Quantitative analysis of DNA-porphyrin interactions

The binding of manganese(III)-tetra(4-N-methylpyridyl)porphyrin (MnTMpyP) with synthetic poly(dA-dT)2, poly(dI-dC)2, and poly(dG-dC)2 DNAs as well as calf thymus (CT) DNA has been quantitatively studied in detail using induced CD (circular dichroism) spectroscopy in the Soret absorption band. The CD spectra, which changed greatly depending on the porphyrin to DNA base-pair molar ratio (r), were normalized with respect to DNA concentration and deconvoluted. Three independent component binding modes (named mode 1, 2, and 3 in the order of increasing r values) were identified, which successfully simulated the observed CD spectra with negligibly small residuals for a wide range of r values. In the case of poly(dA-dT)2, poly (dI-dC)2, and CT DNA, all the three modes appeared, whereas in the case of poly(dG-dC)2 DNA, only modes 1 and 3 appeared in the r range studied. The r dependence of each binding mode, i.e., its relative affinity toward DNA, has been revealed by this analysis. Mode 1, which appeared as a single binding mode at very low r values (r < or = ca. 0.05), was inhibited by the addition of methyl green, a drug that preferentially binds to the major groove of poly (dA-dT)2 DNA. Berenil, a known minor groove binder to poly(dA-dT)2 or poly(dI-dC)2 DNA, inhibited modes 2 and 3. From these inhibition experiments as well as comparison of the component spectra for DNAs of different sequence, a binding site on DNA was proposed for each component binding mode. The number of DNA base pairs covered by a single molecule of porphyrin was estimated.     

Auto-inhibition of the Dbl family protein Tim by an N-terminal helical motif

Dbl-related oncoproteins are guanine nucleotide exchange factors specific for Rho-family GTPases and typically possess tandem Dbl homology (DH) and pleckstrin homology domains that act in concert to catalyze exchange. Because the ability of many Dbl-family proteins to catalyze exchange is constitutively activated by truncations N-terminal to their DH domains, it has been proposed that the activity of Dbl-family proteins is regulated by auto-inhibition. However, the exact mechanisms of regulation of Dbl-family proteins remain poorly understood. Here we show that the Dbl-family protein, Tim, is auto-inhibited by a short, helical motif immediately N-terminal to its DH domain, which directly occludes the catalytic surface of the DH domain to prevent GTPase activation. Similar to the distantly related Vav isozymes, auto-inhibition of Tim is relieved by truncation, mutation, or phosphorylation of the auto-inhibitory helix. A peptide comprising the helical motif inhibits the exchange activity of Tim in vitro. Furthermore, substitutions within the most highly conserved surface of the DH domain designed to disrupt interactions with the auto-inhibitory helix also activate the exchange process.     

Quantitative analysis of the selective pressure exerted on homologous proteins

Evolution analysis is used to locate the regions of a proteinthat are important for its function or structure. The rate ofevolution is generally constant for a given family of homologoussequences. From the starting point of this observation, an algorithmis proposed to establish quantitatively the sequence zones whereselective pressure is maximal. A program that computes thispressure has been written in PASCAL. Analysis of results onsome sequences validate this theoretical approach, and thisknowledge can be used as a starting-point for carrying out site-directedmutagenesis. Received on October 3, 1989; accepted on February 28, 1990     

The phosphoinositide sensitivity of the KV channel family

Recently, we screened several K_V channels for possible dependence on plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). The channels were expressed in tsA-201 cells and the PI(4,5)P₂ was depleted by several manipulations in whole-cell experiments with parallel measurements of channel activity. In contrast to reports on excised-patches using Xenopus laevis oocytes, we found only K_V7, but none of the other tested K_V channels, to be strongly dependent on PI(4,5)P₂. We now have extended our study to K_V1.2 channels, a K_V channel we had not previously tested, because a new published study on excised patches showed regulation of the voltage-dependence of activation by PI(4,5)P₂. In full agreement with those published results, we found a reduction of current amplitude by ~20% after depletion of PI(4,5)P₂ and a small left shift in the activation curve of K_V1.2 channels. We also found a small reduction of K_V11.1 (hERG) currents that was not accompanied by a gating shift. In conclusion, our whole-cell methods yield a PI(4,5)P₂-dependence of K_V1.2 currents in tsA-201 cells that is comparable to findings from excised patches of Xenopus laevis oocytes. We discuss possible physiological rationales for PI(4,5)P₂ sensitivity of some ion channels and insensitivity of others.     

Conservation of residue interactions in a family of Ca-binding proteins

In the TNC family of Ca-binding proteins (calmodulin, parvalbumin, intestinal calcium binding protein and troponin C) approximately 70 well-conserved amino acid sequences and six crystal structures are known. We find a clear correlation between residue contacts in the structures and residue conservation in the sequences: residues with strong sidechain-sidechain contacts in the three-dimenesional structure tend to be the more conserved in the sequence. This is one way to quantify the intuitive notion of the importance of sidechain interactions for maintaining protein three-dimensional structure in evolution and may usefully be taken into account in planning point mutations in protein engineering.     

Protein-protein interactions of proteins from the ESAT-6 family of Mycobacterium tuberculosis

In the present study, we demonstrate that, in analogy with the genes encoding ESAT-6 and CFP-10, the genes rv0287 and rv0288 from the ESAT-6 gene family are cotranscribed. Using Western-Western blotting and protein-print overlay methodologies, we demonstrate that ESAT-6 and CFP-10, as well as the protein pair Rv0288/Rv0287, interact pairwise in a highly specific way. Most notably, the ESAT-6 proteins interact directly with Rv3873, a possible cell envelope component of the ESAT-6 secretion pathway.     

The effect of long-range interactions on the secondary structure formation of proteins

The influence of long-range residue interactions on defining secondary structure in a protein has long been discussed and is often cited as the current limitation to accurate secondary structure prediction. There are several experimental examples where a local sequence alone is not sufficient to determine its secondary structure, but a comprehensive survey on a large data set has not yet been done. Interestingly, some earlier studies denied the negative effect of long-range interactions on secondary structure prediction accuracy. Here, we have introduced the residue contact order (RCO), which directly indicates the separation of contacting residues in terms of the position in the sequence, and examined the relationship between the RCO and the prediction accuracy. A large data set of 2777 nonhomologous proteins was used in our analysis. Unlike previous studies, we do find that prediction accuracy drops as residues have contacts with more distant residues. Moreover, this negative correlation between the RCO and the prediction accuracy was found not only for beta-strands, but also for alpha-helices. The prediction accuracy of beta-strands is lower if residues have a high RCO or a low RCO, which corresponds to the situation that a beta-sheet is formed by beta-strands from different chains in a protein complex. The reason why the current study draws the opposite conclusion from the previous studies is examined. The implication for protein folding is also discussed.     

Two microtubule-associated proteins of the Arabidopsis MAP65 family function differently on microtubules

The organization and dynamics of microtubules are regulated by microtubule-associated proteins, or MAPs. In Arabidopsis (Arabidopsis thaliana), nine genes encode proteins of the evolutionarily conserved MAP65 family. We proposed that different MAP65s might have distinct roles in the interaction with microtubules. In this study, two AtMAP65 proteins, AtMAP65-1 and AtMAP65-6, were chosen to test this hypothesis in vitro. Although both fusion proteins were able to cosediment with microtubules in vitro, different properties on tubulin polymerization and microtubule bundling were observed. AtMAP65-1 was able to promote tubulin polymerization, enhance microtubule nucleation, and decrease the critical concentration for tubulin polymerization. It also induced the formation of large microtubule bundles by forming cross-bridges between microtubules evenly along the whole length of microtubules. In the presence of AtMAP65-1, microtubule bundles were more resistant to cold and dilution treatments. AtMAP65-6, however, demonstrated no activity in promoting tubulin polymerization and stabilizing preformed microtubules. AtMAP65-6 induced microtubules to form a mesh-like network with individual microtubules. Cross-bridge-like interactions were only found at regional sites between microtubules. The microtubule network induced by AtMAP65-6 was more resistant to high concentration of NaCl than the bundles induced by AtMAP65-1. Purified monospecific anti-AtMAP65-6 antibodies revealed that AtMAP65-6 was associated with mitochondria in Arabidopsis cells. It was concluded that these two MAP65 proteins were targeted to distinct sites, thus performing distinct functions in Arabidopsis cells.     

Quantitative analysis of regions of adenovirus E1A products involved in interactions with cellular proteins.

Human adenovirus E1A proteins and oncogene products of several other DNA tumour viruses derive much of their oncogenic potential from interactions with cellular polypeptides. E1A proteins form complexes with p105Rb and a related p107 polypeptide, and with at least three other proteins (p60cycA, p130, and p300); all may be required for cell transformation. Using a series of E1A deletion mutants, we have carried out a quantitative analysis of the binding patterns of cellular proteins to E1A products. Binding of most of the proteins was affected at least partially by mutations within the amino terminal 25 residues, amino acids 36-69 within conserved region 1 (CR1), and residues 121-138 in conserved region 2 (CR2). However, the specific binding characteristics of each protein varied considerably. p300 was the only species for which binding was totally eliminated by deletions at the amino terminus. Removal of regions within CR1 eliminated binding of all species except p107 and p60cycA. Deletion of portions of CR2 reduced or eliminated binding of all proteins except p300. Thus, whereas cellular polypeptides generally were found to interact with the same three regions of E1A proteins, specific interactions varied considerably.     

Molecular architecture and function of the Omp85 family of proteins

Omp85 is a protein found in Gram-negative bacteria where it serves to integrate proteins into the bacterial outer membrane. Members of the Omp85 family of proteins are defined by the presence of two domains: an N-terminal, periplasmic domain rich in POTRA repeats and a C-terminal beta-barrel domain embedded in the outer membrane. The widespread distribution of Omp85 family members together with their fundamental role in outer membrane assembly suggests the ancestral Omp85 arose early in the evolution of prokaryotic cells. Mitochondria, derived from an ancestral bacterial endosymbiont, also use a member of the Omp85 family to assemble proteins in their outer membranes. More distant relationships are seen between the Omp85 family and both the core proteins in two-partner secretion systems and the Toc75 family of protein translocases found in plastid outer envelopes. Aspects of the ancestry and molecular architecture of the Omp85 family of proteins is providing insight into the mechanism by which proteins might be integrated and assembled into bacterial outer membranes.     

Cellular signaling of Dock family proteins in neural function

Dock180-related proteins are genetically conserved from Drosophila and C. elegans to mammals and are atypical types of guanine–nucleotide exchange factors (GEFs) for Rac and/or Cdc42 of small GTPases of the Rho family. Eleven members of the family occur in mammalian cells, each playing key roles in many aspects of essential cellular functions such as regulation of cytoskeletal organization, phagocytosis, cell migration, polarity formation, and differentiation. This review will summarize the newly accumulated findings concerning the Dock180-related proteins' molecular and cellular functions, emphasizing the roles of these proteins in neuronal cells and glial cells as well as their interactions in the central and peripheral nervous systems.     

Quantitative analysis of ligand-receptor interactions in physiological experiments

In isolated smooth muscles of the sea cucumber, the rat intestine, vas deferens and portal vein, and in chick embryonic amnion, contractile responses of smooth muscles to transmitters and their agonists were described with two equations: p = (Pm.A(n))/(EC50n + A(n)) [7] or p = [(Pm1.An1)/(EC50(1)n1 + An1)] + [(Pm2.An2)/(EC50(2)n2 + An2)] [8]. The findings reveal a possibility of ligand-receptor interaction according to several models: a single receptor pool with n = 1 or n not equal to 1; two receptor pools in the same effector system with n1 = n2 or n1 not equal to n2.     

Ras oncoprotein induces CD44 cleavage through phosphoinositide 3-OH kinase and the rho family of small G proteins

CD44 is a cell surface adhesion molecule for several extracellular matrix components. We previously showed that CD44 expressed in cancer cells is proteolytically cleaved at the ectodomain through membrane-anchored metalloproteases and that CD44 cleavage plays a critical role in cancer cell migration. Therefore, cellular signals that promote the migration and metastatic activity of cancer cells may regulate the CD44 ectodomain cleavage. Here, we demonstrate that the expression of the dominant active mutant of Ha-Ras (Ha-Ras(Val-12)) induces redistribution of CD44 to the newly generated membrane ruffling area and CD44 ectodomain cleavage. The migration assay revealed that the CD44 cleavage contributes to the Ha-Ras(Val-12)-induced migration of NIH3T3 cells on hyaluronate substrate. Treatment with LY294002, an inhibitor for phosphoinositide 3-OH kinase (PI3K), significantly inhibits Ha-Ras(Val-12)-induced CD44 cleavage, whereas that with PD98059, an inhibitor for MEK, does not. The active mutant p110 subunit of PI3K has also been shown to enhance the CD44 cleavage, suggesting that PI3K mediates the Ras-induced CD44 cleavage. Moreover, the expression of dominant negative mutants of Cdc42 and Rac1 inhibits the Ha-Ras(Val-12)-induced CD44 cleavage. These results suggest that Ras > PI3K > Cdc42/Rac1 pathway plays an important role in CD44 cleavage and may provide a novel molecular basis to explain how the activated Ras facilitates cancer cell migration.