19 research outputs found

    Caracterizaci贸n de la interacci贸n entre K-Ras y el dominio RBD de Raf-1 humanos

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    [EN] MAPK activation in mammals is a signal transduction pathway which permits cell response to a variety of stimuli. It regulates fundamental processes such as proliferation, differentiation, apoptosis and survival. Impairing this cascade could give rise to different pathologies, which makes MAPK pathway a promising therapeutic target. ERK cascade is one of the most known MAPK pathways, in which Ras and Raf signaling proteins participate. Ras is a small GTPase which functions as a nucleotide- dependent switch between GDP and GTP. Its binding to GTP gives rise to conformational changes which increase its affinity to Raf. Raf is a protein- serine/threonine kinase whose structure is formed by three conserved regions involved in the activation and deactivation of the protein and regulating signal transmission. Most studies of the interaction of Ras and Raf are based on H-Ras and Raf-1. However, mutated K-Ras results in a great incidence in cancer. Characterization of the manner in which K-Ras and Raf-1 interact could provide some insights in the development of new treatments against cancer. Cloning strategy by using recombiant vectors provided the correct tag for purification protocols. The expression system used for these fusion proteins was based on BL21 or BL21 codon plus strains of E.coli. K-Ras and Raf-1 purification by using both affinity chromatography and molecular size exclusion chromatography yielded significant protein levels for the experimental procedure described. Ras binding domain (RBD) involving residues 51 to 131 of Raf-1 was enough to interact with Ras. Binding characterization by using techniques such as pull down or isothermal titration calorimetry (ITC) verified the interaction between K-Ras and Raf-1. Future strategies will include crystallization series in order to structurally define the binding complex between these two proteins.[ES] La v铆a de activaci贸n de las MAPK en mam铆feros es una ruta de transducci贸n de se帽ale que permite a la c茅lula responder frente a un amplio rango de est铆mulos y dirigir procesos como la proliferaci贸n, diferenciaci贸n, apoptosis y supervivencia celular. La desregulaci贸n de esta ruta est谩 implicada en diferentes patolog铆as, lo que la convierte en una diana terap茅utica de gran inter茅s. Una de las v铆as MAPKs m谩s conocidas es la mediada por ERK, que se caracteriza por la intervenci贸n de dos prote铆na se帽alizadoras, Ras y Raf. Ras es un GTPasa peque帽a cuya funci贸n biol贸gica queda regulada a trav茅s de su uni贸n a un nucle贸tido guanina GDP o GTP, de forma que su uni贸n a GTP favorece que se produzca un cambio conformacional que incrementa su afinidad por Raf. Raf es una serina/treonina quinasa formada por tres dominios conservados, los cuales intervienen tanto en los procesos de activaci贸n e inactivaci贸n de la prote铆na como de transmisi贸n de la se帽al. La mayor铆a de los estudios de interacci贸n entre Ras y Raf descritos hasta el momento se basan en H-Ras y Raf-1. Sin emabrgo, la incidencia de c谩ncer cuando K-Ras se encuentra mutada es mayor, por lo que el estudio de su modo de uni贸n con Raf-1 resulta de gran relevancia en el desarrollo de nuevas estrategias de tratamiento. El clonaje en vectores recombinantes permiti贸 la adquisi贸n de etiquetas que facilitar铆an la purificaci贸n posterior. El sistema de expresi贸n utilizado para las prote铆nas de fusi贸n fueron las cepas BL21 o BL21 codon plus de E.coli. La purificaci贸n de K-Ras y Raf-1 por cromatograf铆a l铆quida de afinidad empleando columnas de n铆quel y, posteriormente, por exclusi贸n molecular mostr贸 unos niveles significativos de prote铆na para el sistema experimental descrito. El dominio de uni贸n a Ras (RBD) comprendido por los residuos 51 al 131 de Raf-1 fue suficiente para mediar su interacci贸n con Ras. La caracterizaci贸n del modo de uni贸n mediante t茅cnicas como pull-down o isothermal titration calorimetry (ITC) verificaron la interacci贸n entre K-Ras y Raf-1. Las estrategias futuras comprender谩n la disposici贸n de series de cristalizaci贸n que permitir谩n definir la estructura del complejo de interacci贸n.Valera Alberni Miriam (2014). Caracterizaci贸n de la interacci贸n entre K-Ras y el dominio RBD de Raf-1 humanos. http://hdl.handle.net/10251/40239.Archivo delegad

    Mitochondrial stress management: a dynamic journey

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    Mitochondria undergo continuous challenges in the course of their life, from their generation to their degradation. These challenges include the management of reactive oxygen species, the proper assembly of mitochondrial respiratory complexes and the need to balance potential mutations in the mitochondrial DNA. The detection of damage and the ability to keep it under control is critical to fine-tune mitochondrial function to the organismal energy needs. In this review, we will analyze the multiple mechanisms that safeguard mitochondrial function in light of in crescendo damage. This sequence of events will include initial defense against excessive reactive oxygen species production, compensation mechanisms by the unfolded protein response (UPRmt), mitochondrial dynamics and elimination by mitophagy

    Crosstalk between Drp1 phosphorylation sites during mitochondrial dynamics and metabolic adaptation

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    Mitochondria are highly dynamics organelles that undergo coordinated cycles of fission and fusion, referred to as mitochondrial dynamics. Mitochondrial dynamics regulate mitochondrial bioenergetics and allow cells to adapt to changing cellular stresses and physiological challenges. The balance between different GTPase enzymes dictates the shift from interconnected tubular networks to fragmented individual units. Among them, mitochondrial fission is regulated by the mitochondrial fission orchestrator, Drp1. Drp1 function is modulated by post-translational modifications, of which the phosphorylation at two specific sites have gained most attention. Drp1 phosphorylation at the S579 site results in Drp1 recruitment to mitochondria to promote fission, whereas the research on the role of the Drp1 S600 phosphorylation have yielded contradictory results on whether it promotes fission or fusion. Importantly, the crosstalk and physiological impact of these phosphorylations is poorly understood. In this project, we focused first on elucidating the interplay between Drp1 S600 and Drp1 S579 phosphorylations. We described how Drp1 S600 phosphorylation promotes S579 phosphorylation by protecting against its dephosphorylation. The activation at both S600 and S579 sites is required to, then, promote mitochondrial fragmentation. To explore the physiological relevance of these phosphorylations, we generated a Drp1 S600A knock-in (Drp1KI) mouse model. Drp1KI mice displayed enhanced lipid oxidation rates and respiratory capacity, granting improved glucose tolerance and thermogenic capacity upon high-fat feeding. Housing mice at thermoneutrality blunted these differences, suggesting a role for the brown adipose tissue in the protection of Drp1KI mice against metabolic damage. Therefore, this work unveils for the first time the crosstalk between Drp1 phosphorylation sites and their relationship to impact on mitochondrial architecture. Moreover, we demonstrate that targeting the Drp1 S600 site can grant protection against diet-induced insulin resistance, suggesting that the modulation of these phosphorylations could be used in the treatment and prevention of metabolic diseases

    Circadian Rhythms and Mitochondria: Connecting the Dots

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    Circadian rhythms provide a selective advantage by anticipating organismal nutrient needs and guaranteeing optimal metabolic capacity during active hours. Impairment of circadian rhythms is associated with increased risk of type 2 diabetes and emerging evidence suggests that metabolic diseases are linked to perturbed clock machinery. The circadian clock regulates many transcriptional鈥搕ranslational processes influencing whole cell metabolism and particularly mitochondrial activity. In this review, we survey the current literature related to cross-talks between mitochondria and the circadian clock and unravel putative molecular links. Understanding the mechanisms that link metabolism and circadian responses to transcriptional modifications will provide valuable insights toward innovative therapeutic strategies to combat the development of metabolic disease

    Circadian Rhythms and Mitochondria: Connecting the Dots

    No full text
    Circadian rhythms provide a selective advantage by anticipating organismal nutrient needs and guaranteeing optimal metabolic capacity during active hours. Impairment of circadian rhythms is associated with increased risk of type 2 diabetes and emerging evidence suggests that metabolic diseases are linked to perturbed clock machinery. The circadian clock regulates many transcriptional-translational processes influencing whole cell metabolism and particularly mitochondrial activity. In this review, we survey the current literature related to cross-talks between mitochondria and the circadian clock and unravel putative molecular links. Understanding the mechanisms that link metabolism and circadian responses to transcriptional modifications will provide valuable insights toward innovative therapeutic strategies to combat the development of metabolic disease

    Mfn2 is critical for brown adipose tissue thermogenic function

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    Mitochondrial fusion and fission events, collectively known as mitochondrial dynamics, act as quality control mechanisms to ensure mitochondrial function and fine-tune cellular bioenergetics. Defective mitofusin 2 (Mfn2) expression and enhanced mitochondrial fission in skeletal muscle are hallmarks of insulin-resistant states. Interestingly, Mfn2 is highly expressed in brown adipose tissue (BAT), yet its role remains unexplored. Using adipose-specific Mfn2 knockout (Mfn2-adKO) mice, we demonstrate that Mfn2, but not Mfn1, deficiency in BAT leads to a profound BAT dysfunction, associated with impaired respiratory capacity and a blunted response to adrenergic stimuli. Importantly, Mfn2 directly interacts with perilipin 1, facilitating the interaction between the mitochondria and the lipid droplet in response to adrenergic stimulation. Surprisingly, Mfn2-adKO mice were protected from high-fat diet-induced insulin resistance and hepatic steatosis. Altogether, these results demonstrate that Mfn2 is a mediator of mitochondria to lipid droplet interactions, influencing lipolytic processes and whole-body energy homeostasis

    Endogenous nicotinamide riboside metabolism protects against diet-induced liver damage

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    Supplementation with the NAD(+) precursor nicotinamide riboside (NR) ameliorates and prevents a broad array of metabolic and aging disorders in mice. However, little is known about the physiological role of endogenous NR metabolism. We have previously shown that NR kinase 1 (NRK1) is rate-limiting and essential for NR-induced NAD(+) synthesis in hepatic cells. To understand the relevance of hepatic NR metabolism, we generated whole body and liver-specific NRK1 knockout mice. Here, we show that NRK1 deficiency leads to decreased gluconeogenic potential and impaired mitochondrial function. Upon high-fat feeding, NRK1 deficient mice develop glucose intolerance, insulin resistance and hepatosteatosis. Furthermore, they are more susceptible to diet-induced liver DNA damage, due to compromised PARP1 activity. Our results demonstrate that endogenous NR metabolism is critical to sustain hepatic NAD(+) levels and hinder diet-induced metabolic damage, highlighting the relevance of NRK1 as a therapeutic target for metabolic disorders

    Crosstalk between Drp1 phosphorylation sites during mitochondrial remodeling and their impact on metabolic adaptation

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    Mitochondria constantly undergo fusion and fission events, referred as mitochondrial dynamics, which determine mitochondrial architecture and bioenergetics. Cultured cell studies demonstrate that mitochondrial dynamics are acutely regulated by phosphorylation of the mitochondrial fission orchestrator dynamin-related protein 1 (Drp1) at S579 or S600. However, the physiological impact and crosstalk of these phosphorylation sites is poorly understood. Here, we describe the functional interrelation between S579 and S600 phosphorylation sites in vivo and their role on mitochondrial remodeling. Mice carrying a homozygous Drp1 S600A knockin (Drp1 KI) mutation display larger mitochondria and enhanced lipid oxidation and respiratory capacities, granting improved glucose tolerance and thermogenic response upon high-fat feeding. Housing mice at thermoneutrality blunts these differences, suggesting a role for the brown adipose tissue in the protection of Drp1 KI mice against metabolic damage. Overall, we demonstrate crosstalk between Drp1 phosphorylation sites and provide evidence that their modulation could be used in the treatment and prevention of metabolic diseases
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