The acylation cycle

Innerhalb von Zellen ist die Verteilung von Proteinen häufig präzise, das bedeutet mit spezifischer Anreicherung in verschiedenen intrazellulären Kompartimenten. Als Ursache solcher Verteilungen wurden bereits Interaktionen, Rezeptoren oder auch Signalsequenzen beschrieben. In dieser Arbeit wird die Entstehung einer räumlich inhomogenen Proteinverteilung innerhalb von Zellen beschrieben – "Acylation Cycle" genannt - die auf einem Reaktions-Diffusions-Prozess basiert, welcher die reversible SPalmitoylierung umfasst. Palmitoylierung läuft am Golgi-Apparat ab, wobei die dort lokalisierten Enzyme kaum oder keine Spezifität für ein konkretes Proteinsubstrat zeigen. Anscheinend benötigen DHHC-Palmitoyltransferasen zur Palmitoylierung lediglich einen membrannahen Cysteinrest. Den gerichteten Transport zur Plasmamembran ermöglicht der sekretorische Weg. Der "Acylation Cycle" wirkt der entropie-getriebenen Homogenisierung palmitoylierter Proteine im Zellvolumen entgegen. Weiterhin wird die Aufenthaltsdauer palmitoylierter Proteine, wie z.B. Ras, an der Plasmamembran durch die Kinetik des "Acylation Cycle" beeinflusst, wodurch auch die Menge an Ras, die dem MAPK-Signalweg zur Verfügung steht, verändert wird. Das Unterbrechen des "Acylation Cycle" durch den neuartigen Inhibitor Palmostatin B bewirkt die durch Entropie getriebene Umverteilung von Ras. Durch Palmostatin B-Behandlung wird die Ras-Signalaktivität nach EGF Stimulierung an der Plasmamembran von der Aktivität am Golgi Apparat entkoppelt. Letztendlich führt die Palmostatin B-Behandlung von mit onkogenem HRasG12V transformierten MDCK Zellen zu einer Unterdrückung ihres konstitutiv-aktiven MAPKSignals, welches zur Zellteilung führen kann und verursacht so die Reversion des Phänotyps zu einem der untransformierten MDCK Zellen sehr ähnlichen. Der "Acylation Cycle" ist ein Phänomen, dem viele Proteine unterworfen sind und bietet daher die Gelegenheit die Modulation von Signalaktivitäten therapeutisch auszunutzen

Intravital Dynamic and Correlative Imaging of Mouse Livers Reveals Diffusion-Dominated Canalicular and Flow-Augmented Ductular Bile Flux

Background and Aims Small‐molecule flux in tissue microdomains is essential for organ function, but knowledge of this process is scant due to the lack of suitable methods. We developed two independent techniques that allow the quantification of advection (flow) and diffusion in individual bile canaliculi and in interlobular bile ducts of intact livers in living mice, namely fluorescence loss after photoactivation and intravital arbitrary region image correlation spectroscopy. Approach and Results The results challenge the prevailing “mechano‐osmotic” theory of canalicular bile flow. After active transport across hepatocyte membranes, bile acids are transported in the canaliculi primarily by diffusion. Only in the interlobular ducts is diffusion augmented by regulatable advection. Photoactivation of fluorescein bis‐(5‐carboxymethoxy‐2‐nitrobenzyl)‐ether in entire lobules demonstrated the establishment of diffusive gradients in the bile canalicular network and the sink function of interlobular ducts. In contrast to the bile canalicular network, vectorial transport was detected and quantified in the mesh of interlobular bile ducts. Conclusions The liver consists of a diffusion‐dominated canalicular domain, where hepatocytes secrete small molecules and generate a concentration gradient and a flow‐augmented ductular domain, where regulated water influx creates unidirectional advection that augments the diffusive flux

The Autodepalmitoylating activity of APT maintains the spatial organization of Palmitoylated membrane proteins

The localization and signaling of S-palmitoylated peripheral membrane proteins is sustained by an acylation cycle in which acyl protein thioesterases (APTs) depalmitoylate mislocalized palmitoylated proteins on endomembranes. However, the APTs are themselves reversibly S-palmitoylated, which localizes thioesterase activity to the site of the antagonistc palmitoylation activity on the Golgi. Here, we resolve this conundrum by showing that palmitoylation of APTs is labile due to autodepalmitoylation, creating two interconverting thioesterase pools: palmitoylated APT on the Golgi and depalmitoylated APT in the cytoplasm, with distinct functionality. By imaging APT-substrate catalytic intermediates, we show that it is the depalmitoylated soluble APT pool that depalmitoylates substrates on all membranes in the cell, thereby establishing its function as release factor of mislocalized palmitoylated proteins in the acylation cycle. The autodepalmitoylating activity on the Golgi constitutes a homeostatic regulation mechanism of APT levels at the Golgi that ensures robust partitioning of APT substrates between the plasma membrane and the Golgi.Fil: Vartak, Nachiket. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Papke, Bjoern. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Grecco, Hernan Edgardo. Institut Max Planck Fur Molekulare Physiologie; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rossmannek, Lisaweta. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Waldmann, Herbert. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Hedberg, Christian. Institut Max Planck Fur Molekulare Physiologie; AlemaniaFil: Bastiaens, Philippe I. H.. Institut Max Planck Fur Molekulare Physiologie; Alemani

Prediction of human drug-induced liver injury (DILI) in relation to oral doses and blood concentrations

Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity

Development of Acyl Protein Thioesterase 1 (APT1) Inhibitor Palmostatin B That Revert Unregulated H/N‐Ras Signaling

This chapter describes the combination of bioinformatics, organic synthesis, in vitro inhibition studies, and live-cell imaging (microscopy) to elucidate the function of acyl protein thioesterase 1 (APT1) in regulation of protein palmitoylation. APT1 critically influences the localization and function of several palmitoylated peripheral membrane proteins of the rat sarcoma (Ras) and Rous sarcoma oncogene cellular homolog (Src) family, which themselves have pivotal roles in cancer signaling. The chapter provides a case study in which protein structure similarity clustering (PSSC) was applied to find small-molecule inhibitors of the enzyme APT1. The inhibitors were applied in reverse chemical genetics investigations of Ras localization and signaling in cell-based studies. In order to determine the efficacy of palmostatin B to inhibit APT1 in cells, Fluorescence lifetime imaging microscopy (FLIM) was performed with TAMRA-labeled (tetramethyl-6-carboxyrhodamine) derivative of palmostatin B in cells expressing APT1-GFP

KRas is maintained on the plasma membrane by Arl2GTP-mediated release from PDEδ through the recycling endosome

<p>KRas is a major proto-oncogene product whose signaling activity is determined by its level of enrichment on the<br>plasma membrane (PM). This localization depends on post-translational farnesylation for membrane affinity,<br>while PM specificity is attributed to electrostatic interactions between negatively charged phospholipids in the<br>PM and basic amino-acid residues at the C-terminus of KRas. However, the cellular interior is filled with<br>endomembranes to which the farnesyl-tail of KRas imparts aspecific affinity. Considering the amount of potential<br>binding sites offered to the aspecific farnesyl moiety by the large and densely folded endomembranes, we<br>question if electrostatic interaction is sufficient to maintain the highly asymmetric distribution of KRas at the PM.<br>This electrostatic equilibration also would have to be strong enough to overcome non-equilibrium mixing<br>processes like endocytosis, which tend to redistribute KRas to endomembranes. Inspite of these, more than<br>80% of KRas is enriched on the plasma membrane at steady-state, leaving an open question with regard to how<br>such out-of-equlibrium enrichment is generated in living cells. We developed a 3D-cellular automaton(CA) that<br>models protein mobility and interactions in realistic cellular geometries. Informed with dynamic imaging data, the<br>CA simulation is used to explore the role of electrostatics and endocytosis, and discover a reaction-diffusion<br>cycle that counters equilibration to endomembranes and generate PM enrichment of KRas.</p> <p> </p

Spatial regulation of the acylation cycle by thioesterase auto-depalmitoylation

<p>The acylation cycle is a reaction diffusion mechanism that<br>maintains H/N-Ras localization on the plasma membrane (PM)<br>by countering the equilibration of palmitoylated H/N-Ras<br>proteins to endomembranes. The thioesterases APT1/2<br>perform this critical function of depalmitoylating mislocalized<br>H/N-Ras proteins, thereby allowing them to diffuse rapidly and<br>re-encounter the Golgi apparatus to be repalmitoylated.<br>However, the regulation of APT activity in the endomembranes,<br>Golgi and PM remains unknown. We utilize live-cell imaging to<br>show that APTs are themselves palmitoylated resulting in<br>rapidly interconverting, Golgi-localized and cytoplasmic soluble<br>fractions. By imaging the steady state distribution of APT-<br>substrate catalytic intermediates through Enzyme-Substrate<br>(ES) imaging, we decipher the distinct functions of these<br>fractions in the acylation cycle.</p> <p> </p

The ascending pathophysiology of cholestatic liver disease

In this review we develop the argument that cholestatic liver diseases, particularly primary biliary cholangitis and primary sclerosing cholangitis (PSC), evolve over time with anatomically an ascending course of the disease process. The first and early lesions are in "downstream" bile ducts. This eventually leads to cholestasis, and this causes bile salt (BS)-mediated toxic injury of the "upstream" liver parenchyma. BS are toxic in high concentration. These concentrations are present in the canalicular network, bile ducts, and gallbladder. Leakage of bile from this network and ducts could be an important driver of toxicity. The liver has a great capacity to adapt to cholestasis, and this may contribute to a variable symptom-poor interval that is often observed. Current trials with drugs that target BS toxicity are effective in only about 50%-60% of primary biliary cholangitis patients, with no effective therapy in PSC. This motivated us to develop and propose a new view on the pathophysiology of primary biliary cholangitis and PSC in the hope that these new drugs can be used more effectively. These views may lead to better stratification of these diseases and to recommendations on a more "tailored" use of the new therapeutic agents that are currently tested in clinical trials. Apical sodium- dependent BS transporter inhibitors that reduce intestinal BS absorption lower the BS load and are best used in cholestatic patients. The effectiveness of BS synthesis- suppressing drugs, such as farnesoid X receptor agonists, is greatest when optimal adaptation is not yet established. By the time cytochrome P450 7A1 expression is reduced these drugs may be less effective. Anti- inflammatory agents are probably most effective in early disease, while drugs that antagonize BS toxicity, such as ursodeoxycholic acid and nor- ursodeoxycholic acid, may be effective at all disease stages. Endoscopic stenting in PSC should be reserved for situations of intercurrent cholestasis and cholangitis, not for cholestasis in end- stage disease. These are arguments to consider a step- wise pathophysiology for these diseases, with therapy adjusted to disease stage. An obstacle in such an approach is that disease stage-defining biomarkers are still lacking. This review is meant to serve as a call to prioritize the development of biomarkers that help to obtain a better stratification of these diseases