Biomedical subjects
Publications and source records attributed to D Krylov.
We show how a nonlinear system that supports solitons can be driven to generate exact (regular) Cantor set fractals. As an example, we use numerical simulations to demonstrate the formation of Cantor set fractals by temporal optical solitons. This fractal formation occurs in a cascade of nonlinear optical fibers through the dynamical evolution from a single input soliton.
The protein cross-linking enzyme tissue transglutaminase binds in vitro with high affinity to fibronectin via its 42-kD gelatin-binding domain. Here we report that cell surface transglutaminase mediates adhesion and spreading of cells on the 42-kD fibronectin fragment, which lacks integrin-binding motifs. Overexpression of tissue transglutaminase increases its amount on the cell surface, enhances adhesion and spreading on fibronectin and its 42-kD fragment, enlarges focal adhesions, and amplifies adhesion-dependent phosphorylation of focal adhesion kinase. These effects are specific for tissue transglutaminase and are not shared by its functional homologue, a catalytic subunit of factor XIII. Adhesive function of tissue transglutaminase does not require its cross-linking activity but depends on its stable noncovalent association with integrins. Transglutaminase interacts directly with multiple integrins of beta1 and beta3 subfamilies, but not with beta2 integrins. Complexes of transglutaminase with integrins are formed inside the cell during biosynthesis and accumulate on the surface and in focal adhesions. Together our results demonstrate that tissue transglutaminase mediates the interaction of integrins with fibronectin, thereby acting as an integrin-associated coreceptor to promote cell adhesion and spreading.
Neuronal calcium sensors (NCSs) belong to a family of Ca(2+)-binding proteins, which serve important functions in neurotransmission, and are highly conserved from yeast to humans. Overexpression of the neuronal calcium sensor-1, called frequenin in the fruit fly and in frog, increases the release of neurotransmitters. Studying the functional role of frequenin in mammals and understanding its structural dynamics is critically dependent on the availability of active purified protein. Neuronal calcium sensors like other members of the family share common structural features: they contain four EF-hands as potential binding sites for Ca(2+) and an N-terminal consensus sequence for myristoylation. Previously, recoverin, distantly related to NCSs, has been expressed and purified from Escherichia coli, involving a combination of different chromatographic steps. NCS-1 has earlier been purified adopting a two-step procedure used for recoverin purification. We have overexpressed NCS-1 from rat in its myristoylated and nonmyristoylated form in E. coli and purified it from crude lysates using a single-step hydrophobic interaction chromatography. The purified protein was identified by Western blotting and mass spectrometry and assayed for its ability to bind Ca(2+) using a Ca(2+) shift assay, terbium fluorescence, and Stains-all binding. The present protocol provides a rapid, more efficient and simplified, single-step method for purifying NCS-1 for structural and functional studies. This method can also be applied to purify related proteins of the superfamily.
Guanylate cyclase regulator protein (GCAP)-2 is a Ca2+-binding protein that regulates photoreceptor outer segment membrane guanylate cyclase (RetGC) in a Ca2+-sensitive manner. GCAP-2 activates RetGC at free Ca2+ concentrations below 100 nM, characteristic of light-adapted photoreceptors, and inhibits RetGC when free Ca2+ concentrations are above the 500 nM level, characteristic of dark-adapted photoreceptors. We have mapped functional domains in GCAP-2 by using deletion mutants and chimeric proteins in which parts of GCAP-2 were substituted with corresponding fragments of other closely related recoverin-like proteins that do not regulate RetGC. We find that in addition to the EF-hand Ca2+-binding centers there are three regions that contain GCAP-2-specific sequences essential for regulation of RetGC. 1) The region between Phe78 and Asp113 determines whether GCAP-2 activates outer segment RetGC in low or high Ca2+ concentrations. Substitution of this domain with the corresponding region from neurocalcin causes a paradoxical behavior of the chimeric proteins. They activate RetGC only at high and not at low Ca2+ concentrations. 2) The amino acid sequence of GCAP-2 between Lys29 and Phe48 that includes the EF-hand-related motif EF-1 is essential both for activation of RetGC at low Ca2+ and inhibition at high Ca2+ concentrations. Most of the remaining N-terminal region can be substituted with recoverin or neurocalcin sequences without loss of GCAP-2 function. 3) Region Val171-Asn189, adjacent to the C-terminal EF-4 contributes to activation of RetGC, but it is not essential for the ability of Ca2+-loaded GCAP-2 to inhibit RetGC. Other regions of the molecule can be substituted with the corresponding fragments from neurocalcin or recoverin, or even partially deleted without preventing GCAP-2 from regulating RetGC. Substitution of these three domains in GCAP-2 with corresponding neurocalcin sequences also affects activation of individual recombinant RetGC-1 and RetGC-2 expressed in HEK293 cells.
USF1 and USF2 are basic helix-loop-helix transcription factors implicated in the control of cellular proliferation. In HeLa cells, the USF proteins are transcriptionally active and their overexpression causes marked growth inhibition. In contrast, USF overexpression had essentially no effect on the proliferation of the Saos-2 osteosarcoma cell line. USF1 and USF2 also lacked transcriptional activity in Saos-2 cells when assayed by transient cotransfection with USF-dependent reporter genes. Yet, there was no difference in the expression, subcellular localization, or DNA-binding activity of the USF proteins in HeLa and Saos-2 cells. Furthermore, Gal4-USF1 and Gal4-USF2 fusion proteins activated transcription similarly in both cell lines. Mutational analysis and domain swapping experiments revealed that the small, highly conserved USF-specific region (USR) was responsible for the inactivity of USF in Saos-2 cells. In HeLa, the USR serves a dual function. It acts as an autonomous transcriptional activation domain at promoters containing an initiator element and also induces a conformational change that is required for USF activity at promoters lacking an initiator. Taken together, these results suggest a model in which the transcriptional activity of the USF proteins, and consequently their antiproliferative activity, is tightly controlled by interaction with a specialized coactivator that recognizes the conserved USR domain and, in contrast to USF, is not ubiquitous. The activity of USF is therefore context dependent, and evidence for USF DNA-binding activity in particular cells is insufficient to indicate USF function in transcriptional activation and growth control.
We have generated a transgenic mouse with no white fat tissue throughout life. These mice express a dominant-negative protein, termed A-ZIP/F, under the control of the adipose-specific aP2 enhancer/promoter. This protein prevents the DNA binding of B-ZIP transcription factors of both the C/EBP and Jun families. The transgenic mice (named A-ZIP/F-1) have no white adipose tissue and dramatically reduced amounts of brown adipose tissue, which is inactive. They are initially growth delayed, but by week 12, surpass their littermates in weight. The mice eat, drink, and urinate copiously, have decreased fecundity, premature death, and frequently die after anesthesia. The physiological consequences of having no white fat tissue are profound. The liver is engorged with lipid, and the internal organs are enlarged. The mice are diabetic, with reduced leptin (20-fold) and elevated serum glucose (3-fold), insulin (50- to 400-fold), free fatty acids (2-fold), and triglycerides (3- to 5-fold). The A-ZIP/F-1 phenotype suggests a mouse model for the human disease lipoatrophic diabetes (Seip-Berardinelli syndrome), indicating that the lack of fat can cause diabetes. The myriad of consequences of having no fat throughout development can be addressed with this model.
We have investigated the physical nature of the observed coupling energy (Delta Delta DeltaGint) between the charged side-chains of the three inter-helical g<-->e' (i, i'+5) pairs (E<-->R, E<-->K, and E<-->E) in the leucine zipper coiled coil dimer. Circular dichroism (CD) spectroscopy measured the thermal stability of eight proteins derived from the basic region leucine zipper domain of chicken VBP, the mammalian TEF at seven pHs and three KCl concentrations. Data from these proteins were used to construct double mutant alanine thermodynamic cycles and determine coupling energies (Delta Delta DeltaGint) for the three g<-->e' pairs. The attractive E<-->R coupling energy of -0.6 kcal mol-1 at low salt decreases to -0.2 kcal mol-1 at high salt. The E<-->K coupling energy of -0.5 kcal mol-1 at low salt decreases to -0.1 kcal mol-1 at high salt. The repulsive E<-->E coupling energy of +0.8 kcal mol-1 at low salt drops to +0.4 at high salt. Reducing the pH to 2.2 halved the attractive coupling energy for the E<-->R and E<-->K pairs while abolishing the repulsion of the E<-->E pair. 13C NMR of a protein selectively labeled with [13Cdelta]glutamate that contained three E<-->R and one R<-->E pair identified four glutamates shifted upfield. We suggest that this is due to electronic perturbation of glutamates in inter-helical E<-->R interactions. Taken together, these data indicate that the E<-->R coupling energy of -0.5 kcal mol-1 at pH 7.4 and 150 mM KCl has an electrostatic component.
Several studies have characterized the upstream regulatory region of c-fos, and identified cis-acting elements termed the cyclic AMP (cAMP) response elements (CREs) that are critical for c-fos transcription in response to a variety of extracellular stimuli. Although several transcription factors can bind to CREs in vitro, the identity of the transcription factor(s) that activates the c-fos promoter via the CRE in vivo remains unclear. To help identify the trans-acting factors that regulate stimulus-dependent transcription of c-fos via the CREs, dominant-negative (D-N) inhibitor proteins that function by preventing DNA binding of B-ZIP proteins in a dimerization domain-dependent fashion were developed. A D-N inhibitor of CREB, termed A-CREB, was constructed by fusing a designed acidic amphipathic extension onto the N terminus of the CREB leucine zipper domain. The acidic extension of A-CREB interacts with the basic region of CREB forming a coiled-coil extension of the leucine zipper and thus prevents the basic region of wild-type CREB from binding to DNA. Other D-N inhibitors generated in a similar manner with the dimerization domains of Fos, Jun, C/EBP, ATF-2, or VBP did not block CREB DNA binding activity, nor did they inhibit transcriptional activation of a minimal promoter containing a single CRE in PC12 cells. A-CREB inhibited activation of CRE-mediated transcription evoked by three distinct stimuli: forskolin, which increases intracellular cAMP; membrane depolarization, which promotes Ca2+ influx; and nerve growth factor (NGF). A-CREB completely inhibited cAMP-mediated, but only partially inhibited Ca2+- and NGF-mediated, transcription of a reporter gene containing 750 bp of the native c-fos promoter. Moreover, glutamate induction of c-fos expression in primary cortical neurons was dependent on CREB. In contrast, induction of c-fos transcription by UV light was not inhibited by A-CREB. Lastly, A-CREB attenuated NGF induction of morphological differentiation in PC12 cells. These results suggest that CREB or its closely related family members are general mediators of stimulus-dependent transcription of c-fos and are required for at least some of the long-term actions of NGF.
Green fluorescent protein (GFP) is a valuable marker for intracellular protein localization. However the fusion of GFP with structural proteins can alter their properties, resulting in a loss of fusion protein localization, decreased GFP fluorescence or both. We describe a novel targeting approach based on noncovalent heterodimerization of GFP and cytoplasmic structural proteins. The formation of structural protein/GFP complexes was mediated by modified leucine zipper protein spacers designed to form high-affinity heterodimers. The complexes localized accurately to specific sites within cells, providing selective fluorescence labeling of subcellular structures such as microfilaments or focal contacts.
We describe a method to design dominant-negative proteins (D-N) to the basic helix-loop-helix-leucine zipper (B-HLHZip) family of sequence-specific DNA binding transcription factors. The D-Ns specifically heterodimerize with the B-HLHZip dimerization domain of the transcription factors and abolish DNA binding in an equimolar competition. Thermal denaturation studies indicate that a heterodimer between a Myc B-HLHZip domain and a D-N consisting of a 12-amino acid sequence appended onto the Max dimerization domain (A-Max) is -6.3 kcal.mol-1 more stable than the Myc:Max heterodimer. One molar equivalent of A-Max can totally abolish the DNA binding activity of a Myc:Max heterodimer. This acidic extension also has been appended onto the dimerization domain of the B-HLHZip protein Mitf, a member of the transcription factor enhancer binding subfamily, to produce A-Mitf. The heterodimer between A-Mitf and the B-HLHZip domain of Mitf is -3.7 kcal.mol-1 more stable than the Mitf homodimer. Cell culture studies show that A-Mitf can inhibit Mitf-dependent transactivation both in acidic extension and in a dimerization-dependent manner. A-Max can inhibit Myc-dependent foci formation twice as well as the Max dimerization domain (HLHZip). This strategy of producing D-Ns may be applicable to other B-HLHZip or B-HLH proteins because it provides a method to inhibit the DNA binding of these transcription factors in a dimerization-specific manner.
The energetic contribution of seven amino acids in the d position of a dimeric leucine zipper coiled coil structure was measured by determining the thermal stability. The d position contains the conserved leucines found in the leucine zipper. We used a natural bZIP protein as our host-guest system that remains dimeric when a single d position is mutated. We have determined the thermal stability, monitored by circular dichroism, of 14 proteins which indicate that alanine is 4.6 kcal mol-1 per residue less stabilizing than leucine. The similarly sized amino acid isoleucine is 2.9 kcal mol-1 per residue less stabilizing than leucine, suggesting that leucine is well-packed. Model building indicates that the beta-branched amino acids isoleucine and valine in the d position produced interhelical clashes between the Cgamma2 methyl groups when placed in the favored rotamer conformation. The stabilization by leucine in different d positions is context-dependent; it varies by over 2 kcal mol-1 in the two positions examined. The order of stabilization is L, M, I, V, C, A, and S. Cysteine in the d position can form a disulfide bond which stabilizes the coiled coil.
We describe a dominant negative (DN) to activation protein-1 (AP1) that inhibits DNA binding in an equimolar competition. AP1 is a heterodimer of the oncogenes Fos and Jun, members of the bZIP family of transcription factors. The DN, termed A-Fos, consists of a newly designed acidic amphipathic protein sequence appended onto the N-terminus of the Fos leucine zipper, replacing the normal basic region critical for DNA binding. The acidic extension and the Jun basic region form a heterodimeric coiled coil structure that stabilizes the complex over 3000-fold and prevents the basic region of Jun from binding to DNA. Gel shift assays indicate that A-Fos can inactivate the DNA binding of a Fos:Jun heterodimer in an equimolar competition. Transient transfection assays indicate that A-Fos inhibits Jun-dependent transactivation. Both the acidic extension and the Fos leucine zipper are critical for this inhibition. Expression of A-Fos in mouse fibroblasts inhibits focus formation more than colony formation, reflecting the ability of A-Fos to interfere with the AP1 biological functions in mammalian cells. This reagent is more potent than a deletion of either the Fos or Jun transactivation domain, which has been used previously as a dominant negative to AP1 activity.
Thrombospondin-1 (TSP-1) is a large modular trimeric protein that has been proposed to play a diverse role in biological processes. Newly synthesized TSP-1 either is incorporated into the matrix or binds to the cell surface where it is rapidly internalized and degraded. TSP-1 catabolism is mediated by the low density lipoprotein receptor-related protein (LRP), a large endocytic receptor that is a member of the low density lipoprotein receptor family. Using adenovirus-mediated gene transfer experiments, we demonstrate that the very low density lipoprotein receptor can also bind and internalize TSP-1. An objective of the current investigation was to identify the portion of TSP-1 that binds to these endocytic receptors. The current studies found that the amino-terminal heparin binding domain (HBD, residues 1-214) of mouse TSP-1, when prepared as a fusion protein with glutathione S-transferase (GST), bound to purified LRP with an apparent KD ranging from 10 to 25 nM. Recombinant HBD (rHBD) purified following proteolytic cleavage of GST-HBD, also bound to purified LRP, but with an apparent KD of 830 nM. The difference in affinity was attributed to the fact that GST-HBD exists in solution as a dimer, whereas rHBD is a monomer. Like TSP-1, 125I-labeled GST-HBD or 125I-labeled rHBD were internalized and degraded by wild type fibroblasts that express LRP, but not by fibroblasts that are genetically deficient in LRP. The catabolism of both 125I-labeled GST-HBD and rHBD in wild type fibroblast was blocked by the 39-kDa receptor-associated protein, an inhibitor of LRP function. GST-HBD and rHBD both completely blocked catabolism of 125I-labeled TSP-1 in a dose-dependent manner, as did antibodies prepared against the HBD. Taken together, these data provide compelling evidence that the amino-terminal domain of TSP-1 binds to LRP and thus the recognition determinants on TSP-1 for both LRP and for cell surface proteoglycans reside within the same TSP-1 domain. Further, high affinity binding of TSP-1 to LRP likely results from the trimeric structure of TSP-1.
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Phosphorylation of threonine destabilizes the leucine zipper of a bZIP protein by 4.6 kcal mol-1 dimer-1, which reduces DNA binding 100-fold. This decrease in stability reflects the low alpha-helix forming propensity of a phosphorylated threonine.
We appended a rationally designed acidic amphipathic protein sequence to the N-terminus of a leucine zipper. Circular dichroism data indicate that this engineered polypeptide sequence can 'zipper' up the basic region of a bZIP monomer into a heterodimeric coiled coil. This propagation of the leucine zipper dimerization interface into the basic region can proceed for up to four heptads and stabilizes the heterodimer complex 2.5 kcal/mol or > 100-fold. The acidic nature of the extension is the most critical component of the design, suggesting that the extension is acting as a DNA mimetic. The dimerization prevents the basic region in this heterodimeric coiled coil structure from binding to DNA. Gel-shift, fluorescence and transient transfection assays indicate that the acidic extension appended to a leucine zipper can inactivate the DNA-binding and transactivation properties of the bZIP protein C/EBP. The three bZIP basic regions examined in this study dimerize with similar stability with the acidic extension, suggesting that this N-terminal extension can be used to develop dominant-negatives to other bZIP transcription factors.
The leucine zipper is a dimeric coiled-coil protein structure composed of two amphipathic alpha-helices with the hydrophobic surfaces interacting to create the dimer interface. This structure has been found to mediate the dimerization of two abundant classes of DNA binding proteins: the bZIP and bHLH-Zip proteins. Several workers have reported that amino acids in the e and g positions of the coiled coil can modulate dimerization stability and specificity. Using the bZIP protein VBP as a host molecule, we report a thermodynamic scale (delta delta G) for 27 interhelical interactions in 35 proteins between amino acids in the g and the following e positions (g<==>e') of a leucine zipper coiled coil. We have examined the four commonly occurring amino acids in the e and g positions of bZIP proteins, lysine (K), arginine (R), glutamine (Q), glutamic acid (E), as well as the only other remaining charged amino acid aspartic acid (D), and finally alanine (A) as a reference amino acid. These results indicate that E<==>R is the most stable interhelical pair, being 0.35 kcal/mol more stable than E<==>K. A thermodynamic cycle analysis shows that the E<==>R pair is 1.33 kcal/mol more stable than A<==>A with -1.14 kcal/mol of coupling energy (delta delta Gint) coming from the interaction of E with R. The E<==>K coupling energy is only -0.14 kcal/mol. E interacts with more specificity than Q. The R<==>R pair is less stable than the K<==>K by 0.24 kcal/mol. R interacts with more specificity than K. Q forms more stable pairs with the basic amino acids K and R rather than with E. Changing amino acids in the e position to A creates bZIP proteins that form tetramers.
The interaction of the linker histones H1 and H5 from chicken erythrocyte chromatin with pBR322 was studied as a function of the number of superhelical turns in circular plasmid molecules. Supercoiled plasmid DNA was relaxed with topoisomerase I so that a population with a narrow distribution of topoisomers, containing from zero to five superhelical turns, was obtained. None of the topoisomers contained alternative non-B-DNA structures. Histone-DNA complexes formed at either 25 or 100 mM NaCl final concentration and at histone-DNA molar ratios ranging from 10 to 150 were analyzed by agarose gel electrophoresis. The patterns of disappearance of individual topoisomer bands from the gel were interpreted as an indication of preference of the linker histones for crossovers of double-helical DNA. This preference was observed at both salt concentrations, being more pronounced under conditions of low ionic strength. Isolated H5 globular domain also caused selective disappearance of topoisomers from the gel, but it did so only at very high peptide-DNA molar ratios. The observed preference of the linker histones for crossovers of double-helical DNA is viewed as a part of the mechanism involved in the sealing of the two turns of DNA around the histone octamer.