mTOR under stress

Het ‘mechanistic/ mammalian target of rapamycin complex 1’ (mTORC1) is een belangrijke metabole regulator die signalen van aminozuren, groeifactoren en energie integreert om het cel metabolisme te controleren. Het mTORC1 kinase versterkt vrijwel alle anabole processen en remt katabolisme, om celgroei te stimuleren. Gedurende stress bevordert mTORC1 ook cel overleving. Daarom is mTORC1 activiteit nodig om onder gunstige en ongunstige omstandigheden respectievelijk groei en overleving te faciliteren. Hyperactiviteit van mTORC1 resulteert echter in celdood doordat metabolisme en groei worden losgekoppeld van de regulatoire signalen. Vandaar dat mTORC1 activiteit nauwkeurig geregeld moet worden om (i) anabole processen en celgroei te bevorderen wanneer er voldoende voedingsstoffen aanwezig zijn, en (ii) anabole en katabole processen te balanceren om overleving van de cel bij stress mogelijk te maken. Terwijl de moleculaire mechanismen die anabole signalen naar mTORC1 mediëren uitgebreid beschreven zijn, zijn mechanistische studies over stress-signalering naar mTORC1 en mTORC1-gemedieerde modificatie van cel metabolisme relatief schaars. Dit proefschrift richt zich op de mechanismen die het evenwicht regelen van mTORC1 activiteit en functie gedurende stress. We identificeren nieuwe mechanismen die het evenwicht tussen anabole en catabole processen regelen om tijdens stress cel overleving te ondersteunen. Onze inzichten suggereren een belangrijke ‘Achilleshiel’, wat een therapeutisch doel kan dienen in de behandeling van kanker

Four-dimensional light shaping: manipulating ultrafast spatio-temporal foci in space and time

Spectral dispersion of ultrashort pulses allows simultaneous focusing of light in both space and time creating so-called spatio-temporal foci. Such space-time coupling may be combined with existing holographic techniques to give a further dimension of control when generating focal light fields. It is shown that a phase-only hologram placed in the pupil plane of an objective and illuminated by a spatially chirped ultrashort pulse can be used to generate three dimensional arrays of spatio-temporally focused spots. Exploiting the pulse front tilt generated at focus when applying simultaneous spatial and temporal focusing (SSTF), it is possible to overlap neighbouring foci in time to create a smooth intensity distribution. The resulting light field displays a high level of axial confinement, with experimental demonstrations given through two-photon microscopy and non-linear laser fabrication of glass

Finding new edges:systems approaches to MTOR signaling

Cells have evolved highly intertwined kinase networks to finely tune cellular homeostasis to the environment. The network converging on the mechanistic target of rapamycin (MTOR) kinase constitutes a central hub that integrates metabolic signals and adapts cellular metabolism and functions to nutritional changes and stress. Feedforward and feedback loops, crosstalks and a plethora of modulators finely balance MTOR-driven anabolic and catabolic processes. This complexity renders it difficult - if not impossible - to intuitively decipher signaling dynamics and network topology. Over the last two decades, systems approaches have emerged as powerful tools to simulate signaling network dynamics and responses. In this review, we discuss the contribution of systems studies to the discovery of novel edges and modulators in the MTOR network in healthy cells and in disease

Cardiac PI3K p110 alpha attenuation delays aging and extends lifespan

Phosphoinositide 3-kinase (PI3K) is a key component of the insulin signaling pathway that controls cellular me-tabolism and growth. Loss-of-function mutations in PI3K signaling and other downstream effectors of the insulin signaling pathway extend the lifespan of various model organisms. However, the pro-longevity effect appears to be sex-specific and young mice with reduced PI3K signaling have increased risk of cardiac disease. Hence, it remains elusive as to whether PI3K inhibition is a valid strategy to delay aging and extend healthspan in humans. We recently demonstrated that reduced PI3K activity in cardiomyocytes delays cardiac growth, causing subnormal contractility and cardiopulmonary functional capacity, as well as increased risk of mortality at young age. In stark contrast, in aged mice, experi-mental attenuation of PI3K signaling reduced the age-dependent decline in cardiac function and extended maximal lifespan, suggesting a biphasic effect of PI3K on cardiac health and survival. The cardiac anti-aging effects of reduced PI3K activity coincided with enhanced oxida-tive phosphorylation and required increased autophagic flux. In humans, explanted failing hearts showed in-creased PI3K signaling, as indicated by increased phos-phorylation of the serine/threonine-protein kinase AKT. Hence, late-life cardiac-specific targeting of PI3K might have a therapeutic potential in cardiac aging and related diseases

The Geometry of the Catalytic Active Site in [FeFe]-hydrogenases is Determined by Hydrogen Bonding and Proton Transfer

[FeFe]-hydrogenases are efficient metalloenzymes that catalyze the oxidation and evolution of molecular hydrogen, H2. They serve as a blueprint for the design of synthetic H2-forming catalysts. [FeFe]-hydrogenases harbor a six-iron cofactor that comprises a [4Fe-4S] cluster and a unique diiron site with cyanide, carbonyl, and hydride ligands. To address the ligand dynamics in catalytic turnover and upon carbon monoxide (CO) inhibition, we replaced the native aminodithiolate group of the diiron site by synthetic dithiolates, inserted into wild-type and amino acid variants of the [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii. The reactivity with H2 and CO was characterized using in situ and transient infrared spectroscopy, protein crystallography, quantum chemical calculations, and kinetic simulations. All cofactor variants adopted characteristic populations of reduced species in the presence of H2 and showed significant changes in CO inhibition and reactivation kinetics. Differences were attributed to varying interactions between polar ligands and the dithiolate head group and/or the environment of the cofactor (i.e., amino acid residues and water molecules). The presented results show how catalytically relevant intermediates are stabilized by inner-sphere hydrogen bonding suggesting that the role of the aminodithiolate group must not be restricted to proton transfer. These concepts may inspire the design of improved enzymes and biomimetic H2-forming catalysts

Isolation of a HypC–HypD complex carrying diatomic CO and CN− ligands

The HypC and HypD maturases are required for the biosynthesis of the Fe(CN)2CO cofactor in the large subunit of [NiFe]-hydrogenases. Using infrared spectroscopy we demonstrate that an anaerobically purified, Strep-tagged HypCD complex from Escherichia coli exhibits absorption bands characteristic of diatomic CO and CN− ligands as well as CO2. Metal and sulphide analyses revealed that along with the [4Fe–4S]2+ cluster in HypD, the complex has two additional oxygen-labile Fe ions. We prove that HypD cysteine 41 is required for the coordination of all three ligands. These findings suggest that the HypCD complex carries minimally the Fe(CN)2CO cofactor

The TSC Complex-mTORC1 Axis:From Lysosomes to Stress Granules and Back

The tuberous sclerosis protein complex (TSC complex) is a key integrator of metabolic signals and cellular stress. In response to nutrient shortage and stresses, the TSC complex inhibits the mechanistic target of rapamycin complex 1 (mTORC1) at the lysosomes. mTORC1 is also inhibited by stress granules (SGs), RNA-protein assemblies that dissociate mTORC1. The mechanisms of lysosome and SG recruitment of mTORC1 are well studied. In contrast, molecular details on lysosomal recruitment of the TSC complex have emerged only recently. The TSC complex subunit 1 (TSC1) binds lysosomes via phosphatidylinositol-3,5-bisphosphate [PI(3,5)P2]. The SG assembly factors 1 and 2 (G3BP1/2) have an unexpected lysosomal function in recruiting TSC2 when SGs are absent. In addition, high density lipoprotein binding protein (HDLBP, also named Vigilin) recruits TSC2 to SGs under stress. In this mini-review, we integrate the molecular mechanisms of lysosome and SG recruitment of the TSC complex. We discuss their interplay in the context of cell proliferation and migration in cancer and in the clinical manifestations of tuberous sclerosis complex disease (TSC) and lymphangioleiomyomatosis (LAM)