Biomedical subjects

Irina Zaitseva

Publications and source records attributed to Irina Zaitseva.

5 recordsLinked to original sources

PubMed2007

Membrane-permeable calmodulin inhibitors (e.g. W-7/W-13) bind to membranes, changing the electrostatic surface potential: dual effect of W-13 on epidermal growth factor receptor activation.

Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca2+/CaM) in living cells. If two chemically different inhibitors (e.g. W-7 and trifluoperazine) produce similar effects, investigators often assume the effects are due to CaM inhibition. Zeta potential measurements, however, show that these amphipathic weak bases bind to phospholipid vesicles at the same concentrations as they inhibit Ca2+/CaM; this suggests that they also bind to the inner leaflet of the plasma membrane, reducing its negative electrostatic surface potential. This change will cause electrostatically bound clusters of basic residues on peripheral (e.g. Src and K-Ras4B) and integral (e.g. epidermal growth factor receptor (EGFR)) proteins to translocate from the membrane to the cytoplasm. We measured inhibitor-mediated translocation of a simple basic peptide corresponding to the calmodulin-binding juxtamembrane region of the EGFR on model membranes; W-7/W-13 causes translocation of this peptide from membrane to solution, suggesting that caution must be exercised when interpreting the results obtained with these inhibitors in living cells. We present evidence that they exert dual effects on autophosphorylation of EGFR; W-13 inhibits epidermal growth factor-dependent EGFR autophosphorylation under different experimental conditions, but in the absence of epidermal growth factor, W-13 stimulates autophosphorylation of the receptor in four different cell types. Our interpretation is that the former effect is due to W-13 inhibition of Ca2+/CaM, but the latter results could be due to binding of W-13 to the plasma membrane.

Animals↗

PubMed2006

Membrane-bound basic peptides sequester multivalent (PIP2), but not monovalent (PS), acidic lipids.

Several biologically important peripheral (e.g., myristoylated alanine-rich C kinase substrate) and integral (e.g., the epidermal growth factor receptor) membrane proteins contain clusters of basic residues that interact with acidic lipids in the plasma membrane. Previous measurements demonstrate that the polyvalent acidic lipid phosphatidylinositol 4,5-bisphosphate is bound electrostatically (i.e., sequestered) by membrane-adsorbed basic peptides corresponding to these clusters. We report here three experimental observations that suggest monovalent acidic lipids are not sequestered by membrane-bound basic peptides. 1), Binding of basic peptides to vesicles does not decrease when the temperature is lowered below the fluid-to-gel phase transition. 2), The binding energy of Lys-13 to lipid vesicles increases linearly with the fraction of monovalent acidic lipids. 3), Binding of basic peptides to vesicles produces no self-quenching of fluorescent monovalent acidic lipids. One potential explanation for these results is that membrane-bound basic peptides diffuse too rapidly for the monovalent lipids to be sequestered. Indeed, our fluorescence correlation spectroscopy measurements show basic peptides bound to phosphatidylcholine/phosphatidylserine membranes have a diffusion coefficient approximately twofold higher than that of lipids, and those bound to phosphatidylcholine/phosphatidylinositol 4,5-bisphosphate membranes have a diffusion coefficient comparable to that of lipids.

Amino Acid Sequence↗

PubMed2005

Molecular characterization of virulent Newcastle disease virus isolates from chickens during the 1998 NDV outbreak in Kazakhstan.

Newcastle disease virus (NDV) infects domesticated and wild birds throughout the world and has the possibility to cause outbreaks in chicken flocks in future. To assess the evolutionary characteristics of 10 NDV strains isolated from chickens in Kazakhstan during 1998 we investigated the phylogenetic relationships among these viruses and viruses described previously. For genotyping, fusion (F) gene phylogenetic analysis (nucleotide number 47-421) was performed using sequences of Kazakhstanian isolates as compared to sequences of selected NDV strains from GenBank. Phylogenetic analysis showed that all newly characterized strains belonged to the genetic group designated as VIIb. All strains possessed a virulent fusion cleavage site (RRQRR/F) belonging to velogenic or mesogenic pathotypes with intracerebral pathogenicity indexes (ICPI) varying from 1.05 to 1.87.

Amino Acid Sequence↗

PubMed2005

Reversible - through calmodulin - electrostatic interactions between basic residues on proteins and acidic lipids in the plasma membrane.

The inner leaflet of a typical mammalian plasma membrane contains 20-30% univalent PS (phosphatidylserine) and 1% multivalent PtdIns(4,5)P(2). Numerous proteins have clusters of basic (or basic/hydrophobic) residues that bind to these acidic lipids. The intracellular effector CaM (calmodulin) can reverse this binding on a wide variety of proteins, including MARCKS (myristoylated alanine-rich C kinase substrate), GAP43 (growth-associated protein 43, also known as neuromodulin), gravin, GRK5 (G-protein-coupled receptor kinase 5), the NMDA (N-methyl-D-aspartate) receptor and the ErbB family. We used the first principles of physics, incorporating atomic models and the Poisson-Boltzmann equation, to describe how the basic effector domain of MARCKS binds electrostatically to acidic lipids on the plasma membrane. The theoretical calculations show the basic cluster produces a local positive electrostatic potential that should laterally sequester PtdIns(4,5)P(2), even when univalent acidic lipids are present at a physiologically relevant 100-fold excess; four independent experimental measurements confirm this prediction. Ca(2+)/CaM binds with high affinity (K(d) approximately 10nM) to this domain and releases the PtdIns(4,5)P(2). MARCKS, a major PKC (protein kinase C) substrate, is present at concentrations comparable with those of PtdIns(4,5)P(2) (approx. 10 microM) in many cell types. Thus MARCKS can act as a reversible PtdIns(4,5)P(2) buffer, binding PtdIns(4,5)P(2) in a quiescent cell, and releasing it locally when the intracellular Ca(2+) concentration increases. This reversible sequestration is important because PtdIns(4,5)P(2) plays many roles in cell biology. Less is known about the role of CaM-mediated reversible membrane binding of basic/hydrophobic clusters for the other proteins.

Animals↗

PubMed2004

Electrostatic sequestration of PIP2 on phospholipid membranes by basic/aromatic regions of proteins.

The basic effector domain of myristoylated alanine-rich C kinase substrate (MARCKS), a major protein kinase C substrate, binds electrostatically to acidic lipids on the inner leaflet of the plasma membrane; interaction with Ca2+/calmodulin or protein kinase C phosphorylation reverses this binding. Our working hypothesis is that the effector domain of MARCKS reversibly sequesters a significant fraction of the L-alpha-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PIP2) on the plasma membrane. To test this, we utilize three techniques that measure the ability of a peptide corresponding to its effector domain, MARCKS(151-175), to sequester PIP2 in model membranes containing physiologically relevant fractions (15-30%) of the monovalent acidic lipid phosphatidylserine. First, we measure fluorescence resonance energy transfer from Bodipy-TMR-PIP2 to Texas Red MARCKS(151-175) adsorbed to large unilamellar vesicles. Second, we detect quenching of Bodipy-TMR-PIP2 in large unilamellar vesicles when unlabeled MARCKS(151-175) binds to vesicles. Third, we identify line broadening in the electron paramagnetic resonance spectra of spin-labeled PIP2 as unlabeled MARCKS(151-175) adsorbs to vesicles. Theoretical calculations (applying the Poisson-Boltzmann relation to atomic models of the peptide and bilayer) and experimental results (fluorescence resonance energy transfer and quenching at different salt concentrations) suggest that nonspecific electrostatic interactions produce this sequestration. Finally, we show that the PLC-delta1-catalyzed hydrolysis of PIP2, but not binding of its PH domain to PIP2, decreases markedly as MARCKS(151-175) sequesters most of the PIP2.

Amino Acid Sequence↗