The role of lipids in Wnt secretion and function

Wnt signaling pathways are a set of signal transduction cascades which are activated through the interaction of Wnt proteins with so-called Frizzled receptors [1-3]. These pathways are critically involved in many biological processes such as embryonic development, regeneration, organogenesis, cell division, cellular and tissue homeostasis, among many others [3, 4]. In addition, alterations of these signaling pathways have been linked to various types of diseases such as cancer [5-7], familial tooth agenesis [8], bipolar disease [9], Alzheimer's disease [10], and cardiac valve formation [11]. Wnt signaling components are accordingly promising drug targets to treat these diseases. Wnt pathways are probably among the best characterized receptor-ligand signaling pathways. Wnt proteins are therefore key players in biological signaling and promising drug targets to treat a plethora of diseases. Although several proteins involved in Wnt trafficking and secretion have been identified over the past years, little is known about the contribution of different lipid species into these processes. The trafficking and secretion of Wnts could be modulated by the type and number of acyl species covalently linked to Wnt proteins. Currently, the best described acyl modification is the palmiteoylation of a serine residue located around amino acids 205-215 mediated by the ER-resident O-acyltransferase Porcupine is responsible for this process. This lipid modification has been described for Wnt3a, Wnt5, xWnt8, and Wnt1, and it has been assumed to also take place in other members of the family of Wnt proteins. Despite the extensive data available, the debate around the lipid-modified amino acids in Wnt proteins has not yet reached a consensus. Recent results from O. Voloshanenko (M. Boutros group, DKFZ, Heidelberg, Germany) suggested that there may be other amino acid residues in Wnts that are lipidated, apart from this canonical serine residue. Furthermore, the specific saturation of the acylated chain that binds to Wnt remains inconclusive. In this thesis, I aimed to define other putative acylation types and lipid-modified sites in Wnt proteins and to determine the role of these alternative lipidations in Wnt secretion and signaling. Furthermore, I evaluated the impact of Wnt signaling and Wnt secretion on the lipidome of HEK293T and HCT116 cells. To achieve this, I employed a combination of chemical biology tools, mutagenesis experiments, and mass spectrometric measurements. In particular, I focused on Wnt11 as a working model. I studied its acylation using clickable lipids such as palmitic acid alkyne (cC16:0) and palmitoleic acid alkyne (cC16:1n-7). One of the early observations was that palmitoylation and secretion of Wnt11 were not wholly abolished in Porcupine knockout cells or some mutant variants of Wnt11. However, these observations seem to depend on the type of clickable fatty acid used. Our results suggest a lipid modification of Wnt11 at serine 215 via the monounsaturated fatty acid cC16:1n-7, consistent with the previously predicted models. However, lipid modification with the saturated fatty acid cC16:0 showed variations in the experimental replicates, which did not fully resolve whether Wnt11 contains another modification site. Importantly, our experiments stress that unsaturation is a key feature for Wnt acylation. The relevance of covalent lipid binding for the secretion and signaling activity of Wnts has also been assessed. It was demonstrated that lipidation is essential for the signaling activity of Wnt11 but is not strictly necessary for its secretion. In addition, an impact of Wnt protein expression on the overall cellular lipidome of HEK293T and HCT116 cells has been tested, yielding preliminary observations on the crosstalk between the Wnt signaling and the overall cellular lipid homeostasis. This study is expected to contribute to our understanding of how post-translational lipid modifications influence Wnt cellular secretion, signaling and, conversely, how proteins of the Wnt signaling pathway affect the lipid composition of cells

Localization and ordering of lipids around Aquaporin-0: Proteinand lipid mobility effects.

Hydrophobic matching, lipid sorting, and protein oligomerization are key principles by which lipids and proteins organize in biological membranes. The Aquaporin-0 channel (AQP0), solved by electron crystallography (EC) at cryogenic temperatures, is one of the few protein-lipid complexes of which the structure is available in atomic detail. EC and room-temperature molecular dynamics (MD) of dimyristoylglycerophosphocholine (DMPC) annular lipids around AQP0 show similarities, however, crystal-packing and temperature might affect the protein surface or the lipids distribution. To understand the role of temperature, lipid phase, and protein mobility in the localization and ordering of AQP0-lipids, we used MD simulations of an AQP0-DMPC bilayer system. Simulations were performed at physiological and at DMPC gel-phase temperatures. To decouple the protein and lipid mobility effects, we induced gel-phase in the lipids or restrained the protein. We monitored the lipid ordering effects around the protein. Reducing the system temperature or inducing lipid gel-phase had a marginal effect on the annular lipid localization. However, restraining the protein mobility increased the annular lipid localization around the whole AQP0 surface, resembling EC. The distribution of the inter-phosphate and hydrophobic thicknesses showed that stretching of the DMPC annular layer around AQP0 surface is the mechanism that compensates the hydrophobic mismatch in this system. The distribution of the local area-per-lipid and the acyl-chain order parameters showed particular fluid- and gel-like areas that involved several lipid layers. These areas were in contact with the surfaces of higher and lower protein mobility, respectively. We conclude that the AQP0 surfaces induce specific fluid- and gel-phase prone areas. The presence of these areas might guide the AQP0 lipid sorting interactions with other membrane components, and is compatible with the squared array oligomerization of AQP0 tetramers separated by a layer of annular lipids

MIPModDB: a central resource for the superfamily of major intrinsic proteins

The channel proteins belonging to the major intrinsic proteins (MIP) superfamily are diverse and are found in all forms of life. Water-transporting aquaporin and glycerol-specific aquaglyceroporin are the prototype members of the MIP superfamily. MIPs have also been shown to transport other neutral molecules and gases across the membrane. They have internal homology and possess conserved sequence motifs. By analyzing a large number of publicly available genome sequences, we have identified more than 1000 MIPs from diverse organisms. We have developed a database MIPModDB which will be a unified resource for all MIPs. For each MIP entry, this database contains information about the source, gene structure, sequence features, substitutions in the conserved NPA motifs, structural model, the residues forming the selectivity filter and channel radius profile. For selected set of MIPs, it is possible to derive structure-based sequence alignment and evolutionary relationship. Sequences and structures of selected MIPs can be downloaded from MIPModDB database which is freely available at http://bioinfo.iitk.ac.in/MIPModDB

Verständnis der molekularen Maschinerie von Aquaporinen durch Molekulardynamiksimulationen

Aquaporine sind Proteinkanäle, die den Durchtritt von Wasser und kleinen Molekülen durch biologische Membranen als Reaktion auf osmotische Druckveränderungen erlauben. Das Ziel dieser Arbeit ist es, das Wissen über die molekulare Maschine Aquaporin mittels Molekulardynamiksimulationen und verwandten computergestützten Methoden zu erweitern.Zunächst präsentiere ich den Permeations-Mechanismus für das Plasmodium falciparum Aquaglyceroporin, ein vielversprechendes Drug Target gegen Malaria. Nach diesem Mechanismus bestimmen hydrophobe Barrieren in der Mitte des Kanals die Permeationskinetik von Wasser. Auβerdem wird die Selektivität für Glyzerin und Harnstoff hauptsächlich durch den Wechsel von Wasser-Arginin-Wechselwirkungen zu Lösungsmittel-Arginin-Wechselwirkungen sowie durch die Kompatibilität der Losüngsmittel an der engsten Stelle des Kanals bestimmt.Zweitens untersuchen wir die molekularen Ursachen für Leitfähigkeitsveränderungen von Aquaporinen, die von Organismen genutzt wird um dem schädlichen Effekt von plötzlichen osmotischen Druckveränderungen entgegenzuwirken. Unsere Simulationen sowie strukturelle und funktionelle Experimente legen nahe, dass Hefe Aquaporin-1 durch Phosphorylierung eines Serins sowie durch Mechanoperzeption gesteuert werden kann. Unsere Simulationen von Aquaporinen aus Spinatpflanzen zeigen weiter, dass sich die gesamte Maschinerie zum Öffnen und Schlieβen der Kanäle in dem zytosolischen Loop D befindet. Diese Simulationen stärken jedoch nicht den Vorschlag des Mechanismus, der auf Serin-Phosphorylierung oder Histidin-Protonierung basiert. Auβerdem konnte in silico eine Abhängikeit der Permeabilität von der Membranspannung in humanem AQP4 beobachtet werden, die wir auf den Übergang des Arginins in der engsten Region des Protein zurückführten. Kombiniert mit ähnlichen Beobachtungen für humanes AQP1, legt dies die experimentell zu testende Vermutung nahe, dass Spannungsabhängigkeit eine natürliche Eigenschaft von AQPs sein könnte.Drittens widme ich mich drei wichtigen Prozessen, in denen Aquaporine mit anderen (Bio-)Molekülen interagieren. Die Bildung und Stabilität des AQP2-LIP5 Komplexes, der unerlässlich für den Transport von AQP2 in Nierenzellen ist, wurde untersucht. Es konnten zwei wahrscheinliche Strukturen dieses Komplexes vorhergesagt werden, bei denen sich das Aquaporin-Tetramer in der Membran befindet und durch Bindung von drei Leucinen des C-Terminus von AQP2 in einer hydrophoben Tasche von LIP5 stabilisiert wird. Darüber hinaus konnte beobachtet werden, dass die Wechselwirkungen von AQP0 mit Membranlipiden in erster Linie durch Anpassung der hydrophoben Ketten der Lipide an die Oberfläche des Proteins gegeben sind, wohingegen der Einfluss der Lipid-Kopfgruppen wesentlich geringer ist und Lipide ihre Bulk-Eigenschaften erst mit zunehmendem Abstand von AQP0 wiedergewinnen. Die Daten zeigen auch, dass bestimmte Lipidpositionen, die in einem 2D-Kristall um AQP0 mittels Elektronen Kristallographie beobachtet worden waren, tatsächlich Lipiden entsprechen, die um niedrig konzentriertes AQP0 in eine Doppellipidschicht eingebettet sind. Letztlich zeigen wir, wie Molekular-Dynamik-Simulationen in Verbindung mit Funktionsuntersuchungen und molekularem Docking für die Suche und Optimierung von möglichen AQP9 Blockern genutzt werden können

Molecular driving forces defining lipid positions around aquaporin-0.

Lipid–protein interactions play pivotal roles in biological membranes. Electron crystallographic studies of the lens-specific water channel aquaporin-0 (AQP0) revealed atomistic views of such interactions, by providing high-resolution structures of annular lipids surrounding AQP0. It remained unclear, however, whether these lipid structures are representative of the positions of unconstrained lipids surrounding an individual protein, and what molecular determinants define the lipid positions around AQP0. We addressed these questions by using molecular dynamics simulations and crystallographic refinement, and calculated time-averaged densities of dimyristoyl-phosphatidylcholine lipids around AQP0. Our simulations demonstrate that, although the experimentally determined crystallographic lipid positions are constrained by the crystal packing, they appropriately describe the behavior of unconstrained lipids around an individual AQP0 tetramer, and thus likely represent physiologically relevant lipid positions.While the acyl chains were well localized, the lipid head groups were not. Furthermore, in silico mutations showed that electrostatic inter actions do not play a major role attracting these phospholipids towards AQP0. Instead, the mobility of the protein crucially modulates the lipid localization and explains the difference in lipid density between extracellular and cytoplasmic leaflets. Moreover, our simulations support a general mechanism in which membrane proteins laterally diffuse accompanied by several layers of localized lipids, with the positions of the annular lipids being influenced the most by the protein surface. We conclude that the acyl chains rather than the head groups define the positions of dimyristoylphosphatidylcholine lipids around AQP0. Lipid localization is largely determined by the mobility of the protein surface, whereas hydrogen bonds play an important but secondary role

Temperature dependence of protein-water interactions in a gated yeast aquaporin.

Regulation of aquaporins is a key process of living organisms to counteract sudden osmotic changes. Aqy1, which is a water transporting aquaporin of the yeast Pichia pastoris, is suggested to be gated by chemo-mechanical stimuli as a protective regulatory-response against rapid freezing. Here, we tested the influence of temperature by determining the X-ray structure of Aqy1 at room temperature (RT) at 1.3 angstrom resolution, and by exploring the structural dynamics of Aqy1 during freezing through molecular dynamics simulations. At ambient temperature and in a lipid bilayer, Aqy1 adopts a closed conformation that is globally better described by the RT than by the low-temperature (LT) crystal structure. Locally, for the blocking-residue Tyr31 and the water molecules inside the pore, both LT and RT data sets are consistent with the positions observed in the simulations at room-temperature. Moreover, as the temperature was lowered, Tyr31 adopted a conformation that more effectively blocked the channel, and its motion was accompanied by a temperature-driven rearrangement of the water molecules inside the channel. We therefore speculate that temperature drives Aqy1 from a loosely-to a tightly-blocked state. This analysis provides high-resolution structural evidence of the influence of temperature on membrane-transport channels