1617 Cells respond to external stimuli with transient gene expression changes in order to 18 adapt to environmental alterations. However, the dose response profile of gene 19 induction upon a given stress depends on many intrinsic and extrinsic factors. Here we 20show that the accurate quantification of dose dependent gene expression by live cell 21 luciferase reporters reveals fundamental insights into stress signaling. We make the 22 following discoveries applying this non-invasive reporter technology. (1) Signal 23 transduction sensitivities can be compared and we apply this here to salt, oxidative and 24 xenobiotic stress responsive transcription factors. (2) Stress signaling depends on where 25 and how the damage is generated within the cell. Specifically we show that two ROS-26 generating agents, menadione and hydrogen peroxide, differ in their dependence on 27 mitochondrial respiration. (3) Stress signaling is conditioned by the cells history. We 28 demonstrate here that positive memory or an acquired resistance towards oxidative 29 stress is induced dependent on the nature of the previous stress experience. (4) The 30 metabolic state of the cell impinges on the sensitivity of stress signaling. This is shown 31 here for the shift towards higher stress doses of the response profile for yeast cells 32 moved from complex to synthetic medium. (5) The age of the cell conditions its 33 transcriptional response capacity, which is demonstrated by the changes of the dose 34 response to oxidative stress during both replicative and chronological aging. We 35 conclude that capturing dose dependent gene expression in real time will be of 36 invaluable help to understand stress signaling and its dynamic modulation. 37 38 39 [16]. The yeast response to salt, nutrient or xenobiotic stress includes the simultaneous 57 activation of multiple, often structurally unrelated TFs [17,18,19]. Thus the use of 58 different transcriptional activators could create different gene expression patterns at 59 specific sets of target genes. Additionally, different TFs can form hierarchical networks 60 by regulatory connections between them [20,21,22], which makes it necessary to 61 determine the sensitivities of individual TFs. 62 Stress-activated TFs convert signals into a defined gene expression output by allowing 63RNAPII to engage in active transcription.Here, yet other regulatory mechanisms exist 64 to define the strength and timing of transcriptional activation. Active chromatin 65 remodeling is crucial for efficient stimulus-activated transcription. The nucleosome 66 structure of the inducible upstream region can determine the dynamics of the gene 67 expression at a given genomic locus, which has been reported for different stress and 68 developmental adaptations in yeast [23,24,25]. As a consequence, the response to 69 different stress doses might imply the contribution of distinct chromatin remodeling 70 complexes [26]. Finally the distribution of promoter binding sites and their affinity to 71 GRE2-luciferase reporter pAG413-pGRE...
The mitochondrial Complex I assembly (MCIA) complex is an essential player in the biogenesis of respiratory Complex I (CI), the multiprotein complex responsible for the initiation of oxidative phosphorylation (OXPHOS). It is not well understood how MCIA facilitates the assembly of CI. Here we report the structural basis of the complex formation between the MCIA subunits ECSIT and ACAD9. ECSIT binding induces a major conformational change in the FAD-binding loop of ACAD9, resulting in efflux of the FAD cofactor and redeployment of ACAD9 from fatty acid β-oxidation (FAO) to CI assembly. We identify an adjacent α-helix as a key structural element that specifically enables the CI assembly functionality of ACAD9, distinguishing it from its closely related VLCAD counterpart. Furthermore, we show that ECSIT is phosphorylated in vitro and ex cellulo and provide evidence that phosphorylation downregulates its association with ACAD9. Interestingly, ECSIT has previously been linked to the pathogenesis of Alzheimer's disease and here we show that ECSIT phosphorylation in neuronal cells is reduced upon exposure to amyloid-β (Aβ) oligomers. These findings shed light on the assembly of the MCIA complex and implicate ECSIT as a potential reprogrammer of bioenergetic metabolic pathways that can be altered when mitochondria are affected by Aβ toxicity, a hallmark of Alzheimer's disease.
The MAP kinase p38α is a central component of signalling in inflammation and the immune response, and is therefore an important drug target. Little is known about the molecular mechanism of its activation by double-phosphorylation from MAP2Ks, due to the challenge of trapping a transient and dynamic hetero-kinase complex. Here, we applied a multidisciplinary approach to generate the first structure of p38α in complex with its MAP2K MKK6 and understand the activation mechanism. Integrating cryo-EM with MD simulations and in cellulo experiments, we demonstrate a dynamic, multi-step, phosphorylation mechanism, reveal new catalytically relevant interactions, and show that MAP2K disordered N-termini determine pathway specificity. Our work captures, for the first time, a fundamental step of cell signalling: a kinase phosphorylating its downstream target kinase.
Controlled dehydration experiments have revealed a new crystal form of afamin, a human blood plasma glycoprotein and transporter of hydrophobic molecules. The comparison shows substantial molecular plasticity and amplifies the necessity to examine multiple crystal forms and to refine multiple models, while at the same time the new structure cautions against the interpretation of fatty-acid ligand density in crystals derived from PEGs as major precipitants. An isomorphic low-resolution structure model suggests that afamin is capable of transporting gadoteridol (Gd-DO3A), a magnetic resonance imaging compound.
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