Mono-ubiquitination of phosphoinositide-dependent-kinase 1 (PDK1)

Durch die Aktivität der zentralen Effektorkinase AKT (PKB) spielt der PI3K/AKT Singalweg eine wichtige Rolle bei der Transduktion von anti-apoptotischen Signalen in Zellteilung und Zellproliferation. Weiters besteht ein Zusammenhang zwischen der Deregulation des PI3K/AKT Signalwegs und dem Auftreten zahlreicher maligner Erkrankungen wie Brust- und Prostatakrebs und der Entstehung von Glioblastomen. Dies macht die Komponenten dieses Signaltransduktionsweges zu wichtigen therapeutischen Zielen. Die Aktivierung von AKT ist abhängig von der Kinase phosphoinositide-dependent kinase 1 (PDK1), welche AKT unter Mitwirkung von PIP3 aktiviert, und so als essentieller Hauptaktivator für AKT fungiert. PDK1 aktiviert auch weitere downstream Proteine der AGC Familie, welche eine wichtige Rolle für Metabolismus und Translation spielen. Es ist etabliert, dass PDK1 konstitutiv aktiv ist und Wachstumsfaktor- und Insulinsignaltransduktion indirekt die Fähigkeit von PDK1 beinflussen Substrate zu binden. Die Regulation von PDK1 ist jedoch nicht zur Gänze aufgeklärt. Weiters hat PDK1 im Laufe der letzten Jahre als therapeutisches Zielprotein an Popularität gewonnen und mehrere Inhibitoren befinden sich in klinischer Testphase. Erstmals wird mit der vorliegenden Arbeit gezeigt, dass PDK1 monoubiquitiniert ist. Zudem sollen die Position, die Rolle und die molekularen Antagonisten dieser posttranslationalen Modifikation charakterisiert werden. Ubiquitin ist bekannt für seine Rolle in proteosomaler Degradation, jedoch können verschiedene Typen von konjugierten Ubiquitinepitopen auch Lokalisation, Aktivität und Konformation von ubiqutinierten Proteinen beeinflussen. Die Chrakaterisierung der PDK1 Monoubiquitinierung verspricht neue Einsicht in die Regulationsmechanismen von PDK1 in normalen und transformierten Zellen zu gewinnen. Weiters wird das Ubiquitinsystem zunehmend als therapeutisches Ziel betrachtet, da es auf enzymatischen Reaktionen basiert, die potentiell durch niedermolekulare Substanzen inhibiert werden können. Dies eröffnet die Möglichkeit Monoubiquitinierung als alternativen Winkel auszunutzen um den PI3K/AKT Signalweg über PDK1 zu inhibieren. Dieses Projekt versucht die Position der ubiqutinierten Aminosäure in PDK1 durch Mutagenese, Truncations, und Massenspektrometrie zu identifizieren. Obzwar die genaue Position des ubiquitinierten Aminosäurerests nicht definiert werden konnte, konnte die Monoubiquitinierungsstelle der Kinasendomäne zugeordnet werden. Zudem wurde versucht durch Experimente zu intrazellulärer Lokalisation, katalytischer Aktivität und PIP3 Bindung, die Funktion der Monoubiquitinierung zu ergründen. Den hier beschriebenen Resultaten zufolge ist auszuschließen, dass PDK1 Monoubiquitinierung PIP3 Bindung beeinflusst und dass diese keine 99 Vorraussetzung für die Monoubiquitinierung von PDK1 ist. Die Position von PDK1 und seine Fähigkeit AKT zu phosphorylieren waren auch nicht von Monoubiquitinierung abhängig. Letztlich haben wir einen „Loss of function“ RNA-Interferenzscreen durchgeführt um eine PDK1 spezifische Deubiquitinase (DUB) zu identifizieren. Es wurde allerdings keine DUB gefunden. Obwohl die Charakterisierung der Monoubiquitinierung weitere Arbeit verlangen wird, haben wir die erste post-translationale Modifikation von PDK1 durch Ubiquitin beschrieben.The PI3K/AKT signaling pathway mediates anti-apoptotic signals and drives cell growth and proliferation via the activity of its central effector kinase AKT (PKB). Deregulation of the PI3K/AKT pathway has been linked to a large number of human malignancies such as glioblastoma, breast and prostate cancer, which makes its components interesting targets for therapy. Activation of AKT depends on the action of phosphoinositide-dependent kinase 1 (PDK1), which phosphorylates and activates AKT in a PIP3 dependent manner, and thus acts as an essential master activator of the pathway. PDK1 also activates other downstream targets of the AGC kinase family that drive metabolism and translation. It is established that PDK1 is constitutively active and that insulin and growth factor signaling indirectly affect PDK1's ability to bind its substrates, yet its regulation is incompletely understood. Furthermore PDK1 has gained popularity as a therapeutic target over the last years, and several inhibitors have been identified which are currently being tested in clinical trials. Here we show for the first time that PDK1 is mono-ubiquitinated and have attempted to elucidate the site, role and molecular antagonists of this post translational modification. Ubiquitination is known for its role in proteasomal degradation, but depending on the type of conjugated ubiquitin epitope, can also influence localization, activity and conformation of the ubiquitinated protein. Identifying the role of PDK1 mono-ubiquitination may give novel mechanistic insight into PDK1 regulation in normal and transformed cells. Additionally, the ubiquitin system is increasingly considered as a therapeutic target because it involves enzymatic reactions which can potentially be inhibited with low molecular compounds. This raises the possibility that mono-ubiquitination may be exploited as an alternative angle to target the PI3K/AKT pathway via PDK1 in cancer. In this project we have attempted to map the ubiquitinated lysine residue by mutagenesis, truncation experiments and mass spectrometry. We were able to map the ubiquitination site to the kinase domain, but finding the specific ubiquitinated residue will require further efforts. We have also attempted to identify the function of this post-translational modification by investigating localization, catalytic activity and PIP3 binding. We were able to exclude that PDK1 mono-ubiquitination affects PIP3 binding and that it is dependent on it. Our data also suggests that localization and PDK1’s ability to phosphorylate AKT are not affected by mono-ubiquitin. Finally an RNAi loss of function screen was performed to identify a de-ubiquitinase (DUB), but no DUB was found. Although our efforts to characterize PDK1 mono-ubiquitination will require further work, we have described the first post-translational modification of PDK1 by ubiquitin

Physical linkages between an offshore canyon and surf zone morphologic change

Author Posting. © American Geophysical Union, 2017. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Journal of Geophysical Research: Oceans 122 (2017): 3451–3460, doi:10.1002/2016JC012319.The causes of surf zone morphologic changes observed along a sandy beach onshore of a submarine canyon were investigated using field observations and a numerical model (Delft3D/SWAN). Numerically simulated morphologic changes using four different sediment transport formulae reproduce the temporal and spatial patterns of net cross-shore integrated (between 0 and 6.5 m water depths) accretion and erosion observed in a ∼300 m alongshore region, a few hundred meters from the canyon head. The observations and simulations indicate that the accretion or erosion results from converging or diverging alongshore currents driven primarily by breaking waves and alongshore pressure gradients. The location of convergence or divergence depends on the direction of the offshore waves that refract over the canyon, suggesting that bathymetric features on the inner shelf can have first-order effects on short-term nearshore morphologic change.WHOI-USGS postdoctoral scholarship, NSF, ONR2017-10-2

CLICS² An Improved Database of Cross-Linguistic Colexifications : Assembling Lexical Data with the Help of Cross-Linguistic Data Formats

International audienceThe Database of Cross-Linguistic Colexifications (CLICS), has established a computer-assisted framework for the interactive representation of cross-linguistic colexification patterns. In its current form, it has proven to be a useful tool for various kinds of investigation into cross-linguistic semantic associations , ranging from studies on semantic change, patterns of conceptualization, and linguistic pale-ontology. But CLICS has also been criticized for obvious shortcomings, ranging from the underlying dataset, which still contains many errors, up to the limits of cross-linguistic colexification studies in general. Building on recent standardization efforts reflected in the Cross-Linguistic Data Formats initiative (CLDF) and novel approaches for fast, efficient, and reliable data aggregation, we have created a new database for cross-linguistic colexifications, which not only supersedes the original CLICS database in terms of coverage but also offers a much more principled procedure for the creation, curation and aggregation of datasets. The paper presents the new database and discusses its major features

Incremental nonlinear dynamic data reconciliation

Measurement noise reduction and parameter estimation is a topic of central importance in plant control. The complexity of real world plants and the working conditions in practice require robust real-time algorithms which are easy to implement, simple to use and economic in computer ressources. The state of the art is given by the novel approach of Liebman et al. called the NDDR (nonlinear dynamic data reconciliation) which is based on nonlinear dynamic programming. We present in the present paper a new algorithm based more traditionally on gradient descent methods supplemented with a self control of the parameters of the algorithm. It uses an iterative method for the rectification and correction of state variables and system parameters, what makes it a true on-line algorithm. Despite its simplicity, the perfomance of the new algorithm proved superior to that of the NDDR in the applications considered so far

Matriptase regulates c-Met mediated proliferation and invasion in inflammatory breast cancer.

The poor prognosis for patients with inflammatory breast cancer (IBC) compared to patients with other types of breast cancers emphasizes the need to better understand the molecular underpinnings of this disease with the goal of developing effective targeted therapeutics. Dysregulation of matriptase expression, an epithelial-specific member of the type II transmembrane serine protease family, has been demonstrated in many different cancer types. To date, no studies have assessed the expression and potential pro-oncogenic role of matriptase in IBC. We examined the functional relationship between matriptase and the HGF/c-MET signaling pathway in the IBC cell lines SUM149 and SUM190, and in IBC patient samples. Matriptase and c-Met proteins are localized on the surface membrane of IBC cells and their expression is strongly correlated in infiltrating cancer cells and in the cancer cells of lymphatic emboli in patient samples. Abrogation of matriptase expression by silencing with RNAi or inhibition of matriptase proteolytic activity with a synthetic inhibitor impairs the conversion of inactive pro-HGF to active HGF and subsequent c-Met-mediated signaling, leading to efficient impairment of proliferation and invasion of IBC cells. These data show the potential of matriptase inhibitors as a novel targeted therapy for IBC, and lay the groundwork for the development and testing of such drugs

Team reasoning and intentional cooperation for mutual benefit

This paper proposes a concept of intentional cooperation for mutual benefit. This concept uses a form of team reasoning in which team members aim to achieve common interests, rather than maximising a common utility function, and in which team reasoners can coordinate their behaviour by following pre-existing practices. I argue that a market transaction can express intentions for mutually beneficial cooperation even if, extensionally, participation in the transaction promotes each party’s self-interest

Efficient Sample Tracking With OpenLabFramework

The advance of new technologies in biomedical research has led to a dramatic growth in experimental throughput. Projects therefore steadily grow in size and involve a larger number of researchers. Spreadsheets traditionally used are thus no longer suitable for keeping track of the vast amounts of samples created and need to be replaced with state-of-the-art laboratory information management systems. Such systems have been developed in large numbers, but they are often limited to specific research domains and types of data. One domain so far neglected is the management of libraries of vector clones and genetically engineered cell lines. OpenLabFramework is a newly developed web-application for sample tracking, particularly laid out to fill this gap, but with an open architecture allowing it to be extended for other biological materials and functional data. Its sample tracking mechanism is fully customizable and aids productivity further through support for mobile devices and barcoded labels

Regulating craving by anticipating positive and negative outcomes : a multivariate pattern analysis and network connectivity approach

During self-control, we may resist short-term temptations in order to reach a favorable future (e.g., resisting cake to stay healthy). The neural basis of self-control is typically attributed to “cold,” unemotional cognitive control mechanisms which inhibit affect-related regions via the prefrontal cortex (PFC). Here, we investigate the neural underpinnings of regulating cravings by mentally evoking the positive consequences of resisting a temptation (e.g., being healthy) as opposed to evoking the negative consequences of giving in to a temptation (e.g., becoming overweight). It is conceivable that when using these types of strategies, regions associated with emotional processing [e.g., striatum, ventromedial prefrontal cortex (vmPFC)] are involved in addition to control-related prefrontal and parietal regions. Thirty-one participants saw pictures of unhealthy snacks in the fMRI scanner and, depending on the trial, regulated their craving by thinking of the positive consequences of resisting, or the negative consequences of not resisting. In a control condition, they anticipated the pleasure of eating and thus, allowed the craving to occur (now-condition). In line with previous studies, we found activation of a cognitive control network during self-regulation. In the negative future thinking condition, the insula was more active than in the positive condition, while there were no activations that were stronger in the positive (> negative) future thinking condition. However, additionally, multivariate pattern analysis showed that during craving regulation, information about the valence of anticipated emotions was present in the vmPFC, the posterior cingulate cortex (PCC) and the insula. Moreover, a network including vmPFC and PCC showed higher connectivity during the positive (> negative) future thinking condition. Since these regions are often associated with affective processing, these findings suggest that “hot,” affective processes may, at least in certain circumstances, play a role in self-control