The University of Texas Health Science Center at Houston
McGovern Medical School
Department of Biochemistry and Molecular Biology
Topobiology (lipid and protein topogenesis) and bioconformatics
Lipid-assisted dynamic membrane protein topogenesis.
The goal of the first project in the lab is to investigate the molecular mechanism and physiological significance of lipid-dependent membrane protein topogenesis by testing a novel Charge Balance Rule for different proteins and lipid profiles in vivo and in vitro in order to establish the physiological significance of conformational and topological heterogeneity, co-translational, post-translational and post-insertional dynamic changes in topological organization of membrane proteins in bacterial, “healthy” mammalian cells and its organelles, resting and stressed cancer cells. The subaim of this project is to test the Charge Balance Rule in silico and verify the concept of the funnel-like intermolecular energy landscape of lipid–protein transient interactions by using of a series of in vivo “translocation force” measurements and computational experiments. The aim of this project is also to test Charge Balance Rule for polytopic membrane protein assembly in cancer cells where changes in lipid asymmetry and external environment occur regularly due to development of this severe disease. Whether transmembrane protein topology in cancer cells is affected by the changes in lipid asymmetry and charge distribution across the membrane is still unknown, thus Charge Balance Rule will be tested at first time for eukaryotic membrane protein.
Dynamic membrane asymmetry.
Why cell membrane is (a)symmetric? When proteins and lipids are synthesized in the cell, they are inserted into membrane in an asymmetric fashion. Is this because they are inserted primarily in genetically and structurally predefined asymmetric manner and govern asymmetric distribution of lipids? Or net lipid asymmetry could be in place to help balance the asymmetric charges of membrane proteins and orient them in accordance with Positive Inside Rule?
Lipid asymmetry in membranes is a consequence of multiple factors, including the physical properties of lipids that dictate their ability to spontaneously “flip or not to flip” their polar headgroups through the hydrophobic membrane interior and the presence of active phospholipid pumps (ATP-dependent flippases) and ATP-independent scramblases, which are able to create or dissipate a transmembrane lipid asymmetry and maintains such non-equilibrium thermodynamic state of the membrane. Biogenic membranes contain the enzymes and machineries that continuously synthesize the membrane lipids and proteins respectively but only on one cytosolic leaflet of the bilayer. Thus alternatively lipid asymmetry can be metabolically controlled to balance temporally the net rates of synthesis and flip-flop and adjust bilayer chemical and physical properties as spontaneous response.
We are still far from full understanding of the physiological significance and detailed molecular mechanisms by which membrane phospholipid asymmetry is generated, maintained and modulated. We pioneered methods to interrogate lipid enzymology and individual phospholipid topography (transmembrane sidedness) using novel assays to establish the function of individual lipid enzymes and lipids in such complex processes as translocation of lipids and proteins across the lipid bilayer, folding and topogenesis, cancerogenesis, sickle cell disease and chronic kidney disease. We utilized recently vectoral chemical and fluoresecent probes with different membrane penetrating and chemical properties to report a novel methodology to determine head group and acyl asymmetry of aminophospholipids using radiometric, mass-spectrometric methods and spectrophotometric approaches. This novel approach is suitable for investigation of steady-state distribution and transbilayer dynamics of aminolipid (phosphatidylserine (PS) and phosphatidylethanolamine (PE) in any cellular organelle (erythrocytes, exosomes, apoptotic bodies, mitoplasts etc.) or any single membrane systems including liposomes , inner membrane (IM) or outer membrane (OM) of diderm Gram-negative bacteria using either radiometric, spectrophotometric or mass-spectrometric methods. This novel approach is suitable for investigation of steady-state distribution and transbilayer dynamics of aminolipids (phosphatidylserine (PS) and phosphatidylethanolamine (PE) in any cellular organelle (erythrocytes, exosomes, apoptotic bodies, mitoplasts, etc.) surrounded by a single membrane. We demonstrate that the IM of Escherichia coli and Yersinia pseudotuberculosis is asymmetric with 75%/25% (cytoplasmic/periplasmic leaflet) distribution of PE in rod-shaped cells and an opposite distribution in filamentous cells. Moreover PE and CL are dynamically redistributed across IM to follow or direct the changes in bacterial shape.
Whether such flippase-less or “lipid only” “passive” mechanism of generation and maintenance of lipid asymmetry exists?
What is the reason for the cell membrane to be (a)symmetric in biogenic- and non-biogenic cells, healthy and cancer cells?
The goal of this project is to test a hypothesis that specific lipids (cold and shock lipids in this case) can optimize protein function and conformation and promote proper folding at low or elevated temperatures i.e. act in the way molecular chaperones of protein origin do. The aim of this project is to test a hypothesis that conformation of outer membrane b-barrel porins from pathogenic bacteria may adapt to in vivo changes in phospholipid composition in order to optimize or modulate their activity or folding and assembly pathway. The main hypothesis to be tested is that endogenous lipids with different molecular structure and thermotropic behavior act as molecular chaperones affecting conformational maturation of the model channel-forming oligomeric protein porin YOmpF from Gram-negative psychrotrophic bacteria Y. pseudotuberculosis. The second aim of this project is to investigate the role of glycerophospholipid asymmetry, externalization and remodeling within bacterial envelope in the development of bacterial resistance to antibiotics and ability to resist innate immunity of the host.
Bacterial envelope remodeling and development of resistance to antibiotics and innate immunity system.
The goal of this project is to investigate the physiological significance of the “lipid” genes multiplicity and lipid interchangeability in Gram-negative bacteria and the role of transmembrane phospholipid asymmetry and remodeling within bacterial envelope in the development of resistance to antibiotics, intracellular survival and ability to resist innate immunity of infected host.
Lipids and SecYEG translocon.
The goal of this project to investigate the role of the membrane phospholipids in the translocon-assisted and unassisted protein translocation and insertion of membrane proteins. Membrane phospholipids might exert their effect on nascent protein either directly by affecting its conformation and interacting with topogenic signals distributed throughout of translocating or inserting polypeptide or indirectly by influencing the translocation and insertion machineries.
Keywords: Membrane protein · Phospholipid · Protein Topogenesis · Charge Balance Rule · Protein folding · Lipochaperones · Envelope remodeling · Antibiotic resistance · Lipid asymmetry · Translocon
Education & Training
Ph.D. - USSR Academy of Sciences - 1989