Biology subjects

Falzone, M. E.

Publications and source records attributed to Falzone, M. E..

5 recordsLinked to original sources

bioRxiv2026

Anionic lipids regulate PLCβ membrane recruitment

Phospholipase C-{beta} (PLC{beta}) enzymes are essential effectors of G protein-coupled receptor signaling that hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) at the plasma membrane to generate the second messengers inositol trisphosphate and diacylglycerol. PLC{beta}s play essential roles in diverse physiological processes, including cardiac and neuronal function, macrophage activation, and the pathogenesis of diseases such as hypertrophic cardiomyopathy. PLC{beta} enzymes are unique in that they are aqueous-soluble and must partition onto the membrane surface to access their substrate, making membrane association a critical regulatory step. For example, we recently demonstrated that G{beta}{gamma} activates PLC{beta} by membrane recruitment and orientation of the catalytic core on the membrane surface. Although PLC{beta} membrane recruitment is required for function, the molecular determinants governing this process remain incompletely understood. Using a quantitative membrane partitioning assay, we show that robust membrane association of PLC{beta} requires anionic phospholipids, whereas polar phospholipids cannot substitute. Membrane partitioning exhibits a steep dependence on anionic lipid abundance, which is mediated by electrostatic interactions between negatively charged lipids and basic residues within the distal C-terminal domain of PLC{beta}. We further demonstrate that anionic lipids cooperate with G{beta}{gamma} to regulate PLC{beta} membrane recruitment, such that the magnitude of G{beta}{gamma}-dependent recruitment is dictated by membrane anionic lipid content. These findings reconcile previous discrepancies regarding G{beta}{gamma}-mediated PLC{beta} membrane recruitment and establish membrane electrostatics as a key regulatory input that integrates lipid composition with G protein signaling to regulate PLC{beta} activity. SummaryMembrane association is a crucial component of PLC{beta} function and regulation, yet the mechanisms governing this process are poorly understood. The authors demonstrate that anionic lipids drive PLC{beta} membrane recruitment and influence G{beta}{gamma}-dependent activation, revealing how membrane composition shapes cellular signal transduction.

biophysics↗

bioRxiv2023

The mechanism of Gαq regulation of PLCβ3-catalyzed PIP2 hydrolysis.

PLC{beta} enzymes cleave PIP2 producing IP3 and DAG. PIP2 modulates the function of many ion channels, while IP3 and DAG regulate intracellular Ca2+ levels and protein phosphorylation by protein kinase C, respectively. PLC{beta} enzymes are under the control of GPCR signaling through direct interactions with G proteins G{beta}{gamma} and Gq and have been shown to be coincidence detectors for dual stimulation of Gq and Gi coupled receptors. PLC{beta}s are aqueous-soluble cytoplasmic enzymes, but partition onto the membrane surface to access their lipid substrate, complicating their functional and structural characterization. Using newly developed methods, we recently showed that G{beta}{gamma} activates PLC{beta}3 by recruiting it to the membrane. Using these same methods, here we show that Gq increases the catalytic rate constant, kcat, of PLC{beta}3. Since stimulation of PLC{beta}3 by Gq depends on an autoinhibitory element (the X-Y linker), we propose that Gq produces partial relief of the X-Y linker autoinhibition through an allosteric mechanism. We also determined membrane-bound structures of the PLC{beta}3-Gq, and PLC{beta}3-G{beta}{gamma}(2)-Gq complexes, which show that these G proteins can bind simultaneously and independently of each other to regulate PLC{beta}3 activity. The structures rationalize a finding in the enzyme assay, that co-stimulation by both G proteins follows a product rule of each independent stimulus. We conclude that baseline activity of PLC{beta}3 is strongly suppressed, but the effect of G proteins, especially acting together, provides a robust stimulus upon G protein stimulation. Significance StatementFor certain cellular signaling processes, the background activity of signaling enzymes must be minimal and stimulus-dependent activation robust. Nowhere is this truer than in signaling by PLC{beta}3, whose activity regulates intracellular Ca2+, phosphorylation by Protein Kinase C, and the activity of numerous ion channels and membrane receptors. In this study we show how PLC{beta}3 enzymes are regulated by two kinds of G proteins, G{beta}{gamma} and Gq. Enzyme activity studies and structures on membranes show how these G proteins act by separate, independent mechanisms, leading to a product rule of co-stimulation when they act together. The findings explain how cells achieve robust stimulation of PLC{beta}3 in the setting of very low background activity, properties essential to cell health and survival.

biophysics↗

bioRxiv2022

Gβγ Activates PIP2 Hydrolysis by Recruiting and Orienting PLCβ on the Membrane Surface

PLC{beta}s catalyze the hydrolysis of PIP2 into IP3 and DAG. PIP2 regulates the activity of many membrane proteins, while IP3 and DAG lead to increased intracellular Ca2+ levels and activate PKC, respectively. PLC{beta}s are regulated by GPCRs through direct interaction with Gq and G{beta}{gamma}. This study addresses the mechanism by which G{beta}{gamma} activates PLC{beta}3. We show that PLC{beta}3 functions as a slow Michaelis-Menten enzyme (kcat~2 sec-1, KM~0.43 mol%) on membrane surfaces. Its partition coefficient (Kx~2.9 * 104) is such that only a small quantity of PLC{beta}3 exists in the membrane in the absence of G{beta}{gamma}. When G{beta}{gamma} is present, equilibrium binding (Keq~0.009 mol%) increases PLC{beta}3 in the membrane, increasing Vmax in proportion. Atomic structures on membrane vesicle surfaces show that two G{beta}{gamma} anchor PLC{beta}3 with its catalytic site oriented toward the membrane surface. This principle of activation explains rapid stimulated catalysis with low background catalysis.

biophysics↗

bioRxiv2018

The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K

Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase and ion channel activity, or specific chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident calcium-regulated lipid scramblase. Our crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional structures solved by cryo-EM reveal extensive conformational changes extending from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity. Our results suggest mechanisms by which missense variants of TMEM16K could cause SCAR10 ataxia, providing new hypotheses to explore for therapy.

biochemistry↗

bioRxiv2018

Cryo-EM structures reveal bilayer remodeling during Ca2+ activation of a TMEM16 scramblase

The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several members of the TMEM16 family have been identified as Ca2+-activated scramblases but the mechanisms underlying their Ca2+-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs, revealing large Ca2+-dependent conformational changes of the protein as well as significant, function dependent membrane reorganization.

biochemistry↗