Mario J. Rebecchi, Ph.D.

e-mail: Mario.Rebecchi@stonybrook.edu
Phone: (631) 444-8178
Fax: (631) 444-2907

Current members of the Rebecchi Lab:
Srinivas Pentyala, Ph.D. Pharmacology
Edward Tall, B.S. Physics (Ph.D. candidate, Biophysics Program)
Laura Cipp, M.S. Pharmacology
Shobha Mathew, B.S. Biology  (M.S. candidate)

Summary of Research Interests
Our group is working on two main questions: (1) How do inositol lipids and their metabolites operate as intracellular signals? and (2) How do volatile anesthetics affect allosteric proteins?   To address these issues we study the protein and lipid players from the molecular to the cellular level.  Currently we use a number of sophisticated technologies including isothermal titration calorimetry, fluorescence spectroscopy, epi and confocal fluorescence microscopy, and the latest recombinant DNA methodology.  We are currently looking for well-qualified candidates at post-doctoral, graduate and undergraduate levels to take part in these projects.

Phospholipases and inositol lipids  Our research is aimed at understanding the molecular basis for the actions and control of intracellular phospholipases.  The efforts are focused on phospholipase C (PLC), an enzyme found in every eukaryotic organism, from yeast to man.  This soluble enzyme catalyzes the hydrolysis of a rare lipid, phosphatidylinositol-4,5- bisphosphate (PIP2), generating the universal intracellular second messengers, inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG).  InsP3 is a soluble product that opens an internal InsP3-gated cation channel permitting the flow of calcium from intracellular stores into the cytosol.  The other second messenger, DAG, is a lipid that diffuses laterally in the membrane bilayer where it activates a serine/ threonine protein kinase, PKC, that phosphorylates (and regulates) other signaling proteins.  Activation of PKC and the rise in cytoplasmic calcium triggers cellular responses ranging from secretion to cell division.

Artificial and biological membrane bilayers are used to study PLC enzymology. Substrate analogs are used to understand the structure of the membrane anchoring and active sites. Effects of lipid packing and surface potential are also investigated.   A combination of physical, chemical and molecular biological approaches are utilized to understand how the enzyme absorbs to membrane surfaces and to identify those features important in processive hydrolysis of substrate.

On a cellular level the distribution and dynamics of PLC and its substrate (PIP2) are examined, in vivo, by fluorescence microscopy utilizing green and red fluorescent protein tags, and the latest digital imaging technology.  Our work indicates that one form of PLC, PLC-d1, and its substrate, PIP2, are concentrated in actin-supported structures (left figure below, arrows).  We propose that calcium itself stimulates this PLC isoform degrading PIP2 locally, leading to collapse of the dendritic actin network that underpins these extensions of the plasma membrane.  Click here to play an animated GIF series.

Touching on the fields of developmental and cancer biology, the results of this project should help us understand how cells exert control of their own shape and motility.   This work is supported by a grant from the National Institutes of Health: GM R01-43422.

Volatile anesthetics and GTP-binding proteins  A very different project addresses the question of how volatile anesthetics interact with allosteric proteins.    Discovered over 150 years ago, these agents (such as ether and halothane) produce immobility, analgesia and amnesia, yet the mechanism by which they work has yet to be elucidated.  Because they partition so readily into the lipid bilayer, previous theories of anesthesia have postulated that general anesthetics affect membrane electrical excitability by changing the way lipids interact with channels.   Nice idea, but there's not much data to support it.

Alternatively these drugs may partition into voids or crevices in transmembrane, and even soluble proteins, thereby affecting their stability and/or conformation.  Based on the work of Franks and Leib (Imperial College) and Eckenhoff (University of Pennsylvania), it is clear that volatile anesthetics bind directly to proteins.  The left panel shows the anesthetic bromoform bound to luciferase, a soluble model protein target.  The drug binds in a pocket with a suprising degree of polarity.  In our own work, we have explored the actions of these drugs on a well understood allosteric enzyme, G-alpha-i1, which, along with appropriate beta and gamma subunits, forms the familiar the heterotrimeric GTP-binding protein (right panel) that couples heptahelical receptors to their downstream effectors, including various second messenger generating enzymes and ion channels.

Dr. Pentyala, a senior scientist in our group, has discovered that some GTP-binding proteins are very sensitive to volatile anesthetics, particularly to members of the haloalkane and haloether classes.  Specifically these agents inhibit GDP/GTP exchange on G-alpha-i1, alpha-i2 and alpha-i3 subunits, but not the closely related alpha-o.  These same volatile anesthetics promote the association of alpha-i, but not alpha-o with G-betagamma subunits, independently of the guanine nucleotide bound - that is they appear to stabilize association of the betagamma heterodimer with the G-alpha subunit, even when GTP is bound.

Because the three-dimensional structures of some G-proteins are known, it is possible to propose a molecular hypothesis as to how anesthetics enhance the binding of betagamma to G-alpha subunits.  The right-hand panel (above) is a molecular cartoon borrowed from an article by Henry Bourne.  It depicts the alpha subunit in three states: the inactive GDP, active GTP and an empty transition state that is specifically stabilized by agonist-bound receptor.  All alpha subunits are divided into a well-conserved ras domain, and an alpha helical domain unique to G-alpha subunits. The guanine nucleotide, shown in red, binds to the cleft between these two domains.  Binding of GTP and a single Mg ion orders several alpha helices and loops known as the switch regions, shown in green.  The GTP-bound conformation of the switch regions are recognized by effector enzymes such as PLC and adenylyl cyclase.  GTP hydrolysis leads to the retention of strongly bound GDP, but disorders most of the switch region.  The switch conformations (GTP or GDP state) are coupled to formation of an activation/recognition surface for G-betagamma heterodimer and and agonist-activated receptor.   This recognition surface is stabilized in the GDP state, but disordered when GTP is bound.   By stabilizing this otherwise disordered microdomain, anesthetics allow the GTP-state to mimic the GDP form thereby promoting the binding of G-betagamma and activated receptor.  This could effectively uncouple agonist binding from downstream signaling pathways, accounting for at least some of the neurophysiologic actions of general anesthetic agents.   To test these ideas further, fluorescence, thermodynamic, and crystallographic studies of the G-protein/anesthetic complex are under way.  This work is supported by a separate grant from the National Institutes of Health: GM-R01-60376.

Guo Y, Rebecchi M, Scarlata S. Phospholipase Cβ2 binds to and inhibits phospholipase Cδ1. J Biol Chem. 2005 Jan 14;280(2):1438-47.

Stallings JD, Tall EG, Pentyala S, Rebecchi MJ. Nuclear translocation of phospholipase C-δ1 is linked to the cell cycle and nuclear phosphatidylinositol 4,5-bisphosphate. J Biol Chem. 2005 Apr 4

Sawas A, Pentyala S, Rebecchi M Binding of volatile anesthetics to serum albumin: measurements of enthalpy and solvent contributions. Biochemistry 2004 Oct 5;43(39):12675-8

Pentyala S, Halpern-Lewis J, Sawas A, Rosa D, Sawas Ay, Rebecchi M, Vitkun S. Intravenous anesthetics reduce airway resistance by their action on plasma membrane calcium pump. In: Molecular and Basic Mechanisms of Anesthesia, BW Urban and M Barann, eds., Pabst Scientific Publishers, Lengerich, Germany

Rebecchi MJ, Pentyala SN. Anaesthetic actions on other targets:protein kinase C and guanine nucleotide-binding proteins. Br. J. Anaesth. 2002 89: 62-78.

Galneder R, Kahl V, Arbuzova A, Rebecchi M, Radler JO, McLaughlin S. Microelectrophoresis of a Bilayer-Coated Silica Bead in an Optical Trap: Application to Enzymology. Biophys J. 2001 May;80(5):2298-2309

Khan RN, Tall EG, Rebecchi M, Ramsdell JS, Pentyala S. Effect of maitotoxin on guanine nucleotide interaction with G-protein alpha subunits.  Int J Toxicol. 2001 Jan-Feb;20(1):39-44

Pentyala SN, Lee J, Hsieh K, Waltzer WC, Trocchia A, Musacchia L, Rebecchi MJ, Khan SA. Prostate cancer: a comprehensive review. Med Oncol. 2000 May;17(2):85-105

Rebecchi MJ, Pentyala SN.  Structure, function, and control of phosphoinositide-specific phospholipase C.
Physiol Rev. 2000 Oct;80(4):1291-335.  [Abstract]

Tall EG, Spector I, Pentyala SN, Bitter I, Rebecchi MJ. Dynamics of phosphatidylinositol 4,5-bisphosphate in actin-rich structures. Curr Biol. 2000 Jun 15;10(12):743-746. [Abstract]

Wang T, Dowal L, El-Maghrabi MR, Rebecchi M, Scarlata S. The pleckstrin homology domain of phospholipase C-beta(2) links the binding of gbetagamma to activation of the catalytic core. J Biol Chem. 2000 Mar 17;275(11):7466-9. [Abstract]

Rebecchi MJ, Bonhomme M, Scarlata S.
Role of lipid packing in the activity of phospholipase C-delta1 as determined by real time hydrostatic pressure measurements.
Biochem. J. 341:571-576. 1999. [abstract]

Wang T, Pentyala S, Elliott J, Dowal L, Gupta E, Rebecchi M, Scarlata S. Selective Interaction of the C2 domains of phospholipase C-b1 and b2 with activated Gaq subunits: A novel function for C2 signaling modules. Proc. Natl. Acad. Sci., 96:7843-7846, 1999.
[abstract]

Wang T, Pentyala S, Gupta E, Rebecchi M, Scarlata S. Membrane association of C2 domain of Phosphoinositide-specific phospholipase C b1, b2 and d1. Biophys. J. 76:A212,1999. [Abstract]

Wang T, Pentyala S, Rebecchi MJ, Scarlata S.  Differential association of the pleckstrin homology domains of phospholipases C-beta 1, C-beta 2, and C-delta 1 with lipid bilayers and the beta gamma subunits of heterotrimeric G proteins. Biochemistry 38: 1517-1524, 1999.

Wang,T.; Pentyala,S.; Rebecchi,M.J.; Scarlata,S.
Differential association of the pleckstrin homology domains of phospholipases C beta1, C- beta2, and C-delta 1 with lipid bilayers and the beta gamma subunits of heterotrimeric G proteins.
Tristate G-protein Workshop, Mount Sinai School of Medicine, NY, 1998

Pentyala S, Sung K-Y, Chowdhury A, Rebecchi MR. Volatile anesthetics modulate the binding of guanine nucleotides to the alpha subunits of heterotrimeric GTP binding proteins. European Jounal of Pharmacology 384: 213-222 1999.[Abstract]

Pentyala, S.; Moller,D.; Chowdhury,A.; Sung,K.Y.; Rebecchi,M.J.
Effects of inhalational anesthetics on alpha 2 adrenergic signaling in isolated platelets.
Toxicology Letters 100-101:115-120, 1998

Pentyala,S.N.; Sung,K.Y.; Chowdhury,A.; Rebecchi,M.J.
Volatile anesthetics modulate the interaction of guanine nucleotides with GTP binding proteins.
AUA 45th Annual Meeting Report :57, 1998

Pentyala S, Wang T, Rebecchi MJ, Scarlata S.
Expression and characterization of the pleckstrin homology domains of phospholipase-C isozymes.
Intl. Congress of Immunology Abstracts, 1998.

Rebecchi,M.J.; Scarlata,S.
Pleckstrin homology domains: a common fold with diverse functions.
Ann. Rev. Biophys. Biomol. Struct. 27:503-528, 1998

Tall E. Dorman G. Garcia P. Runnels L. Shah S. Chen J. Profit A. Gu QM. Chaudhary A. Prestwich GD. Rebecchi MJ.
Phosphoinositide binding specificity among phospholipase C isozymes as determined by photo-cross-linking to novel substrate and product analogs.
Biochemistry36:7239-7248, 1997

Scarlata S. Gupta R. Garcia P. Keach H. Shah S. Kasireddy CR. Bittman R. Rebecchi MJ.
Inhibition of phospholipase C-d₁ catalytic activity by sphingomyelin.
Biochemistry 35:14882-14888, 1996

Garcia, P., Gupta, R., Shah, S., Morris, A., Rudge, S., Scarlata, S., Petrova, V., McLaughlin, S. and Rebecchi, M.
The pleckstrin homology domain of phospholipase C-d₁ binds with high affinity to phosphatidylinositol 4,5-bisphosphate in bilayer membranes.
Biochemistry 34: 16228-16234, 1995.

Cifuentes, M.E., Delaney, T. and Rebecchi, M.J.
D-myo-iositol 1,4,5-trisphosphate inhibits binding of phospholipase C-d₁ to bilayer membranes.
Journal of Biological Chemistry 269:1945-1948, 1994.

Boguslavsky, V., Rebecchi, M.J., Morris, A.J., Jhon, D.-Y., Rhee, S.G. and McLaughlin, S.
Effect of monolayer surface pressure on the activities of phosphoinositide-specific phospholipase C-b₁, -g₁ and -d₁.
Biochemistry 33: 3032-3037, 1994.

Rebecchi, M.J., Eberhardt, R., Delaney, T., Ali, S., and Bittman, R.
Hydrolysis of short acyl-chain inositol lipids by phospholipase C-d₁.
Journal of Biological Chemistry 268: 1735-1741, 1993.

Cifuentes, M., Honkanen, L., and Rebecchi, M. J.
Proteolytic fragments of phosphoinositide-specific phospholipase C-d₁: Their catalytic and membrane binding properties.
Journal of Biological Chemistry 268:11586-11593, 1993.