Showing papers by "Richard M. Epand published in 2019"

Cholesterol-Recognition Motifs in Membrane Proteins

Abstract: The impact of cholesterol on the structure and function of membrane proteins was recognized several decades ago, but the molecular mechanisms underlying these effects have remained elusive. There appear to be multiple mechanisms by which cholesterol interacts with proteins. A complete understanding of cholesterol-sensing motifs is still undergoing refinement. Initially, cholesterol was thought to exert only non-specific effects on membrane fluidity. It was later shown that this lipid could specifically interact with membrane proteins and affect both their structure and function. In this article, we have summarized and critically analyzed our evolving understanding of the affinity, specificity and stereoselectivity of the interactions of cholesterol with membrane proteins. We review the different computational approaches that are currently used to identify cholesterol binding sites in membrane proteins and the biochemical logic that governs each type of site, including CRAC, CARC, SSD and amphipathic helix motifs. There are physiological implications of these cholesterol-recognition motifs for G-protein coupled receptors (GPCR) and ion channels, in membrane trafficking and membrane fusion (SNARE) proteins. There are also pathological implications of cholesterol binding to proteins involved in neurological disorders (Alzheimer, Parkinson, Creutzfeldt-Jakob) and HIV fusion. In each case, our discussion is focused on the key molecular aspects of the cholesterol and amino acid motifs in membrane-embedded regions of membrane proteins that define the physiologically relevant crosstalk between the two. Our understanding of the factors that determine if these motifs are functional in cholesterol binding will allow us enhanced predictive capabilities.

Atomic resolution map of the soluble amyloid beta assembly toxic surfaces

Abstract: Soluble amyloid beta assemblies (Aβn) are neurotoxic and play a central role in the early phases of the pathogenesis cascade leading to Alzheimer's disease. However, the current knowledge about the molecular determinants of Aβn toxicity is at best scant. Here, we comparatively analyze Aβn prepared in the absence or presence of a catechin library that modulates cellular toxicity. By combining solution NMR with dynamic light scattering, fluorescence spectroscopy, electron microscopy, wide-angle X-ray diffraction and cell viability assays, we identify a cluster of unique molecular signatures that distinguish toxic vs. nontoxic Aβ assemblies. These include the exposure of a hydrophobic surface spanning residues 17–28 and the concurrent shielding of the highly charged N-terminus. We show that the combination of these two dichotomous structural transitions promotes the colocalization and insertion of β-sheet rich Aβn into the membrane, compromising membrane integrity. These previously elusive toxic surfaces mapped here provide an unprecedented foundation to establish structure-toxicity relationships of Aβ assemblies.

Plasmalogen loss caused by remodeling deficiency in mitochondria

Abstract: Lipid homeostasis is crucial in human health. Barth syndrome (BTHS), a life-threatening disease typically diagnosed with cardiomyopathy and neutropenia, is caused by mutations in the mitochondrial transacylase tafazzin. By high-resolution 31P nuclear magnetic resonance (NMR) with cryoprobe technology, recently we found a dramatic loss of choline plasmalogen in the tafazzin-knockdown (TAZ-KD) mouse heart, besides observing characteristic cardiolipin (CL) alterations in BTHS. In inner mitochondrial membrane where tafazzin locates, CL and diacyl phosphatidylethanolamine are known to be essential via lipid–protein interactions reflecting their cone shape for integrity of respiratory chain supercomplexes and cristae ultrastructure. Here, we investigate the TAZ-KD brain, liver, kidney, and lymphoblast from patients compared with controls. We identified common yet markedly cell type–dependent losses of ethanolamine plasmalogen as the dominant plasmalogen class therein. Tafazzin function thus critically relates to homeostasis of plasmalogen, which in the ethanolamine class has conceivably analogous and more potent molecular functions in mitochondria than diacyl phosphatidylethanolamine. The present discussion of a loss of plasmalogen–protein interaction applies to other diseases with mitochondrial plasmalogen loss and aberrant forms of this organelle, including Alzheimer9s disease.

Specificity of Acyl Chain Composition of Phosphatidylinositols.

Abstract: Phosphatidylinositol (PI) lipids have a predominance of a single molecular species present through the organism. In healthy mammals this molecular species is 1-stearoyl-2-arachidonoyl (18:0/20:4) PI. Although the importance of PI lipids for cell physiology has long been appreciated, less is known about the biological role of enriching PI lipids with 18:0/20:4 acyl chains. In conditions with dysfunctional lipid metabolism, the predominance of 18:0/20:4 acyl chains is lost. Recently, molecular mechanisms underpinning the enrichment or alteration of these acyl chains in PI lipids have begun to emerge. In the majority of the cases a common feature is the presence of enzymes bearing substrate acyl chain specificity. However, in cancer cells, it has been shown that one (not the only) of the mechanisms responsible for the loss in this acyl chain enrichment is mutation on the transcription factor p53 gene, which is one of the most highly mutated genes in cancers. There is a compelling need for a global picture of the specificity of the acyl chain composition of PIs. This can be possible once high-resolution spatio-temporal information is gathered in a cellular context; which can ultimately lead to potential novel targets to combat conditions with altered PI acyl chain profiles.

Anionic Lipid Clustering Model

Abstract: Many molecular features contribute to the antimicrobial activity of peptides. One aspect that contributes to the antimicrobial activity of a peptide, in many cases, results from the fact that many antimicrobial peptides are polycationic and the lipids on the surface of bacteria are often anionic. In certain cases this can result in the clustering of anionic lipids as a result of the binding of the cationic peptide to the surface of the bacterial membrane. This lipid clustering can be detrimental to the viability of the bacteria to which the peptide binds. Several factors, including the charge, size, and conformational flexibility of the peptide, will determine the efficiency of lipid clustering. In addition, the lipid composition of the bacterial membrane is very variable, and it plays a critical role in this mechanism. As a result, one can test the importance of this factor by determining the species specificity of the antimicrobial activity of the peptide. The molecular mechanism by which lipid clustering affects bacterial viability is uncertain in many cases. This phenomenon can be used to increase the antimicrobial potency of peptides in some case and can also predict the bacterial species specificity of some agents.

Membrane remodeling by the lytic fragment of sticholysin II: implications for the toroidal pore model

Abstract: Sticholysins are pore-forming toxins of biomedical interest and represent a prototype of proteins acting through the formation of protein-lipid or toroidal pores. Peptides spanning the N-terminus of sticholysins can mimic their permeabilizing activity and together with the full-length toxins have been used as a tool to understand the mechanism of pore formation in membranes. However, the lytic mechanism of these peptides and the lipid shape modulating their activity are not completely clear. In this paper, we combine molecular dynamics (MD) simulations and experimental biophysical tools to dissect different aspects of the pore-forming mechanism of StII1-30, a peptide derived from the N-terminus of sticholysin II. With this combined approach, membrane curvature induction and flip-flop movement of the lipids were identified as two important membrane remodeling steps mediated by StII1-30-pore forming activity. Pore-formation by this peptide was enhanced by the presence of the negatively-curved lipid phosphatidylethanolamine (PE) in membranes. This lipid emerged not only as a facilitator of membrane interactions but also as a structural element of the StII1-30-pore that is recruited to the pore ring upon its assembly. Collectively, these new findings support a toroidal model for the architecture of the pore formed by this peptide and provide new molecular insight into the role of PE as a membrane component that easily accommodates into the ring of toroidal pores aiding in its stabilization. This study contributes to a better understanding of the molecular mechanism underlying the permeabilizing activity of StII1-30 and peptides or proteins acting via a toroidal pore mechanism and offers an informative framework for the optimization of the biomedical application of this and similar molecules. State of significance We provide evidence about the ability of StII1-30 to form toroidal pores. Due to pore assembly, StII1-30-pore induces membrane curvature and facilitates flip-flop movement of the lipids. The negatively-curved lipid PE relocates from the membrane into the pore ring, being also a structural element of the pore StII1-30 forms. This peptide emerged as a new tool, together with the full-length toxin, to understand the mechanism of toroidal pore formation in membranes. This study provides new molecular insight into the role of curved lipids as co-factors of toroidal pores, which could aid in its stabilization by easily accommodating into the ring. This framework could underpin strategies for the rational use of peptides or proteins acting via toroidal pores.

Phosphatidylinositol Cycle Disruption is Central to Atypical Hemolytic-Uremic Syndrome Caused by Diacylglycerol Kinase Epsilon Deficiency

Abstract: Background Loss-of-function mutations in diacylglycerol kinase epsilon (DGKE) cause a rare form of atypical hemolytic-uremic syndrome (aHUS) for which there is no treatment besides kidney transplantation. Highly expressed in kidney endothelial cells, DGKE is a lipid kinase that phosphorylates diacylglycerol (DAG) to phosphatic acid (PA). Specifically, DGKE’s preferred substrate is 38:4-DAG, that is DAG containing stearic acid (18:0) and arachidonic acid (20:4). DAG is produced when phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is cleaved by phospholipase C (PLC). A better understanding of how DGKE deficiency impacts the endothelial lipid landscape is critical to developing a treatment for this condition. Methods We used orthogonal methods to compare the lipid levels in two novel models of DGKE deficiency to their respective controls: an immortalized human umbilical vein endothelial cell (iHUVEC) engineered with CRISPR/Cas9 and a blood outgrowth endothelial cell (BOEC) from an affected patient. Methods included mass spectrometry lipidomics, radiolabeling of phosphoinositides with [3H]myo-inositol, and live-tracking of a transfected fluorescent PtdIns(4,5)P2 biosensor. Results Unexpectedly, mass spectrometry lipidomics data revealed that high 38:4-DAG was not observed in the two DGKE-deficient models. Instead, a reduction in 38:4-PtdIns(4,5)P2 was the major abnormality.These results were confirmed with the other two methods in DGKE-deficient iHUVEC. Conclusion Reduced 38:4-PtdIns(4,5)P2—but not increased 38:4-DAG—is likely to be key to the pro-thrombotic phenotype exhibited by patients with DGKE aHUS. TRANSLATIONAL STATEMENT Mutations in DGKE cause a severe renal thrombotic microangiopathy that affects young children and leads to end-stage renal disease before adulthood. DGKE preferentially phosphorylates diacylglycerol to its corresponding phosphatidic acid (PA), which is then used to synthesize PtdIns(4,5)P2 via the phosphatidylinositol cycle. Understanding the disease pathophysiology is necessary to develop a treatment to prevent this outcome. This paper describes how we applied mass spectrometry lipidomics to two novel models of DGKE deficiency to investigate how this defect impacts the levels of diacylglycerol, PA and related phosphoinositides in endothelia. Unexpectedly, our data show that the critical abnormality caused by DGKE deficiency is not high diacylglycerol, but rather low PtdIns(4,5)P2. Restoring endothelial PtdIns(4,5)P2 homeostasis may be the cornerstone to treat these patients.