Phospholipases and Membrane Curvature: What Is Happening at the Surface?

In this revision work, we emphasize the close relationship between the action of phospholipases and the modulation of membrane curvature and curvature stress resulting from this activity. The alteration of the tridimensional structure of membranes upon the action of phospholipases is analyzed based on studies on model lipid membranes. The transient unbalance of both compositional and physical membrane properties between the hemilayers upon phospholipase activity lead to curvature tension and the catalysis of several membrane-related processes. Several proteins' membrane-bound and soluble forms are susceptible to regulation by the curvature stress induced by phospholipase action, which has important consequences in cell signaling. Additionally, the modulation of membrane fusion by phospholipase products regulates membrane dynamics in several cellular scenarios. We commented on vesicle fusion in the Golgi-endoplasmic system, synaptic vesicle fusion to the plasma membrane, viral membrane fusion to host cell plasma membrane and gametes membrane fusion upon acrosomal reaction. Furthermore, we explored the modulation of membrane fusion by the asymmetric adsorption of amphiphilic drugs. A deep understanding of the relevance of lipid membrane structure, particularly membrane curvature and curvature stress, on different cellular events leads to the challenge of its regulation, which may become a powerful tool for pharmacological therapy.

Miltefosine inhibits the membrane remodeling caused by phospholipase action by changing membrane physical properties.

Miltefosine (hexadecylphosphocholine or HePC) is an alkylphosphocholine approved for the treatment of visceral and cutaneous Leishmaniasis. HePC exerts its effect by interacting with lipid membranes and affecting membrane-dependent processes. The molecular geometry of HePC suggests that the pharmacological function of HePC is to alter membrane curvature. As a model system, we studied the enzyme production in model membranes of diacylglycerol (DAG) or ceramide (CER), lipids involved in cell signaling which alter the structure of membranes. Here, we studied the effect of HePC on changes in phospholipase activity and on the effect that the lipid products have on the curvature and fusogenicity of membranes where they accumulate. Our results indicate that HePC inhibits the long-time restructuring of membranes, characteristic of the DAG and CER enzyme formation processes. In addition, the drug also reduces the fusogenicity of phospholipase-derived products. We postulate that the effect of HePC is due to a non-specific geometric compensation of HePC to the inverted cone-shape of DAG and CER products, acting as a relaxation agent of membrane curvature stress. These data are important for understanding the mechanism of action by which HePC regulates the lipid metabolism and signal transduction pathways in which these enzymes are involved.

The influence of myristoylation, liposome surface charge and nucleic acid interaction in the partition properties of HIV-1 Gag-N-terminal peptides to membranes.

The group-specific antigen (GAG) polyprotein of HIV-1 is the main coordinator of the virus assembly process at the plasma membrane (PM) and is directed by its N-terminal matrix domain (MA). MA is myristoylated and possess a highly basic region (HBR) responsible for the interaction with the negative lipids of the PM, especially with PIP2. In addition, MA binds RNA molecules proposed as a regulatory step of the assembly process. Here we study the interaction of a synthetic peptide (N-terminal 21 amino acids of MA) and liposomes of different compositions using a variety of biophysical techniques. Particularly, we use the fluorescence properties of the single tryptophan of the peptide to analyze its partition to membranes, where we harness for first time the analytical ability of spectral phasors method to study this interaction. We found that electrostatic interactions play an important role for peptide partition to membranes and myristoylation reduces the free energy of the process. Interestingly, we observe that while the presence of PIP2 does not cause measurable changes on the peptide-membrane interaction, the interaction is favored by cholesterol. Additionally, we found that the partition process goes through a transition state involving peptide disaggregation and changes in the peptide secondary structure. On the other hand, we found that the presence of oligonucleotides competes with the interaction with lipids by increasing peptide solubility. In summary, we think that our results, in context of the current knowledge of the role of HIV-1 MA, contribute to a better molecular understanding of the membrane association process.

Detecting phospholipase activity with the amphipathic lipid packing sensor motif of ArfGAP1.

The amphipathic lipid packing sensor (ALPS) motif of ArfGAP1 brings this GTPase activating protein to membranes of high curvature. Phospholipases are phospholipid-hydrolyzing enzymes that generate different lipid products that alter the lateral organization of membranes. Here, we evaluate by fluorescence microscopy how in-situ changes of membrane lipid composition driven by the activity of different phospholipases promotes the binding of ALPS. We show that the activity of phospholipase A2, phospholipase C and phospholipase D drastically enhances the binding of ALPS to the weakly-curved membrane of giant liposomes. Our results suggest that the enzymatic activity of phospholipases can modulate the ArfGAP1-mediated intracellular traffic and that amphiphilic peptides such as the ALPS motif can be used to study lipolytic activities at lipid membranes.