Notes

Journal Membership: Usefulness as well as Safety involving Voclosporin Versus Placebo regarding Lupus Nephritis (AURORA One): The Double-Blind, Randomized, Multicenter, Placebo-Controlled, Period Three Trial.
Following their generation by lipid kinases and phosphatases, phosphoinositides regulate important biological processes such as cytoskeleton rearrangement, membrane remodeling/trafficking, and gene expression through the interaction of their phosphorylated inositol head group with a variety of protein domains such as PH, PX, and FYVE. Therefore, it is important to determine the specificity of phosphoinositides toward effector proteins to understand their impact on cellular physiology. Several methods have been developed to identify and characterize phosphoinositide effectors, and liposomes-based methods are preferred because the phosphoinositides are incorporated in a membrane, the composition of which can mimic cellular membranes. In this report, we describe the experimental setup for liposome flotation assay and a recently developed method called protein-lipid interaction by fluorescence (PLIF) for the characterization of phosphoinositide-binding specificities of proteins.Phosphoinositides interact with proteins to fulfill various functions in the cell. In many cases, they specifically recruit peripheral membrane proteins to biological membranes. The analysis of their interactions with proteins is therefore essential for understanding the underlying processes. Native mass spectrometry (MS) preserves noncovalent interactions in the gas phase of a mass spectrometer and is therefore well-suited to study protein-phosphoinositide interactions. In this protocol, we describe the application of native MS to integral and peripheral membrane proteins and their interactions with lipids. We discuss sample and instrumental requirements, the realization of experiments, and the data analysis workflow. We further describe a biochemical assay to proof interactions of peripheral membrane proteins with lipids.It is now clear that organelles of a mammalian cell can be distinguished by phospholipid profiles, both as ratios of common phospholipids and by the absence or presence of certain phospholipids. Organelle-specific phospholipids can be used to provide a specific shape and fluidity to the membrane and/or used to recruit and/or traffic proteins to the appropriate subcellular location and to restrict protein function to this location. Studying the interactions of proteins with specific phospholipids using soluble derivatives in isolation does not always provide useful information because the context in which the headgroups are presented almost always matters. Our laboratory has shown this circumstance to be the case for a viral protein binding to phosphoinositides in solution and in membranes. The system we have developed to study protein-phospholipid interactions in the context of a membrane benefits from the creation of tailored membranes in a channel of a microfluidic device, with a fluorescent lipid in the membrane serving as an indirect reporter of protein binding. This system is amenable to the study of myriad interactions occurring at a membrane surface as long as a net change in surface charge occurs in response to the binding event of interest.Proximity ligation assay (PLA) is a well-established method for detecting in situ interactions between two epitopes with high resolution and specificity. Notably, PLA is not only a robust method for studying protein-protein interaction but also an efficient approach to characterize and validate protein posttranslational modifications (PTM) using one antibody against the core protein and one against the PTM residue. Therefore, it could be applied as a powerful approach to detect specific interactions of endogenous phosphoinositides and their binding proteins within cells. Importantly, we have specifically detected the PLA signal between PtdIns(4,5)P2 and its binding effector p53 in the nucleus. This cutting-edge method fully complements other conventional approaches for studying phosphoinositide-protein interactions and provides important localization signals and robust quantitation of the detected interactions. Here, we present the PLA fluorescence protocol for detecting in situ phosphoinositide-protein interactions in cultured cells and is semiquantitative for interactions that are regulated by cellular signaling.Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is an enzyme that converts phosphatidylinositol 4-phosphate [PI4P] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. PIP5K plays a key role in the regulation of vesicular transport, cytoskeleton reorganization, and cell division. In general, to investigate an enzymatic activity of PIP5K, the amount of incorporated [P32] ATP into PI(4,5)P2 fraction is measured in in vitro reconstitution experiments. However, tools to monitor dynamic changes in its activity in real time have been lacking. Recently, we have developed a novel PIP5K assay using fluorescence spectroscopy. Compared to conventional methods in which lipids extraction steps are needed, our method is easy and quick to perform and enables a real-time analysis. This chapter provides a protocol to set up and perform the novel PIP5K assay we have recently established.Chemical dimerization systems have been used to drive acute depletion of polyphosphoinsitides (PPIns). They do so by inducing subcellular localization of enzymes that catabolize PPIns. read more By using this approach, all seven PPIns can be depleted in living cells and in real time. The rapid permeation of dimerizer agents and the specific expression of recruiter proteins confer great spatial and temporal resolution with minimal cell perturbation. In this chapter, we provide detailed instructions to monitor and induce depletion of PPIns in live cells.Phosphoinositides make up only a small fraction of cellular phospholipids yet control cell function in a fundamental manner. Through protein interactions, phosphoinositides define cellular organelle identity and regulate protein function and organization and recruitment at the cytosol-membrane interface. As a result, perturbations on phosphoinositide metabolism alter cell physiology and lead to a wide range of human diseases, including cancer and diabetes. Among seven phosphoinositide members, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2, also known as PI(4,5)P2 or PIP2) is abundant in the plasma membrane. Besides its role in the second messenger pathway of phospholipase C that cleaves PtdIns(4,5)P2 to form diacylglycerol and inositol-1,4,5-trisphosphate (IP3), PtdIns(4,5)P2 regulates membrane trafficking and the function of the cytoskeleton, ion channels, and transporters. The nanoscale organization of PtdIns(4,5)P2 in the plasma membrane becomes essential to understand cellular signaling specificity in time and space.
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