BBA - Molecular and Cell Biology of Lipids (v.1841, #8)

Tools to study lipid functions by Britta Brügger; Alex Brown (1021).

Fat & fabulous: Bifunctional lipids in the spotlight by Per Haberkant; Joost C.M. Holthuis (1022-1030).
Understanding biological processes at the mechanistic level requires a systematic charting of the physical and functional links between all cellular components. While protein–protein and protein–nucleic acid networks have been subject to many global surveys, other critical cellular components such as membrane lipids have rarely been studied in large-scale interaction screens. Here, we review the development of photoactivatable and clickable lipid analogues–so-called bifunctional lipids–as novel chemical tools that enable a global profiling of lipid–protein interactions in biological membranes. Recent studies indicate that bifunctional lipids hold great promise in systematic efforts to dissect the elaborate crosstalk between proteins and lipids in live cells and organisms. This article is part of a Special Issue entitled Tools to study lipid functions.
Keywords: Photoaffinity labeling; Protein–lipid interaction; Proteomics; Imaging; Click chemistry;

Multiple bonds for the lipid interest by Lars Kuerschner; Christoph Thiele (1031-1037).
Polyene lipids and alkyne lipids allow study of lipid organization, dynamics and metabolism. Both types of lipids contain multiple bonds as the essential functional group, leading to minimal disturbance of the hydrophobic properties on which the characteristic behavior of lipids is based. Polyene lipids can directly be traced due to their intrinsic fluorescence, while alkyne lipids need the copper-catalyzed click reaction to an azido-reporter for detection. This review describes recent developments in synthesis and application of both types of lipid analogs with emphasis on metabolic tracing and microscopy imaging. This article is part of a Special Issue entitled Tools to study lipid functions.
Keywords: Polyene lipid; Alkyne lipid; Click lipid; Click-reaction; Analog; Probe;

Avanti lipid tools: Connecting lipids, technology, and cell biology by Kacee H. Sims; Ewan M. Tytler; John Tipton; Kasey L. Hill; Stephen W. Burgess; Walter A. Shaw (1038-1048).
Lipid research is challenging owing to the complexity and diversity of the lipidome. Here we review a set of experimental tools developed for the seasoned lipid researcher, as well as, those who are new to the field of lipid research. Novel tools for probing protein–lipid interactions, applications for lipid binding antibodies, enhanced systems for the cellular delivery of lipids, improved visualization of lipid membranes using gold-labeled lipids, and advances in mass spectrometric analysis techniques will be discussed. Because lipid mediators are known to participate in a host of signal transduction and trafficking pathways within the cell, a comprehensive lipid toolbox that aids the science of lipidomics research is essential to better understand the molecular mechanisms of interactions between cellular components. This article is part of a Special Issue entitled Tools to study lipid functions.
Keywords: Lipid toolbox; Protein–lipid interactions; Lipid-binding antibodies; Gold-conjugated lipids; Lipidomics; Mass spectrometry;

Validity and applicability of membrane model systems for studying interactions of peripheral membrane proteins with lipids by Aleksander Czogalla; Michał Grzybek; Walis Jones; Ünal Coskun (1049-1059).
The cell membrane serves, at the same time, both as a barrier that segregates as well as a functional layer that facilitates selective communication. It is characterized as much by the complexity of its components as by the myriad of signaling process that it supports. And, herein lays the problems in its study and understanding of its behavior — it has a complex and dynamic nature that is further entangled by the fact that many events are both temporal and transient in their nature. Model membrane systems that bypass cellular complexity and compositional diversity have tremendously accelerated our understanding of the mechanisms and biological consequences of lipid–lipid and protein–lipid interactions. Concurrently, in some cases, the validity and applicability of model membrane systems are tarnished by inherent methodical limitations as well as undefined quality criteria. In this review we introduce membrane model systems widely used to study protein–lipid interactions in the context of key parameters of the membrane that govern lipid availability for peripheral membrane proteins. This article is part of a Special Issue entitled Tools to study lipid functions.Display Omitted
Keywords: Phospholipid; Membrane; Protein–lipid interaction; Model membrane system; Lipid presentation; Lipid identity;

Chemical modulation of glycerolipid signaling and metabolic pathways by Sarah A. Scott; Thomas P. Mathews; Pavlina T. Ivanova; Craig W. Lindsley; H. Alex Brown (1060-1084).
Thirty years ago, glycerolipids captured the attention of biochemical researchers as novel cellular signaling entities. We now recognize that these biomolecules occupy signaling nodes critical to a number of physiological and pathological processes. Thus, glycerolipid-metabolizing enzymes present attractive targets for new therapies. A number of fields—ranging from neuroscience and cancer to diabetes and obesity—have elucidated the signaling properties of glycerolipids. The biochemical literature teems with newly emerging small molecule inhibitors capable of manipulating glycerolipid metabolism and signaling. This ever-expanding pool of chemical modulators appears daunting to those interested in exploiting glycerolipid-signaling pathways in their model system of choice. This review distills the current body of literature surrounding glycerolipid metabolism into a more approachable format, facilitating the application of small molecule inhibitors to novel systems. This article is part of a Special Issue entitled Tools to study lipid functions.
Keywords: Glycerolipid; Lipase; Metabolism; Inhibitor; Phospholipase; Autotaxin; Lipid kinase; Lipin; Fatty acyltransferase;

Caged lipids as tools for investigating cellular signaling by Doris Höglinger; André Nadler; Carsten Schultz (1085-1096).
Lipid derivatives that can be activated by light, often referred to as ‘caged’ lipids, are useful tools to manipulate intact cells non-invasively. Here we focus on experimental approaches that have made use of caged lipids. Apart from summarizing the recent advances and available tools in the field, we strive to highlight the experimental challenges that arise from lipid-specific biophysical properties and the abundance of an enormous diversity of distinct molecular lipid species in cells. This article is part of a Special Issue entitled Tools to study lipid functions.
Keywords: Phosphoinositides; Lipid signaling; Lipid second messenger; Cell entry; Photoactivation;

Genome sequencing efforts have identified many uncharacterized lipase/esterase enzymes that have potential to be drug targets for metabolic diseases such as obesity, diabetes, and atherosclerosis. However, sequence information and associated structural predictions provide only a loose framework for linking enzyme function to disease risk. We are now confronted with the challenge of functionally annotating a large number of uncharacterized lipases, with the goal of generating new therapies for metabolic diseases. This daunting challenge involves gathering not only sequence-driven predictions, but also more importantly structural, biochemical (substrates and products), and physiological data. At the center of such drug discovery efforts are accurately identifying physiologically relevant substrates and products of individual lipases, and determining whether newly identified substrates/products can modulate disease in appropriate preclinical animal model systems. This review describes the importance of coupling in vivo metabolite profiling to in vitro enzymology as a powerful means to assign lipase function in disease specific contexts using animal models. In particular, we highlight recent examples using this multidisciplinary approach to functionally annotate genes within the α/β hydrolase fold domain (ABHD) family of enzymes. These new discoveries within the ABHD enzyme family serve as powerful examples of linking novel lipase function to human disease. This article is part of a Special Issue entitled Tools to study lipid functions.
Keywords: ABHD3; ABHD6; ABHD12; Enzymology; Lysophospholipase; Mass spectrometry;

The advent of mass spectrometric methods has facilitated the determination of multiple molecular species of cellular lipid classes including the polyphosphoinositides, though to date methods to analyse and quantify each of the individual three PtdInsP and three PtdInsP2 species are lacking. The use of imaging methods has allowed intracellular localization of the phosphoinositide classes but this methodology does not determine the acyl structures. The range of molecular species suggests a greater complexity in polyphosphoinositide signaling than yet defined but elucidating this will require further method development to be achieved. This article is part of a Special Issue entitled Tools to study lipid functions.
Keywords: Mass spectrometry; Polyphosphoinositide; Lipid binding domain; Imaging; Acyl chains; Membranes;

Imaging lipids with secondary ion mass spectrometry by Mary L. Kraft; Haley A. Klitzing (1108-1119).
This review discusses the application of time-of-flight secondary ion mass spectrometry (TOF-SIMS) and magnetic sector SIMS with high lateral resolution performed on a Cameca NanoSIMS 50(L) to imaging lipids. The similarities between the two SIMS approaches and the differences that impart them with complementary strengths are described, and various strategies for sample preparation and to optimize the quality of the SIMS data are presented. Recent reports that demonstrate the new insight into lipid biochemistry that can be acquired with SIMS are also highlighted. This article is part of a Special Issue entitled Tools to study lipid functions.
Keywords: NanoSIMS; TOF-SIMS; Cell membrane; Model membrane; Sample preparation; Lipid distribution;

Shedding new light on lipid functions with CARS and SRS microscopy by Yong Yu; Prasanna V. Ramachandran; Meng C. Wang (1120-1129).
Modern optical microscopy has granted biomedical scientists unprecedented access to the inner workings of a cell, and revolutionized our understanding of the molecular mechanisms underlying physiological and disease states. In spite of these advances, however, visualization of certain classes of molecules (e.g. lipids) at the sub-cellular level has remained elusive. Recently developed chemical imaging modalities – Coherent Anti-Stokes Raman Scattering (CARS) microscopy and Stimulated Raman Scattering (SRS) microscopy – have helped bridge this gap. By selectively imaging the vibration of a specific chemical group, these non-invasive techniques allow high-resolution imaging of individual molecules in vivo, and circumvent the need for potentially perturbative extrinsic labels. These tools have already been applied to the study of fat metabolism, helping uncover novel regulators of lipid storage. Here we review the underlying principle of CARS and SRS microscopy, and discuss the advantages and caveats of each technique. We also review recent applications of these tools in the study of lipids as well as other biomolecules, and conclude with a brief guide for interested researchers to build and use CARS/SRS systems for their own research. This article is part of a Special Issue entitled Tools to study lipid functions.
Keywords: Lipid metabolism; Optical imaging;

Glycolipids are the most complex lipid type in cell membranes, characterized by a great diversity of different structures and functions. The underlying atomistic/molecular interactions and mechanisms associated with these functions are not well understood. Here we discuss how atomistic and molecular simulations can be used to shed light on the role of glycolipids in membrane structure and dynamics, receptor function, and other phenomena related to emergence of diseases such as Parkinson's. The cases we discuss highlight the challenge to understand how glycolipids function in cell membranes, and the significant added value that one would gain by bridging molecular simulations with experiments. This article is part of a Special Issue entitled Tools to study lipid functions.
Keywords: Molecular dynamics simulation; Computer simulation; Carbohydrate; Lipid; Glyco; Glycosphingolipid;

The appropriate lipid environment is crucial for the proper function of membrane proteins. There is a tremendous variety of lipid molecules in the membrane and so far it is often unclear which component of the lipid matrix is essential for the function of a respective protein. Lipid molecules and proteins mutually influence each other; parameters such as acyl chain order, membrane thickness, membrane elasticity, permeability, lipid-domain and annulus formation are strongly modulated by proteins. More recent data also indicates that the influence of proteins goes beyond a single annulus of next-neighbor boundary lipids. Therefore, a mesoscopic approach to membrane lipid–protein interactions in terms of elastic membrane deformations has been developed. Solid-state NMR has greatly contributed to the understanding of lipid–protein interactions and the modern view of biological membranes. Methods that detect the influence of proteins on the membrane as well as direct lipid–protein interactions have been developed and are reviewed here. Examples for solid-state NMR studies on the interaction of Ras proteins, the antimicrobial peptide protegrin-1, the G protein-coupled receptor rhodopsin, and the K+ channel KcsA are discussed. This article is part of a Special Issue entitled Tools to study lipid functions.
Keywords: Magic-angle spinning; Order parameter; Magnetization transfer; Chemical shift; Dipolar coupling;

The unraveling of sphingolipid metabolism and function in the last 40 years relied on the extensive study of inherited human disease and specifically-tailored mouse models. However, only few of the achievements made so far would have been possible without chemical biology tools, such as fluorescent and/or radio-labeled and other artificial substrates, (mechanism-based) enzyme inhibitors, cross-linking probes or artificial membrane models. In this review we provide an overview over chemical biology tools that have been used to gain more insight into the molecular basis of sphingolipid-related biology. Many of these tools are still of high relevance for the investigation of current sphingolipid-related questions, others may stimulate the tailoring of novel probes suitable to address recent and future issues in the field. This article is part of a Special Issue entitled Tools to study lipid functions.
Keywords: Radiolabeled sphingolipid; Fluorescent sphingolipid; Biotinylated ganglioside; Photoactivatable ganglioside; Spin-labeled ganglioside;

Evolving concepts in cancer therapy through targeting sphingolipid metabolism by Jean-Philip Truman; Mónica García-Barros; Lina M. Obeid; Yusuf A. Hannun (1174-1188).
Traditional methods of cancer treatment are limited in their efficacy due to both inherent and acquired factors. Many different studies have shown that the generation of ceramide in response to cytotoxic therapy is generally an important step leading to cell death. Cancer cells employ different methods to both limit ceramide generation and to remove ceramide in order to become resistant to treatment. Furthermore, sphingosine kinase activity, which phosphorylates sphingosine the product of ceramide hydrolysis, has been linked to multidrug resistance, and can act as a strong survival factor. This review will examine several of the most frequently used cancer therapies and their effect on both ceramide generation and the mechanisms employed to remove it. The development and use of inhibitors of sphingosine kinase will be focused upon as an example of how targeting sphingolipid metabolism may provide an effective means to improve treatment response rates and reduce associated treatment toxicity. This article is part of a Special Issue entitled Tools to study lipid functions.
Keywords: Cancer; Ceramide; Chemoresistance; Sphingosine kinase; Sphingosine-1-phosphate; Inhibitor;

From sheep to mice to cells: Tools for the study of the sphingolipidoses by Hila Zigdon; Anna Meshcheriakova; Anthony H. Futerman (1189-1199).
The sphingolipidoses are a group of inherited lysosomal storage diseases in which sphingolipids accumulate due to the defective activity of one or other enzymes involved in their degradation. For most of the sphingolipidoses, little is known about the molecular mechanisms that lead to disease, which has negatively impacted attempts to develop therapies for these devastating human diseases. Use of both genetically-modified animals, ranging from mice to larger mammals, and of novel cell culture systems, is of utmost importance in delineating the molecular mechanisms that cause pathophysiology, and in providing tools that enable testing the efficacy of new therapies. In this review, we discuss eight sphingolipidoses, namely Gaucher disease, Fabry disease, metachromatic leukodystrophy, Krabbe disease, Niemann–Pick diseases A and B, Farber disease, GM1 gangliosidoses, and GM2 gangliosidoses, and describe the tools that are currently available for their study. This article is part of a Special Issue entitled Tools to study lipid functions.
Keywords: Lysosomal storage disorder; Sphingolipid; Mouse model; Large animal model; Cell culture;

Lipid quantification method using FTIR spectroscopy applied on cancer cell extracts by Allison Derenne; Olivier Vandersleyen; Erik Goormaghtigh (1200-1209).
Reprogramming energy metabolism constitutes one of the hallmarks of cancer. Changes in lipid composition of cell membranes also appear early in carcinogenesis. Quantification of various molecules such as lipids evidences the modifications in the metabolism of tumour cells and can serve as potential markers for cancer diagnosis and treatment. Fourier Transform Infrared (FTIR) spectroscopy is a powerful tool used for the detection and characterization of various types of molecules. This technique remains an attractive approach as it is cheap (equipment and reagents), does not require high grade solvents or expensive internal standards, equipment is widely available in standard laboratories and the method is robust and suitable for routine analyses. In this work we established partial least square (PLS) models based on FTIR spectra able to quantify lipids in complex mixtures such as cell extracts. In the first part, we attempted to build PLS models with FTIR spectra of 53 mixtures of 8 well-characterized pure lipids. Second, the PLS models were verified using FTIR spectra of mixtures that did not contribute to the calibration. The third step was the validation of the models on lipid cell extracts. In order to obtain reference values for cell extracts, high performance liquid chromatography was carried out by AVANTI. The lipid distribution were globally similar with both techniques, PLS models and chromatography. Finally, the models were applied to determine the lipid composition of cells exposed to four treatments. We could not evidence significant changes in the lipid composition of cell extracts after treatment, in terms of polar head groups. However, the models established in this study appear reliable and could be applied for high throughput measurements. This article is part of a Special Issue entitled Tools to study lipid functions.
Keywords: FTIR; Lipid quantification; PLS;

Polyunsaturated fatty acids (PUFAs) have been found to be effective inhibitors of cell signaling in numerous contexts, and we find that acute addition of micromolar PUFAs such as linoleic acid effectively inhibit of Ca2 + responses in mast cells stimulated by antigen-mediated crosslinking of FcεRI or by the SERCA pump inhibitor, thapsigargin. In contrast, the saturated fatty acid, stearic acid, with the same carbon chain length as linoleic acid does not inhibit these responses. Consistent with this inhibition of store-operated Ca2 + entry (SOCE), linoleic acid inhibits antigen-stimulated granule exocytosis to a similar extent. Using the fluorescently labeled plasma membrane Ca2 + channel protein, AcGFP–Orai1, together with the labeled ER Ca2 + sensor protein, STIM1–mRFP, we monitor stimulated coupling of these proteins that is essential for SOCE with a novel spectrofluorimetric resonance energy transfer method. We find effective inhibition of this stimulated coupling by linoleic acid that accounts for the inhibition of SOCE. Moreover, we find that linoleic acid induces some STIM1–STIM1 association, while inhibiting stimulated STIM1 oligomerization that precedes STIM1–Orai1 coupling. We hypothesize that linoleic acid and related PUFAs inhibit STIM1–Orai1 coupling by a mechanism that involves perturbation of ER membrane structure, possibly by disrupting electrostatic interactions important in STIM1 oligomerization. Thisarticle is part of a Special Issue entitled Tools to study lipid functions.
Keywords: Store-operated calcium entry (SOCE); IgE receptors (FcεRI); Linoleic acid; Fluorescence resonance energy transfer (FRET);