RESUMEN
The ATPase Associated with diverse cellular Activities (AAA+) family of proteases play crucial roles in cellular proteolysis and stress responses. Like other AAA+ proteases, the Lon protease is known to be allosterically regulated by nucleotide and substrate binding. Although it was originally classified as a DNA binding protein, the impact of DNA binding on Lon activity is unclear. In this study, we characterize the regulation of Lon by single-stranded DNA (ssDNA) binding and serendipitously identify general activation strategies for Lon. Upon binding to ssDNA, Lon's ATP hydrolysis rate increases due to improved nucleotide binding, leading to enhanced degradation of protein substrates, including physiologically important targets. We demonstrate that mutations in basic residues that are crucial for Lon's DNA binding not only reduces ssDNA binding but result in charge-specific consequences on Lon activity. Introducing negative charge at these sites induces activation akin to that induced by ssDNA binding, whereas neutralizing the charge reduces Lon's activity. Based on single molecule measurements we find that this change in activity is correlated with changes in Lon oligomerization. Our study provides insights into the complex regulation of the Lon protease driven by electrostatic contributions from either DNA binding or mutations.
RESUMEN
The LonA (or Lon) protease is a central post-translational regulator in diverse bacterial species. In Vibrio cholerae, LonA regulates a broad range of behaviors including cell division, biofilm formation, flagellar motility, c-di-GMP levels, the type VI secretion system (T6SS), virulence gene expression, and host colonization. Despite LonA's role in cellular processes critical for V. cholerae's aquatic and infectious life cycles, relatively few LonA substrates have been identified. LonA protease substrates were therefore identified through comparison of the proteomes of wild-type and ΔlonA strains following translational inhibition. The most significantly enriched LonA-dependent protein was TfoY, a known regulator of motility and the T6SS in V. cholerae. Experiments showed that TfoY was required for LonA-mediated repression of motility and T6SS-dependent killing. In addition, TfoY was stabilized under high c-di-GMP conditions and biochemical analysis determined direct binding of c-di-GMP to LonA results in inhibition of its protease activity. The work presented here adds to the list of LonA substrates, identifies LonA as a c-di-GMP receptor, demonstrates that c-di-GMP regulates LonA activity and TfoY protein stability, and helps elucidate the mechanisms by which LonA controls important V. cholerae behaviors.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Cólera/microbiología , GMP Cíclico/análogos & derivados , Proteasa La/antagonistas & inhibidores , Vibrio cholerae/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Mutación , Proteasa La/genética , Proteasa La/aislamiento & purificación , Proteasa La/metabolismo , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Proteolisis , Proteómica , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Sistemas de Secreción Tipo VI/genética , Sistemas de Secreción Tipo VI/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/patogenicidad , Virulencia/genéticaRESUMEN
Day length regulates the effects of gonadal steroids on gonadotropin secretion and behavior in seasonal breeders. To determine whether this influence of photoperiod results from changes in androgen receptor expression in Siberian hamster brain regions that regulate neuroendocrine function, androgen receptor immunostaining was examined in castrated animals given either no androgen replacement or one of three doses of testosterone (T) resulting in physiological serum concentrations. Half of the animals were housed under inhibitory photoperiod conditions, and immunostaining was quantified 11 days later. Measurement of serum gonadotropin and prolactin concentrations confirmed that androgen exerted graded effects on pituitary function but that the animals were killed before photoperiodic influences had fully developed. T significantly increased the numbers of androgen receptor-immunoreactive cells in every brain region examined. Photoperiod exerted no significant influence on androgen receptor-immunoreactive cell number in the arcuate nucleus, bed nucleus of the stria terminalis (BNST), medial preoptic nucleus, or in medial amygdala. An interaction between T and photoperiod was observed in the BNST and in the rostral and middle portions of the arcuate nucleus. Although increasing concentrations of T resulted in more intense cellular immunostaining in the BNST and arcuate, this effect was not influenced by day length. These results indicate that relatively short-duration (11 days) exposure to inhibitory photoperiod triggers localized and regionally specific changes in androgen receptor expression.
Asunto(s)
Fotoperiodo , Prosencéfalo/efectos de la radiación , Receptores Androgénicos/metabolismo , Testículo/efectos de la radiación , Testosterona/fisiología , Análisis de Varianza , Animales , Castración , Ritmo Circadiano , Cricetinae , Hormona Folículo Estimulante/sangre , Inmunohistoquímica , Iluminación , Hormona Luteinizante/sangre , Masculino , Sistemas Neurosecretores/metabolismo , Sistemas Neurosecretores/efectos de la radiación , Phodopus , Prolactina/sangre , Prosencéfalo/metabolismo , Receptores Androgénicos/efectos de la radiación , Reproducción/fisiología , Testículo/fisiologíaRESUMEN
In Drosophila melanogaster, apoptosis is controlled by the integrated actions of the Grim-Reaper (Grim-Rpr) and Drosophila Inhibitor of Apoptosis (DIAP) proteins (reviewed in refs 1 4). The anti-apoptotic DIAPs bind to caspases and inhibit their proteolytic activities. DIAPs also bind to Grim-Rpr proteins, an interaction that promotes caspase activity and the initiation of apoptosis. Using a genetic modifier screen, we identified four enhancers of grim-reaper-induced apoptosis that all regulate ubiquitination processes: uba-1, skpA, fat facets (faf), and morgue. Strikingly, morgue encodes a unique protein that contains both an F box and a ubiquitin E2 conjugase domain that lacks the active site Cys required for ubiquitin linkage. A reduction of morgue activity suppressed grim-reaper-induced cell death in Drosophila. In cultured cells, Morgue induced apoptosis that was suppressed by DIAP1. Targeted morgue expression downregulated DIAP1 levels in Drosophila tissue, and Morgue and Rpr together downregulated DIAP1 levels in cultured cells. Consistent with potential substrate binding functions in an SCF ubiquitin E3 ligase complex, Morgue exhibited F box-dependent association with SkpA and F box-independent association with DIAP1. Morgue may thus have a key function in apoptosis by targeting DIAP1 for ubiquitination and turnover.
Asunto(s)
Apoptosis/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Ligasas/química , Neuropéptidos/metabolismo , Péptidos/metabolismo , Proteínas Ligasas SKP Cullina F-box , Secuencia de Aminoácidos , Animales , Células Cultivadas , Drosophila , Proteínas de Drosophila/química , Proteínas del Ojo/química , Regulación Enzimológica de la Expresión Génica , Proteínas Inhibidoras de la Apoptosis , Proteínas de Insectos/metabolismo , Ligasas/genética , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Ubiquitina/metabolismo , Enzimas Ubiquitina-ConjugadorasRESUMEN
The Drosophila genes reaper, head involution defective (hid), and grim all reside at 75C on chromosome three and encode related proteins that have crucial functions in programmed cell death (reviewed in ). In this report, we describe a novel grim-reaper gene, termed sickle, that resides adjacent to reaper. The sickle gene, like reaper and grim, encodes a small protein which contains an RHG motif and a Trp-block. In wild-type embryos, sickle expression was detected in cells of the developing central nervous system. Unlike reaper, hid, and grim, the sickle gene is not removed by Df(3L)H99, and strong ectopic sickle expression was detected in the nervous system of this cell death mutant. sickle very effectively induced cell death in cultured Spodoptera Sf-9 cells, and this death was antagonized by the caspase inhibitors p35 or DIAP1. Strikingly, unlike the other grim-reaper genes, targeted sickle expression did not induce cell death in the Drosophila eye. However, sickle strongly enhanced the eye cell death induced by expression of either an r/grim chimera or reaper.