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S Zozulya

Publications and source records attributed to S Zozulya.

9 recordsLinked to original sources
PubMed2001

The human olfactory receptor repertoire.

BACKGROUND: The mammalian olfactory apparatus is able to recognize and distinguish thousands of structurally diverse volatile chemicals. This chemosensory function is mediated by a very large family of seven-transmembrane olfactory (odorant) receptors encoded by approximately 1,000 genes, the majority of which are believed to be pseudogenes in humans. RESULTS: The strategy of our sequence database mining for full-length, functional candidate odorant receptor genes was based on the high overall sequence similarity and presence of a number of conserved sequence motifs in all known mammalian odorant receptors as well as the absence of introns in their coding sequences. We report here the identification and physical cloning of 347 putative human full-length odorant receptor genes. Comparative sequence analysis of the predicted gene products allowed us to identify and define a number of consensus sequence motifs and structural features of this vast family of receptors. A new nomenclature for human odorant receptors based on their chromosomal localization and phylogenetic analysis is proposed. We believe that these sequences represent the essentially complete repertoire of functional human odorant receptors. CONCLUSIONS: The identification and cloning of all functional human odorant receptor genes is an important initial step in understanding receptor-ligand specificity and combinatorial encoding of odorant stimuli in human olfaction.

Amino Acid Motifs↗
PubMed1999

Mapping signal transduction pathways by phage display.

Rapid identification of proteins that interact with a novel gene product is an important element of functional genomics. Here we describe a phage display-based technique for interaction screening of complex cDNA libraries using proteins or synthetic peptides as baits. Starting with the epidermal growth factor receptor (EGFR) cytoplasmic tail, we identified known protein interactions that link EGFR to the Ras/MAP kinase signal transduction cascade and several novel interactions. This approach can be used as a rapid and efficient tool for elucidating protein networks and mapping intracellular signal transduction pathways.

3T3 Cells↗
PubMed1998

The effect of recombinant recoverin on the photoresponse of truncated rod photoreceptors.

Recoverin is a heterogeneously acylated calcium-binding protein thought to regulate visual transduction. Its effect on the photoresponse was investigated by dialyzing the recombinant protein into truncated salamander rod outer segments. At high Ca2+ (Ca), myristoylated recoverin (Ca-recoverin) prolonged the recovery phase of the bright flash response but had less effect on the dim flash response. The prolongation of recovery had an apparent Kd for Ca of 13 microM and a Hill coefficient of 2. The prolongation was shown to be mediated by inhibition of rhodopsin deactivation. After a sudden imposed drop in Ca concentration, the effect of recoverin switched off with little lag. The myristoyl (C14:0) modification of recoverin increased its activity 12-fold, and the C12:0 or C14:2 acyl group gave similar effects. These experiments support the notion that recoverin mediates Ca-dependent inhibition of rhodopsin phosphorylation and thereby controls light-triggered phosphodiesterase activity, particularly at high light levels.

Ambystoma↗
PubMed1996

Differentiation induced by the c-Mpl cytokine receptor is blocked by mutant Shc adaptor protein.

c-Mpl, a member of the cytokine receptor superfamily, induces both proliferative and differentiation responses when stimulated with its ligand thrombopoietin (TPO). To examine signal transduction pathways associated with differentiation versus proliferation, 32D clone 3 cells, a murine interleukin 3-(IL-3)-dependent cell line capable of granulocytic differentiation, were engineered to express human c-Mpl (designated 32DM.2). Human TPO-containing medium was produced by transient transfection of 293 cells. Treatment of 32DM.2 cells with human TPO induced cellular aggregates within 12 h of exposure to ligand. 32DM.2 cells maintained in the presence of TPO did not change in cell number over a 72-h period and acquired characteristics of granulocytic differentiation as evidenced by metamyelocytic cellular morphology. The differentiation effect of TPO was observed in the absence and presence of the mitogen IL-3. Evaluation of protein tyrosine phosphorylation following exposure to ligand revealed that TPO stimulation induced an elevated level of tyrosine phosphorylation of the adaptor protein Shc when compared with IL-3. However, treatment of 32DM.2 cells with TPO did not result in the phosphorylation of mitogen-activated protein kinase (MAPK). To evaluate the potential role of Shc in c-Mpl differentiation, we transfected 32DM.2 cells with a mutant Shc gene that lacked the region coding for the phosphotyrosine interaction domain (delta PI-Shc). Expression of the delta PI-Shc protein in 32DM.2 cells blocked the TPO differentiation response with no effect on IL-3-stimulated proliferation. These studies demonstrate that c-Mpl-induced differentiation results from the activation of signal transduction pathways that are dominant to the IL-3 proliferative response and independent of the Ras/MAPK signal transduction pathway. The ability of the delta PI-Shc protein to block TPO-induced differentiation implicates Shc as a mediator of signal transduction pathways leading to differentiation, which is distinct from its role as a mediator in activating the Ras/MAPK pathway.

Animals↗
PubMed1993

Three-dimensional structure of recoverin, a calcium sensor in vision.

Recoverin, a recently discovered member of the EF hand superfamily, serves as a calcium sensor in vision. We report here the crystal structure of recombinant unmyristoylated recoverin at 1.9 A resolution. The four EF hands of the protein are arranged in a compact array that contrasts with the dumbbell shape of calmodulin and troponin C. A calcium ion is bound to EF hand 3, while EF hand 2 can bind samarium but not calcium in this crystal form. The other two EF hands have novel structural features that prevent or impair calcium binding. A concave hydrophobic surface formed by EF hands 1 and 2 may participate in the read out of calcium signals by recoverin and its homologs.

Amino Acid Sequence↗
PubMed1992

Calcium-myristoyl protein switch.

Recoverin, a recently discovered member of the EF-hand superfamily of Ca(2+)-binding proteins, serves as a Ca2+ sensor in vision. The amino terminus of the protein from retinal rod cells contains a covalently attached myristoyl or related N-acyl group. We report here studies of unmyristoylated and myristoylated recombinant recoverin designed to delineate the biological role of this hydrophobic unit. Ca2+ induces the binding of both the unmyristoylated and myristoylated proteins to phenyl-agarose, a hydrophobic support. Binding was half-maximal at 1.1 and 1.0 microM Ca2+, respectively. The Hill coefficients of 1.8 and 1.7, respectively, indicate that binding was cooperative. In contrast, Ca2+ induced the binding of myristoylated but not of unmyristoylated recoverin to rod outer segment membranes. Binding to these membranes was half-maximal at 2.1 microM Ca2+, and the Hill coefficient was 2.4. Likewise, myristoylated but not unmyristoylated recoverin exhibited Ca(2+)-induced binding to phosphatidylcholine vesicles. These findings suggest that the binding of Ca2+ to recoverin has two effects: (i) hydrophobic surfaces are exposed, allowing the protein to interact with complementary nonpolar sites, such as the aromatic rings of phenyl-agarose; and (ii) the myristoyl group is extruded, enabling recoverin to insert into a lipid bilayer membrane. The myristoyl group is likely to be an active participant in Ca2+ signaling by recoverin and related EF-hand proteins such as visinin and neurocalcin.

Amino Acid Sequence↗
PubMed1992

The NH2 terminus of retinal recoverin is acylated by a small family of fatty acids.

Recoverin is a recently identified Ca(2+)-binding protein that imparts Ca2+ sensitivity to vertebrate photoreceptor guanylate cyclase. In response to photo-induced depletion of intracellular cGMP and Ca2+, recoverin stimulates resynthesis of cGMP. Bovine retinal recoverin has now been analyzed by electrospray mass spectrometry (ESI-MS) for post-translational modifications that might influence its activity. Heterogeneous acylation was detected at the NH2 terminus of bovine retinal recoverin. The NH2-terminal glycine of each retinal recoverin molecule is linked to one of four different types of acyl groups. The most abundant is myristoleate (14:1), but 14:0, 14:2, and 12:0 acyl residues are also present.

Acylation↗
PubMed1992

Cloning, expression, and crystallization of recoverin, a calcium sensor in vision.

Recoverin, a recently discovered 23-kDa calcium-binding protein, activates retinal rod guanylate cyclase when the calcium level is lowered in the submicromolar range. We report here the cloning and sequencing of a cDNA for recoverin from a bovine retinal expression library. The recoverin coding sequence was inserted into a pET-11a expression vector under control of the T7 phage promoter. A second expression system, in which the coding sequence was placed under control of the lambda phage PR promoter, gave 10-fold higher yields (10 mg of purified recoverin per liter of Escherichia coli culture). The finding that retinal recoverin is myristoylated at its amino terminus led us to coexpress the recombinant protein and N-myristoyltransferase (EC 2.3.1.97). Myristoylated recombinant recoverin formed in this way in E. coli is like retinal recoverin in exhibiting a large calcium-induced shift in its tryptophan fluorescence emission spectrum. The availability of abundant protein enabled us to crystallize unmyristoylated recombinant recoverin and initiate x-ray studies. The space group of tetragonal crystals obtained from 75% saturation ammonium sulfate is I4 with unit cell dimensions a = 85.1 A and c = 59.8 A. These crystals of the calcium-bound form of the protein diffracted to a resolution of 2.2 A. The expression systems described here open the door to high-resolution x-ray crystallographic and nuclear magnetic resonance studies of this new member of the EF-hand superfamily and to the elucidation of its precise mode of action as a calcium switch.

Amino Acid Sequence↗