosómicas/genética , Alineación de Secuencia , Homología de SecuenciaRESUMEN
LACTB is a mammalian mitochondrial protein sharing sequence similarity to the beta-lactamase/penicillin-binding protein family of serine proteases that are involved in bacterial cell wall metabolism. The physiological role of LACTB is unclear. In this study we have subcloned the cDNA of mouse LACTB (mLACTB) and produced recombinant mLACTB protein in Escherichia coli. When mLACTB was expressed as an N-terminal GST fusion protein (GST-mLACTB), full-length GST-mLACTB protein was recovered by glutathione-agarose affinity chromatography as determined by MALDI-TOF mass spectrometry and immunoblotting. Expression of mLACTB as a C-terminal GST fusion protein or with either an N- or C-terminal His6-tag resulted in proteolytic degradation of the protein and we were not able to detect full-length mLACTB. Analysis of GST-mLACTB by Fourier transform infrared spectrometry revealed the presence of alpha-helices, beta-sheets and turns, consistent with a well-defined secondary structure. These results show that mLACTB can be expressed as a GST fusion protein in E. coli and suggest that GST-mLACTB was properly folded.
