Статті в журналах: "Transient dimerization"

1

Lin, C. L., Y. T. Huang, and J. D. Richter. "Transient CPEB dimerization and translational control." RNA 18, no. 5 (March 28, 2012): 1050–61. http://dx.doi.org/10.1261/rna.031682.111.

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2

Wendler, Christian, and Hartmut Oehme. "The Synthesis and Dimerization of Transient 1-Silabutadienes." Zeitschrift f�r anorganische und allgemeine Chemie 622, no. 5 (May 1996): 801–6. http://dx.doi.org/10.1002/zaac.19966220509.

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3

Hartl, Maximilian J., Kristian Schweimer, Martin H. Reger, Stephan Schwarzinger, Jochen Bodem, Paul Rösch, and Birgitta M. Wöhrl. "Formation of transient dimers by a retroviral protease." Biochemical Journal 427, no. 2 (March 29, 2010): 197–203. http://dx.doi.org/10.1042/bj20091451.

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Retroviral proteases have been shown previously to be only active as homodimers. They are essential to form the separate and active proteins from the viral precursors. Spumaretroviruses produce separate precursors for Gag and Pol, rather than a Gag and a Gag–Pol precursor. Nevertheless, processing of Pol into a PR (protease)–RT (reverse transcriptase) and integrase is essential in order to obtain infectious viral particles. We showed recently that the PR–RT from a simian foamy virus, as well as the separate PRshort (protease) domain, exhibit proteolytic activities, although only monomeric forms could be detected. In the present study, we demonstrate that PRshort and PR–RT can be inhibited by the putative dimerization inhibitor cholic acid. Various other inhibitors, including darunavir and tipranavir, known to prevent HIV-1 PR dimerization in cells, had no effect on foamy virus protease in vitro. 1H-15N HSQC (heteronuclear single quantum coherence) NMR analysis of PRshort indicates that cholic acid binds in the proposed PRshort dimerization interface and appears to impair formation of the correct dimer. NMR analysis by paramagnetic relaxation enhancement resulted in elevated transverse relaxation rates of those amino acids predicted to participate in dimer formation. Our results suggest transient PRshort homodimers are formed under native conditions but are only present as a minor transient species, which is not detectable by traditional methods.

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4

Pawar, Aiswarya B., Sneha A. Deshpande, Srinivasa M. Gopal, Tsjerk A. Wassenaar, Chaitanya A. Athale, and Durba Sengupta. "Thermodynamic and kinetic characterization of transmembrane helix association." Physical Chemistry Chemical Physics 17, no. 2 (2015): 1390–98. http://dx.doi.org/10.1039/c4cp03732d.

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The transient dimerization of transmembrane proteins is an important event in several cellular processes and here we use coarse-grain and meso-scale modeling methods to quantify their underlying dynamics.

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5

Nakasone, Yusuke, Taka-aki Ono, Asako Ishii, Shinji Masuda, and Masahide Terazima. "Transient Dimerization and Conformational Change of a BLUF Protein: YcgF." Journal of the American Chemical Society 129, no. 22 (June 2007): 7028–35. http://dx.doi.org/10.1021/ja065682q.

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6

Engelke, Ray, and Normand C. Blais. "Chemical dimerization of crystalline anthracene produced by transient high pressure." Journal of Chemical Physics 101, no. 12 (December 15, 1994): 10961–72. http://dx.doi.org/10.1063/1.467846.

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7

Schröder, Jan, Daniel Himmel, Daniel Kratzert, Valentin Radtke, Sabine Richert, Stefan Weber, and Tobias Böttcher. "Isolation of a stable pyridine radical anion." Chemical Communications 55, no. 9 (2019): 1322–25. http://dx.doi.org/10.1039/c8cc09700c.

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For almost 150 years, pyridine radical anions have been described as elusive transient species that cannot be isolated due to dimerization and/or subsequent decomposition reactions. In this work the first example of a stable pyridine radical anion is presented.

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8

El-Sayed, Ibrahim, Rita Grøbaek Hazell, Jørgen Øgaard Madsen, Per-Ola Norrby, and Alexander Senning. "An Unprecedented [2+3] Cycloadditive Dimerization of a Transient ThiocarbonylS-Ylide." European Journal of Organic Chemistry 2005, no. 2 (January 2005): 457. http://dx.doi.org/10.1002/ejoc.200400835.

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9

Cuquerella, M. Consuelo, Virginie Lhiaubet-Vallet, Miguel A. Miranda, and Francisco Bosca. "Drug–DNA complexation as the key factor in photosensitized thymine dimerization." Physical Chemistry Chemical Physics 19, no. 7 (2017): 4951–55. http://dx.doi.org/10.1039/c6cp08485k.

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The crucial role of photosensitizer@DNA complexation in the formation of cyclobutane pyrimidine dimers (CPDs) has been demonstrated using femtosecond and nanosecond transient absorption and emission measurements in combination with in vitro DNA damage assays.

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10

Li, Zhenzhen, Jianbang Wang, Zhixin Zhou, Michael P. O’Hagan, and Itamar Willner. "Gated Transient Dissipative Dimerization of DNA Tetrahedra Nanostructures for Programmed DNAzymes Catalysis." ACS Nano 16, no. 3 (February 20, 2022): 3625–36. http://dx.doi.org/10.1021/acsnano.1c06117.

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11

El-Sayed, Ibrahim, Rita Grønbæk Hazell, Jørgen Øgaard Madsen, Per-Ola Norrby, and Alexander Senning. "An Unprecedented [2+3] Cycloadditive Dimerization of a Transient Thiocarbonyl S-Ylide." European Journal of Organic Chemistry 2003, no. 5 (March 2003): 813–15. http://dx.doi.org/10.1002/ejoc.200390123.

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12

Zhou, Peng, Rinshi S. Kasai, Koichiro M. Hirosawa, Alexey Yudin, Yuki M. Shirai, Takahiro K. Fujiwara, and Akihiro Kusumi. "Transient Hetero-Dimerization of Opioid Receptors (GPCRS) Revealed by Single-Molecule Tracking." Biophysical Journal 114, no. 3 (February 2018): 202a. http://dx.doi.org/10.1016/j.bpj.2017.11.1128.

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13

Işbilir, Ali, Jan Möller, Marta Arimont, Vladimir Bobkov, Cristina Perpiñá-Viciano, Carsten Hoffmann, Asuka Inoue, et al. "Advanced fluorescence microscopy reveals disruption of dynamic CXCR4 dimerization by subpocket-specific inverse agonists." Proceedings of the National Academy of Sciences 117, no. 46 (November 4, 2020): 29144–54. http://dx.doi.org/10.1073/pnas.2013319117.

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Although class A G protein−coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer−dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.

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14

Duffy, Ian R., та William J. Leigh. "Fast kinetics studies of the Lewis acid–base complexation of transient stannylenes with σ- and π-donors in solution". Physical Chemistry Chemical Physics 20, № 31 (2018): 20555–70. http://dx.doi.org/10.1039/c8cp03580f.

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The complexation of SnMe₂ and SnPh₂ with a variety of σ- and π-donors and its effects on their dimerization reactions has been studied by laser flash photolysis and DFT methods, and compared to reported data for the Si and Ge homologues.

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15

Wang, Shu, and Huai-hu Chuang. "C-terminal Dimerization Activates the Nociceptive Transduction Channel Transient Receptor Potential Vanilloid 1." Journal of Biological Chemistry 286, no. 47 (September 16, 2011): 40601–7. http://dx.doi.org/10.1074/jbc.m111.256669.

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16

Curcio-Morelli, Cyntia, Balazs Gereben, Ann Marie Zavacki, Brian W. Kim, Stephen Huang, John W. Harney, P. Reed Larsen, and Antonio C. Bianco. "In Vivo Dimerization of Types 1, 2, and 3 Iodothyronine Selenodeiodinases." Endocrinology 144, no. 3 (March 1, 2003): 937–46. http://dx.doi.org/10.1210/en.2002-220960.

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The goal of the present investigation was to test the hypothesis that types 1, 2, and 3 iodothyronine selenodeiodinases (D1, D2, and D3) can form homodimers. The strategy included transient coexpression of wild-type (wt) deiodinases (target), and FLAG-tagged alanine or cysteine mutants (bait) in human embryonic kidney epithelial cells. SDS-PAGE of the immunoprecipitation pellet of 75Se-labeled cell lysates using anti-FLAG antibody revealed bands of the correct sizes for the respective wt enzymes, which corresponded to approximately 2–5% of the total deiodinase protein in the cell lysate. Western blot analysis with anti-FLAG antibody of lysates of cells transiently expressing individual FLAG-tagged-cysteine deiodinases revealed specific monomeric bands for each deiodinase and additional minor bands of relative molecular mass (Mr) of 55,000 for D1, Mr 62,000 for D2, and Mr 65,000 for D3, which were eliminated by 100 mm dithiothreitol at 100 C. Anti-FLAG antibody immunodepleted 10% of D1 and 38% of D2 activity from lysates of cells coexpressing inactive FLAG-tagged Ala mutants and the respective wt enzymes (D1 or D2) but failed to immunodeplete wtD3 activity. D1 or D2 activities were present in these respective pellets. We conclude 1) that overexpressed selenodeiodinases can homodimerize probably through disulfide bridges; and 2) at least for D1 and D2, monomeric forms are catalytically active, demonstrating that only one wt monomer partner is required for catalytic activity of these two deiodinases.

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17

Antonawich, Francis J., Stanislaw Krajewski, John C. Reed, and James N. Davis. "Bcl-x1 Bax Interaction after Transient Global Ischemia." Journal of Cerebral Blood Flow & Metabolism 18, no. 8 (August 1998): 882–86. http://dx.doi.org/10.1097/00004647-199808000-00008.

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Five minutes of bilateral common carotid artery occlusion in the Mongolian gerbil results in a selective, delayed death of CA1 pyramidal neurons. Although Bcl-2 appears to protect a variety of cells from cell death, the precise role of this apoptosis-regulating protein is complicated. We used immunoblots to estimate levels of Bcl-2, Bcl-xl, and Bax at various times after carotid occlusion. Rather than Bcl-2, Bcl-xl appears to be the predominant neuroprotective form of this family of proto-oncogenes in the gerbil hippocampus. After transient ischemia, Bcl-2 and Bcl-xl protein levels remained the same. However, Bax levels were dramatically increased at 6 hours after ischemia, compared with sham-operated animals, and were still elevated at 72 hours after ischemia. To monitor dimerization interactions among theses apoptosis-regulating molecules, immunoprecipitation studies were conducted. These studies demonstrated that Bcl-xl association with Bax increases after ischemia. Therefore, Bax may disrupt the more favorable Bcl-xl (Bcl-2) interactions necessary for normal neuronal functioning and thus promote transient ischemic death.

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18

Matias, Ana C., Paula C. Ramos, and R. Jürgen Dohmen. "Chaperone-assisted assembly of the proteasome core particle." Biochemical Society Transactions 38, no. 1 (January 19, 2010): 29–33. http://dx.doi.org/10.1042/bst0380029.

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The 26S proteasome is a non-lysosomal protease in the cytosol and nucleus of eukaryotic cells. Its main function is to mediate ubiquitin-dependent proteolysis. The 26S proteasome is a multimeric complex composed by the 20S proteasome CP (core particle) and the 19S RPs (regulatory particles). Although the atomic structure of the 26S proteasome has not yet been determined, high-resolution structures are available for its CP. Studies on the complicated assembly pathway of the proteasome have revealed that it involves an unprecedented number of dedicated chaperones. Assembly of the CP alone involves three conserved proteasome-assembly chaperones [PAC1–PAC2, PAC3–PAC4 and UMP1 (ubiquitin-mediated proteolysis 1)]. Whereas the two heterodimeric PACs have been implicated in the formation of rings of the seven distinct α subunits, UMP1 is important for the formation and dimerization of proteasome precursor complexes containing β subunits. Dimerization coincides with the incorporation of the last β subunit (β7). Additional modules important for the assembly of precursor complexes and their dimerization reside in the β subunits themselves, either as transient or as permanent extensions. Particularly important domains are the propeptide of β5 and the C-terminal extensions of β2 and β7. Upon maturation of the active sites by autocatalytic processing, UMP1 is degraded by the native proteasome.

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19

Jara, Oscar, Rodrigo Acuña, Isaac E. García, Jaime Maripillán, Vania Figueroa, Juan C. Sáez, Raúl Araya-Secchi, et al. "Critical role of the first transmembrane domain of Cx26 in regulating oligomerization and function." Molecular Biology of the Cell 23, no. 17 (September 2012): 3299–311. http://dx.doi.org/10.1091/mbc.e11-12-1058.

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To identify motifs involved in oligomerization of the gap junction protein Cx26, we studied individual transmembrane (TM) domains and the full-length protein. Using the TOXCAT assay for interactions of isolated TM α-helices, we found that TM1, a Cx26 pore domain, had a strong propensity to homodimerize. We identified amino acids Val-37–Ala-40 (VVAA) as the TM1 motif required for homodimerization. Two deafness-associated Cx26 mutations localized in this region, Cx26V37I and Cx26A40G, differentially affected dimerization. TM1-V37I dimerized only weakly, whereas TM1-A40G did not dimerize. When the full-length mutants were expressed in HeLa cells, both Cx26V37I and Cx26A40G formed oligomers less efficiently than wild-type Cx26. A Cx26 cysteine substitution mutant, Cx26V37C formed dithiothreitol-sensitive dimers. Substitution mutants of Val-37 formed intercellular channels with reduced function, while mutants of Ala-40 did not form functional gap junction channels. Unlike wild-type Cx26, neither Cx26V37I nor Cx26A40G formed functional hemichannels in low extracellular calcium. Thus the VVAA motif of Cx26 is critical for TM1 dimerization, hexamer formation, and channel function. The differential effects of VVAA mutants on hemichannels and gap junction channels imply that inter-TM interactions can differ in unapposed and docked hemichannels. Moreover, Cx26 oligomerization appears dependent on transient TM1 dimerization as an intermediate step.

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20

Schneppenheim, Reinhard, Ulrich Budde, Tobias Obser, Jacqueline Brassard, Kerstin Mainusch, Zaverio M. Ruggeri, Sonja Schneppenheim, Rainer Schwaab, and Johannes Oldenburg. "Expression and characterization of von Willebrand factor dimerization defects in different types of von Willebrand disease." Blood 97, no. 7 (April 1, 2001): 2059–66. http://dx.doi.org/10.1182/blood.v97.7.2059.

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Abstract Dimerization defects of von Willebrand factor (vWF) protomers underlie von Willebrand disease (vWD) type 2A, subtype IID (vWD 2A/IID), and corresponding mutations have been identified at the 3′ end of the vWF gene in exon 52. This study identified and expressed 2 additional mutations in this region, a homozygous defect in a patient with vWD type 3 (C2754W) and a heterozygous frameshift mutation (8566delC) in a patient with vWD type 2A, subtype IIE. Both mutations involve cysteine residues that we propose are possibly essential for dimerization. To prove this hypothesis, transient recombinant expression of each of the 2 mutations introduced in the carboxy-terminal vWF fragment II and in the complete vWF complementary DNA, respectively, were carried out in COS-7 cells and compared with expression of vWD 2A/IID mutation C2773R and the wild-type (WT) sequence in COS-7 cells. Recombinant WT vWF fragment II assembled correctly into a dimer, whereas recombinant mutant fragments were monomeric. Homozygous expression of recombinant mutant full-length vWF resulted in additional dimers, probably through disulfide bonding at the amino-terminal multimerization site, whereas recombinant WT vWF correctly assembled into multimers. Coexpression of recombinant mutant and recombinant WT vWF reproduced the multimer patterns observed in heterozygous individuals. Our results suggest that a common defect of vWF biosynthesis—lack of vWF dimerization—may cause diverse types and subtypes of vWD. We also confirmed previous studies that found that disulfide bonding at the vWF amino-terminal is independent of dimerization at the vWF carboxy-terminal.

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21

Silvennoinen, O., H. Nishigaki, A. Kitanaka, M. Kumagai, C. Ito, F. Malavasi, Q. Lin, M. E. Conley, and D. Campana. "CD38 signal transduction in human B cell precursors. Rapid induction of tyrosine phosphorylation, activation of syk tyrosine kinase, and phosphorylation of phospholipase C-gamma and phosphatidylinositol 3-kinase." Journal of Immunology 156, no. 1 (January 1, 1996): 100–107. http://dx.doi.org/10.4049/jimmunol.156.1.100.

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Abstract Ligation of CD38 inhibits proliferation and induces apoptosis of human immature B cells, but the molecular mechanisms underlying this function are unknown. We found that CD38 dimerization with the specific mAbs T16 and IB4 induces rapid and transient tyrosine phosphorylation of several intracellular proteins in the immature B cell lines RS4;11, REH, 380, Nalm6, and OP-1. This effect could be markedly reduced by incubating cells with the tyrosine kinase inhibitors genistein, staurosporine, and herbimycin A. CD38 dimerization induced tyrosine phosphorylation of the protein kinase syk and increased syk kinase activity. CD38 dimerization also induced tyrosine phosphorylation of phospholipase C-gamma and of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-K). The latter was accompanied by a distinct increase in PI 3-kinase activity in the immunoprecipitates obtained with an anti-phosphotyrosine Ab. In contrast to the signaling triggered by surface Ig engagement in B lymphocytes, CD38 ligation did not appear to induce tyrosine phosphorylation of the src-like protein tyrosine kinases lyn, fyn, and btk, or of vav- and ras-GTPase-activating protein, nor did it induce detectable changes in cytosolic CA2+ concentrations. CD38 signaling also differed from cytokine-induced signaling in that it did not cause tyrosine phosphorylation of Jak1 and Jak2. Finally, CD38 ligation did not inhibit IL-3-induced tyrosine phosphorylation of Jak2. These results identify CD38 as a cell surface receptor with signal transduction properties activated by dimerization. Induction of signal transduction by CD38 ligation implies the existence of a yet unidentified natural ligand of CD38.

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22

Raab, Monika, Yves Matthess, Christopher A. Raab, Niklas Gutfreund, Volker Dötsch, Sven Becker, Mourad Sanhaji, and Klaus Strebhardt. "A dimerization-dependent mechanism regulates enzymatic activation and nuclear entry of PLK1." Oncogene 41, no. 3 (November 10, 2021): 372–86. http://dx.doi.org/10.1038/s41388-021-02094-9.

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AbstractPolo-like kinase 1 (PLK1) is a crucial regulator of cell cycle progression. It is established that the activation of PLK1 depends on the coordinated action of Aurora-A and Bora. Nevertheless, very little is known about the spatiotemporal regulation of PLK1 during G2, specifically, the mechanisms that keep cytoplasmic PLK1 inactive until shortly before mitosis onset. Here, we describe PLK1 dimerization as a new mechanism that controls PLK1 activation. During the early G2 phase, Bora supports transient PLK1 dimerization, thus fine-tuning the timely regulated activation of PLK1 and modulating its nuclear entry. At late G2, the phosphorylation of T210 by Aurora-A triggers dimer dissociation and generates active PLK1 monomers that support entry into mitosis. Interfering with this critical PLK1 dimer/monomer switch prevents the association of PLK1 with importins, limiting its nuclear shuttling, and causes nuclear PLK1 mislocalization during the G2-M transition. Our results suggest a novel conformational space for the design of a new generation of PLK1 inhibitors.

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23

Nakasone, Yusuke, Takeshi Eitoku, Daisuke Matsuoka, Satoru Tokutomi, and Masahide Terazima. "Kinetic Measurement of Transient Dimerization and Dissociation Reactions of Arabidopsis Phototropin 1 LOV2 Domain." Biophysical Journal 91, no. 2 (July 2006): 645–53. http://dx.doi.org/10.1529/biophysj.106.084772.

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24

Snow, Mark S., Brian J. Howard, Luca Evangelisti, and Walther Caminati. "From Transient to Induced Permanent Chirality in 2-Propanol upon Dimerization: A Rotational Study." Journal of Physical Chemistry A 115, no. 1 (January 13, 2011): 47–51. http://dx.doi.org/10.1021/jp1107944.

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25

Rennie, Martin L., Siri A. McKelvie, Esther M. M. Bulloch, and Richard L. Kingston. "Transient Dimerization of Human MxA Promotes GTP Hydrolysis, Resulting in a Mechanical Power Stroke." Structure 22, no. 10 (October 2014): 1433–45. http://dx.doi.org/10.1016/j.str.2014.08.015.

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26

Richard, Robert E., Brent Wood, Hui Zeng, Liqing Jin, Thalia Papayannopoulou, and C. Anthony Blau. "Expansion of genetically modified primary human hemopoietic cells using chemical inducers of dimerization." Blood 95, no. 2 (January 15, 2000): 430–36. http://dx.doi.org/10.1182/blood.v95.2.430.

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The inability to deliver a therapeutic gene to a sufficient percentage of hematopoietic stem cells is the major obstacle to using gene therapy to treat blood disorders. Providing genetically corrected stem cells with a reversible growth advantage could solve this problem. To this end we have employed small synthetic molecules that can reversibly dimerize and activate fusion proteins which contain a growth factor receptor signaling domain. We have shown that the thrombopoietin receptor (mpl) signaling domain can be used in this system to expand transduced multipotential progenitor cells from mouse bone marrow. In the present study we tested a similar retroviral vector in human CD34-selected cord blood cells. Following transduction, cells cultured in the presence of the dimerizing molecule AP1903 expanded 13.8- to 186-fold relative to cells cultured in the absence of AP1903. The cell type that emerged in suspension culture was erythroid. Contrary to our results in the murine system, cell expansion was transient. Activation of mpl caused the disappearance of BFU-E followed by a transient increase in CFU-E. In contrast, mpl activation had no discernable effect on transduced myeloid progenitor cells. AP1903-mediated expansion was restricted to transduced cells, as demonstrated by immunohistochemical staining. These findings indicate that synthetic dimerizing molecules can be used to expand primary human hematopoietic cells.

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27

Steinmüller, L., and G. Thiel. "Regulation of Gene Transcription by a Constitutively Active Mutant of Activating Transcription Factor 2 (ATF2)." Biological Chemistry 384, no. 4 (April 10, 2003): 667–72. http://dx.doi.org/10.1515/bc.2003.074.

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AbstractActivating transcription factor 2 (ATF2) belongs to the family ofbasic region leucinezipper (bZIP) proteins that are characterized by the presence of a basic domain that functions as the DNAbinding domain and a leucine zipper domain that is required for dimerization. Together with bZIP proteins of the Fos and Jun families, ATF2 constitutes the AP-1 transcription factor complex. The biological activity of ATF2 is controlled by phosphorylation of two threonine residues within the N-terminal activation domain. Unphosphorylated ATF2 is trancriptionally silent, excluding simple overexpression studies to identify transcriptional targets of ATF2. We therefore decided to construct a constitutively active ATF2 mutant that would allow us to uncouple the investigation of transcriptional targets and biological functions of ATF2 from the variety of signaling pathways that lead to an activation of ATF2. We exchanged the phosphorylationdependent activation domain of ATF2 with the constitutively active transcriptional activation domain of the transcription factor CREB2. In transient transfection experiments, this constitutively active ATF2 mutant stimulated c-jun, tumor necrosis factor α, and Fas ligand promoter activities. The transcriptional activity of the constitutively active ATF2 mutant could be impaired by dominantnegative forms of ATF2 or c-jun, indicating that ATF2 and c-jun utilize a similar dimerization code. In contrast, a dominantnegative CREB2 mutant did not impair ATF2-mediated transcriptional activation, suggesting that CREB2 exhibits a different dimerization specificity than ATF2 or c-jun.

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28

Titolo, S., K. Brault, J. Majewski, P. W. White, and J. Archambault. "Characterization of the Minimal DNA Binding Domain of the Human Papillomavirus E1 Helicase: Fluorescence Anisotropy Studies and Characterization of a Dimerization-Defective Mutant Protein." Journal of Virology 77, no. 9 (May 1, 2003): 5178–91. http://dx.doi.org/10.1128/jvi.77.9.5178-5191.2003.

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ABSTRACT The E1 helicase of papillomaviruses is required for replication of the viral double-stranded DNA genome, in conjunction with cellular factors. DNA replication is initiated at the viral origin by the assembly of E1 monomers into oligomeric complexes that have unwinding activity. In vivo, this process is catalyzed by the viral E2 protein, which recruits E1 specifically at the origin. For bovine papillomavirus (BPV) E1 a minimal DNA-binding domain (DBD) has been identified N-terminal to the enzymatic domain. In this study, we characterized the DBD of human papillomavirus 11 (HPV11), HPV18, and BPV E1 using a quantitative DNA binding assay based on fluorescence anisotropy. We found that the HPV11 DBD binds DNA with an affinity and sequence requirement comparable to those of the analogous domain of BPV but that the HPV18 DBD has a higher affinity for nonspecific DNA. By comparing the DNA-binding properties of a dimerization-defective protein to those of the wild type, we provide evidence that dimerization of the HPV11 DBD occurs only on two appropriately positioned E1 binding-sites and contributes approximately a 10-fold increase in binding affinity. In contrast, the HPV11 E1 helicase purified as preformed hexamers binds DNA with little sequence specificity, similarly to a dimerization-defective DBD. Finally, we show that the amino acid substitution that prevents dimerization reduces the ability of a longer E1 protein to bind to the origin in vitro and to support transient HPV DNA replication in vivo, but has little effect on its ATPase activity or ability to oligomerize into hexamers. These results are discussed in light of a model of the assembly of replication-competent double hexameric E1 complexes at the origin.

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29

Bashir, Qamar, Zhong Li, Hongmin Li, David M. LeMaster, and Griselda Hernández. "Crystal structure and transient dimerization for the FKBP12 protein from the pathogenic fungus Candida auris." Biochemical and Biophysical Research Communications 525, no. 4 (May 2020): 1103–8. http://dx.doi.org/10.1016/j.bbrc.2020.03.059.

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30

Farah, M. H., J. M. Olson, H. B. Sucic, R. I. Hume, S. J. Tapscott, and D. L. Turner. "Generation of neurons by transient expression of neural bHLH proteins in mammalian cells." Development 127, no. 4 (February 15, 2000): 693–702. http://dx.doi.org/10.1242/dev.127.4.693.

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Basic helix-loop-helix (bHLH) transcription factors are known to function during mammalian neurogenesis. Here we show that transient transfection of vectors expressing neuroD2, MASH1, ngn1 or related neural bHLH proteins, with their putative dimerization partner E12, can convert mouse P19 embryonal carcinoma cells into differentiated neurons. Transfected cells express numerous neuron-specific proteins, adopt a neuronal morphology and are electrically excitable. Thus, the expression of neural bHLH proteins is sufficient to confer a neuronal fate on uncommitted mammalian cells. Neuronal differentiation of transfected cells is preceded by elevated expression of the cyclin-dependent kinase inhibitor p27(Kip1) and cell cycle withdrawal. This demonstrates that the bHLH proteins can link neuronal differentiation to withdrawal from the cell cycle, possibly by activating the expression of p27(Kip1). The ability to generate mammalian neurons by transient expression of neural bHLH proteins should create new opportunities for studying neurogenesis and devising neural repair strategies.

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Di Antonio, Veronica, Giorgio Palù, and Gualtiero Alvisi. "Live-Cell Analysis of Human Cytomegalovirus DNA Polymerase Holoenzyme Assembly by Resonance Energy Transfer Methods." Microorganisms 9, no. 5 (April 26, 2021): 928. http://dx.doi.org/10.3390/microorganisms9050928.

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Human cytomegalovirus (HCMV) genome replication is a complex and still not completely understood process mediated by the highly coordinated interaction of host and viral products. Among the latter, six different proteins form the viral replication complex: a single-stranded DNA binding protein, a trimeric primase/helicase complex and a two subunit DNA polymerase holoenzyme, which in turn contains a catalytic subunit, pUL54, and a dimeric processivity factor ppUL44. Being absolutely required for viral replication and representing potential therapeutic targets, both the ppUL44–pUL54 interaction and ppUL44 homodimerization have been largely characterized from structural, functional and biochemical points of view. We applied fluorescence and bioluminescence resonance energy transfer (FRET and BRET) assays to investigate such processes in living cells. Both interactions occur with similar affinities and can take place both in the nucleus and in the cytoplasm. Importantly, single amino acid substitutions in different ppUL44 domains selectively affect its dimerization or ability to interact with pUL54. Intriguingly, substitutions preventing DNA binding of ppUL44 influence the BRETmax of protein–protein interactions, implying that binding to dsDNA induces conformational changes both in the ppUL44 homodimer and in the DNA polymerase holoenzyme. We also compared transiently and stably ppUL44-expressing cells in BRET inhibition assays. Transient expression of the BRET donor allowed inhibition of both ppUL44 dimerization and formation of the DNA polymerase holoenzyme, upon overexpression of FLAG-tagged ppUL44 as a competitor. Our approach could be useful both to monitor the dynamics of assembly of the HCMV DNA polymerase holoenzyme and for antiviral drug discovery.

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32

Hoffmann, Douglas, Thoralf Gross, Rhett Kempe, and Hartmut Oehme. "Head-to-head versus head-to-tail dimerizations of transient silenes — the generation and dimerization behavior of 2-(2-dimethylaminoaryl)-1,1-bis(trimethylsilyl)silenes." Journal of Organometallic Chemistry 598, no. 2 (April 2000): 395–402. http://dx.doi.org/10.1016/s0022-328x(99)00740-8.

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33

Monsonego-Ornan, E., R. Adar, T. Feferman, O. Segev, and A. Yayon. "The Transmembrane Mutation G380R in Fibroblast Growth Factor Receptor 3 Uncouples Ligand-Mediated Receptor Activation from Down-Regulation." Molecular and Cellular Biology 20, no. 2 (January 15, 2000): 516–22. http://dx.doi.org/10.1128/mcb.20.2.516-522.2000.

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ABSTRACT A point mutation, Gly380Arg, in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3) leads to achondroplasia, the most common form of genetic dwarfism in humans. This substitution was suggested to enhance mutant receptor dimerization, leading to constitutive, ligand-independent activation. We found that dimerization and activation of the G380R mutant receptor are predominantly ligand dependent. However, using both transient and stable transfections, we found significant overexpression only of the mutant receptor protein. Metabolic pulse-chase experiments, cell surface labeling, and kinetics of uptake of radiolabeled ligand demonstrated a selective delay in the down-regulation of the mutant receptor. Moreover, this receptor was now resistant to ligand-mediated internalization, even at saturating ligand concentrations. Finally, transgenic mice expressing the human G380R mutant receptor under the mouse receptor transcriptional control demonstrated a markedly expanded area of FGFR3 immunoreactivity within their epiphyseal growth plates, compatible with an in vivo defect in receptor down-regulation. We propose that the achondroplasia mutation G380R uncouples ligand-mediated receptor activation from down-regulation at a site where the levels and kinetics of FGFR3 signals are crucial for chondrocyte maturation and bone formation.

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34

Abankwa, Daniel, and Alemayehu A. Gorfe. "Mechanisms of Ras Membrane Organization and Signaling: Ras Rocks Again." Biomolecules 10, no. 11 (November 6, 2020): 1522. http://dx.doi.org/10.3390/biom10111522.

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Ras is the most frequently mutated oncogene and recent drug development efforts have spurred significant new research interest. Here we review progress toward understanding how Ras functions in nanoscale, proteo-lipid signaling complexes on the plasma membrane, called nanoclusters. We discuss how G-domain reorientation is plausibly linked to Ras-nanoclustering and -dimerization. We then look at how these mechanistic features could cooperate in the engagement and activation of RAF by Ras. Moreover, we show how this structural information can be integrated with microscopy data that provide nanoscale resolution in cell biological experiments. Synthesizing the available data, we propose to distinguish between two types of Ras nanoclusters, an active, immobile RAF-dependent type and an inactive/neutral membrane anchor-dependent. We conclude that it is possible that Ras reorientation enables dynamic Ras dimerization while the whole Ras/RAF complex transits into an active state. These transient di/oligomer interfaces of Ras may be amenable to pharmacological intervention. We close by highlighting a number of open questions including whether all effectors form active nanoclusters and whether there is an isoform specific composition of Ras nanocluster.

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35

Dalle Vedove, Andrea, Anna Paola Lucarelli, Valentina Nardone, Angelica Matino, and Emilio Parisini. "The X-ray structure of human P-cadherin EC1-EC2 in a closed conformation provides insight into the type I cadherin dimerization pathway." Acta Crystallographica Section F Structural Biology Communications 71, no. 4 (March 20, 2015): 371–80. http://dx.doi.org/10.1107/s2053230x15003878.

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Cadherins are a large family of calcium-dependent proteins that mediate cellular adherens junction formation and tissue morphogenesis. To date, the most studied cadherins are those classified as classical, which are further divided into type I or type II depending on selected sequence features. Unlike other members of the classical cadherin family, a detailed structural characterization of P-cadherin has not yet been fully obtained. Here, the high-resolution crystal structure determination of the closed form of human P-cadherin EC1-EC2 is reported. The structure shows a novel, monomeric packing arrangement that provides a further snapshot in the yet-to-be-achieved complete description of the highly dynamic cadherin dimerization pathway. Moreover, this is the first multidomain cadherin fragment to be crystallized and structurally characterized in its closed conformation that does not carry any extra N-terminal residues before the naturally occurring aspartic acid at position 1. Finally, two clear alternate conformations are observed for the critical Trp2 residue, suggestive of a transient, metastable state. The P-cadherin structure and packing arrangement shown here provide new and valuable information towards the complete structural characterization of the still largely elusive cadherin dimerization pathway.

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36

Liu, Tina Y., Xin Bian, Fabian B. Romano, Tom Shemesh, Tom A. Rapoport, and Junjie Hu. "Cis and trans interactions between atlastin molecules during membrane fusion." Proceedings of the National Academy of Sciences 112, no. 15 (March 30, 2015): E1851—E1860. http://dx.doi.org/10.1073/pnas.1504368112.

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Atlastin (ATL), a membrane-anchored GTPase that mediates homotypic fusion of endoplasmic reticulum (ER) membranes, is required for formation of the tubular network of the peripheral ER. How exactly ATL mediates membrane fusion is only poorly understood. Here we show that fusion is preceded by the transient tethering of ATL-containing vesicles caused by the dimerization of ATL molecules in opposing membranes. Tethering requires GTP hydrolysis, not just GTP binding, because the two ATL molecules are pulled together most strongly in the transition state of GTP hydrolysis. Most tethering events are futile, so that multiple rounds of GTP hydrolysis are required for successful fusion. Supported lipid bilayer experiments show that ATL molecules sitting on the same (cis) membrane can also undergo nucleotide-dependent dimerization. These results suggest that GTP hydrolysis is required to dissociate cis dimers, generating a pool of ATL monomers that can dimerize with molecules on a different (trans) membrane. In addition, tethering and fusion require the cooperation of multiple ATL molecules in each membrane. We propose a comprehensive model for ATL-mediated fusion that takes into account futile tethering and competition between cis and trans interactions.

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37

Evans, Nathan J., and Jeffery W. Walker. "Sustained Ca2+ signaling and delayed internalization associated with endothelin receptor heterodimers linked through a PDZ fingerThis article is one of a selection of papers published in the special issue (part 2 of 2) on Forefronts in Endothelin." Canadian Journal of Physiology and Pharmacology 86, no. 8 (August 2008): 526–35. http://dx.doi.org/10.1139/y08-050.

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G protein-coupled receptors (GPCRs), including endothelin receptor A (ETA) and B (ETB), may form dimers or higher-order oligomers that profoundly influence signaling. Here we examined a PDZ finger motif within the C-terminus of ETA and its role in heterodimerization with ETB, and in homodimerization with itself, when expressed in HEK293 cells. Receptor dimerization was monitored by (i) fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) (FRET donor) and tetracysteine/FlAsH (FRET acceptor) fused to the C-termini of ET receptors, and (ii) coimmunoprecipitation of ET receptors after mild detergent solubilization. Mutations in a PDZ finger motif at threonine403/serine404 eliminated FRET and reduced coimmunoprecipitation of heterodimers and homodimers. Functional consequences were evaluated by measuring mobilization of intracellular Ca2+ and internalization of receptors in response to a 10 nmol/L ET-1 challenge. PDZ mutations converted a sustained Ca2+ signal mediated by ETA:ETB heterodimers into a transient response, similar to that observed for homodimers or monomers. Heterodimers containing PDZ mutations were seen to internalize in a similar time domain (approximately 5 min) to the transient Ca2+ elevation and with similar kinetics to internalization of ETA homodimers or monomers. Without the PDZ mutations, heterodimers did not internalize over 15 min, suggesting the intriguing possibility that sustained Ca2+ signaling was a consequence (at least in part) of delayed internalization. The results are consistent with structural models of ETA-receptor dimerization that place threonine403/serine404 of the PDZ finger motif at the interaction interface between heterodimers and homodimers. Sustained Ca2+ signaling and delayed endocytosis of ETA:ETB heterodimers argues strongly for a unique dimer interface that impacts transmembrane signaling and internalization.

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38

Muriaux, Delphine, Hugues De Rocquigny, Bernard-Pierre Roques, and Jacques Paoletti. "NCp7 Activates HIV-1LaiRNA Dimerization by Converting a Transient Loop-Loop Complex into a Stable Dimer." Journal of Biological Chemistry 271, no. 52 (December 27, 1996): 33686–92. http://dx.doi.org/10.1074/jbc.271.52.33686.

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39

Otzen, Daniel E., Simona Miron, Mikael Akke, and Mikael Oliveberg. "Transient Aggregation and Stable Dimerization Induced by Introducing an Alzheimer Sequence into a Water-Soluble Protein†." Biochemistry 43, no. 41 (October 2004): 12964–78. http://dx.doi.org/10.1021/bi048509k.

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40

Pillon, Monica, Vignesh Babu, Mark Sutton, and Alba Guarne. "Structural characterization of transient interactions in DNA mismatch repair." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C418. http://dx.doi.org/10.1107/s2053273314095813.

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DNA mismatch repair (MMR) is a conserved pathway that safeguards genome integrity by correcting replication errors. The initiation of MMR is orchestrated by two proteins –MutS and MutL. MutS detects replication errors and recruits MutL, a key regulator in coordinating downstream MMR events. The processivity clamp, typically known to tether the replicative polymerase to DNA during DNA synthesis, also has a role in several steps in MMR. We have previously shown that MutL transiently interacts with the clamp and that this complex is important for MMR in vivo. The role of the clamp in eukaryotes and most bacteria is believed to license MutL endonuclease activity. In bacterial organisms where MutL does not have endonuclease activity, such as in Escherichia coli, the clamp also interacts with MutL and this interaction is also important for MMR activity. However, the transient nature of this complex prevents its functional and structural characterization. Here, we develop a method to stabilize the E. coli MutL-clamp complex by engineering a disulfide bond at the known protein complex interface and characterize its structure using small angle X-ray scattering (SAXS). MutL binds the clamp through a consensus motif found in its dimerization domain. Using this domain (MutL-CTD) we monitor complex formation with the clamp. We observe two complexes using SAXS. In one complex the MutL-CTD occupies a single hydrophobic cleft of the clamp, while the other occupies both hydrophobic clefts simultaneously. To identify the physiological complex, we used the full length MutL protein to impose further constraints. Analysis of complex formation suggests that full length MutL binds a single cleft on the clamp. Altogether, our data reveals how MutL interacts with the clamp in the early steps of MMR and this approach could be implemented to structurally characterize other transient complexes, an aspect of structural biology that is largely unexplored.

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41

Sagar, G. D. Vivek, Balázs Gereben, Isabelle Callebaut, Jean-Paul Mornon, Anikó Zeöld, Wagner S. da Silva, Cristina Luongo, et al. "Ubiquitination-Induced Conformational Change within the Deiodinase Dimer Is a Switch Regulating Enzyme Activity." Molecular and Cellular Biology 27, no. 13 (April 23, 2007): 4774–83. http://dx.doi.org/10.1128/mcb.00283-07.

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ABSTRACT Ubiquitination is a critical posttranslational regulator of protein stability and/or subcellular localization. Here we show that ubiquitination can also regulate proteins by transiently inactivating enzymatic function through conformational change in a dimeric enzyme, which can be reversed upon deubiquitination. Our model system is the thyroid hormone-activating type 2 deiodinase (D2), an endoplasmic reticulum-resident type 1 integral membrane enzyme. D2 exists as a homodimer maintained by interacting surfaces at its transmembrane and globular cytosolic domains. The D2 dimer associates with the Hedgehog-inducible ubiquitin ligase WSB-1, the ubiquitin conjugase UBC-7, and VDU-1, a D2-specific deubiquitinase. Upon binding of T4, its natural substrate, D2 is ubiquitinated, which inactivates the enzyme by interfering with D2's globular interacting surfaces that are critical for dimerization and catalytic activity. This state of transient inactivity and change in dimer conformation persists until deubiquitination. The continuous association of D2 with this regulatory protein complex supports rapid cycles of deiodination, conjugation to ubiquitin, and enzyme reactivation by deubiquitination, allowing tight control of thyroid hormone action.

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42

Guo, Xiangxue, Robert W. Harrison, and Phang C. Tai. "Nucleotide-Dependent Dimerization of the C-Terminal Domain of the ABC Transporter CvaB in Colicin V Secretion." Journal of Bacteriology 188, no. 7 (April 1, 2006): 2383–91. http://dx.doi.org/10.1128/jb.188.7.2383-2391.2006.

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ABSTRACT The cytoplasmic membrane proteins CvaB and CvaA and the outer membrane protein TolC constitute the bacteriocin colicin V secretion system in Escherichia coli. CvaB functions as an ATP-binding cassette transporter, and its C-terminal domain (CTD) contains typical motifs for the nucleotide-binding and Walker A and B sites and the ABC signature motif. To study the role of the CvaB CTD in the secretion of colicin V, a truncated construct of this domain was made and overexpressed. Different forms of the CvaB CTD were found during purification and identified as monomer, dimer, and oligomer forms by gel filtration and protein cross-linking. Nucleotide binding was shown to be critical for CvaB CTD dimerization. Oligomers could be converted to dimers by nucleotide triphosphate-Mg, and nucleotide release from dimers resulted in transient formation of monomers, followed by oligomerization and aggregation. Site-directed mutagenesis showed that the ABC signature motif was involved in the nucleotide-dependent dimerization. The spatial proximity of the Walker A site and the signature motif was shown by disulfide cross-linking a mixture of the A530C and L630C mutant proteins, while the A530C or L630C mutant protein did not dimerize on its own. Taken together, these results indicate that the CvaB CTD formed a nucleotide-dependent head-to-tail dimer.

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43

Prodromou, C. "The ATPase cycle of Hsp90 drives a molecular clamp' via transient dimerization of the N-terminal domains." EMBO Journal 19, no. 16 (August 15, 2000): 4383–92. http://dx.doi.org/10.1093/emboj/19.16.4383.

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44

Mirodatos, C., A. Holmen, R. Mariscal, and G. A. Martin. "Methane activation in exchange and oxidative dimerization reactions : a study using isotope steady-state and transient techniques." Catalysis Today 6, no. 4 (February 1990): 601–10. http://dx.doi.org/10.1016/0920-5861(90)85057-u.

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45

Boned del Río, Isabel, Lucy C. Young, Sibel Sari, Greg G. Jones, Benjamin Ringham-Terry, Nicole Hartig, Ewa Rejnowicz, et al. "SHOC2 complex-driven RAF dimerization selectively contributes to ERK pathway dynamics." Proceedings of the National Academy of Sciences 116, no. 27 (June 18, 2019): 13330–39. http://dx.doi.org/10.1073/pnas.1902658116.

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Despite the crucial role of RAF kinases in cell signaling and disease, we still lack a complete understanding of their regulation. Heterodimerization of RAF kinases as well as dephosphorylation of a conserved “S259” inhibitory site are important steps for RAF activation but the precise mechanisms and dynamics remain unclear. A ternary complex comprised of SHOC2, MRAS, and PP1 (SHOC2 complex) functions as a RAF S259 holophosphatase and gain-of-function mutations in SHOC2, MRAS, and PP1 that promote complex formation are found in Noonan syndrome. Here we show that SHOC2 complex-mediated S259 RAF dephosphorylation is critically required for growth factor-induced RAF heterodimerization as well as for MEK dissociation from BRAF. We also uncover SHOC2-independent mechanisms of RAF and ERK pathway activation that rely on N-region phosphorylation of CRAF. In DLD-1 cells stimulated with EGF, SHOC2 function is essential for a rapid transient phase of ERK activation, but is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and CRAF. Whereas redundant SHOC2-dependent and -independent mechanisms of RAF and ERK activation make SHOC2 dispensable for proliferation in 2D, KRAS mutant cells preferentially rely on SHOC2 for ERK signaling under anchorage-independent conditions. Our study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for selective ERK pathway inhibition by targeting the SHOC2 holophosphatase.

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46

Schmohl, Kathleen, Matthias Blach, Helmut Reinke, Rhett Kempe, and Hartmut Oehme. "Head-to-Head versus Head-to-Tail Dimerizations of Transient Silenes – The Solvent-Dependent Regiospecifity of the Dimerization of 2-(2-Methoxyphenyl)-1,1-bis(trimethylsilyl)silene." European Journal of Inorganic Chemistry 1998, no. 11 (November 1998): 1667–72. http://dx.doi.org/10.1002/(sici)1099-0682(199811)1998:11<1667::aid-ejic1667>3.0.co;2-u.

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47

Nekipelova, T. D. "Phototransformations of non-toxic antioxidants, the derivatives of 1,2-dihydroquinolines, in homogeneous and micellar solutions." International Journal of Photoenergy 1, no. 1 (1999): 31–34. http://dx.doi.org/10.1155/s1110662x99000069.

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Reactions of transient species photogenerated from 6-R-2,2,4-trimethyl-1,2-dihydroquinolines (TMDQ) are very sensitive to medium variation. In anhydrous organic solvents, aminyl radicals were generated. They decay in the reaction of dimerization with the second-order rate constant decreasing in a row heptane>benzene>2-propanol. When passing from organic solvents to water, methanol, and water-alcohol solutions, the kinetics and the direction of the reaction crucially change. As a result of the photolysis, the product of the addition of a solvent to the double bond of heterocycle, 4-hydroxy- or 4-methoxy-6-R-2,2,4- tetramethyl-1,2,3,4-tetrahydroquinoline is formed in water and methanol, respectively. The transformation is a complex reaction, and the formation of excited transient species is followed by a sequence of first-order and pseudo-first-order reactions. Unlike the photolysis in anhydrous organic solvents, the reaction in water and methanol does not involve aminyl radicals. In aqueous solutions, the first-order rate constants for the decay of transient species are higher in acidic and neutral solutions. At the pH close to pKa of the transient species, it drops, indicating that the neutral form is less reactive. The same product is formed over the whole range of pH. For the anionic surfactant (SDS) in acidic and alkaline solutions, the apparent rate constant in the micellar solutions is lower than that in the aqueous (negative micellar catalysis). At the medium pH, a positive micellar catalysis is observed, and the rate constant of the decay depends linearly on the concentration of TMDQ in the micelles, indicative of the direct reaction between TMDQ and the cationic transient species.

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48

Kiyatkin, Anatoly, Iris K. van Alderwerelt van Rosenburgh, Daryl E. Klein, and Mark A. Lemmon. "Kinetics of receptor tyrosine kinase activation define ERK signaling dynamics." Science Signaling 13, no. 645 (August 18, 2020): eaaz5267. http://dx.doi.org/10.1126/scisignal.aaz5267.

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In responses to activation of receptor tyrosine kinases (RTKs), crucial cell fate decisions depend on the duration and dynamics of ERK signaling. In PC12 cells, epidermal growth factor (EGF) induces transient ERK activation that leads to cell proliferation, whereas nerve growth factor (NGF) promotes sustained ERK activation and cell differentiation. These differences have typically been assumed to reflect distinct feedback mechanisms in the Raf-MEK-ERK signaling network, with the receptors themselves acting as simple upstream inputs. We failed to confirm the expected differences in feedback type when investigating transient versus sustained signaling downstream of the EGF receptor (EGFR) and NGF receptor (TrkA). Instead, we found that ERK signaling faithfully followed RTK dynamics when receptor signaling was modulated in different ways. EGFR activation kinetics, and consequently ERK signaling dynamics, were switched from transient to sustained when receptor internalization was inhibited with drugs or mutations, or when cells expressed a chimeric receptor likely to have impaired dimerization. In addition, EGFR and ERK signaling both became more sustained when substoichiometric levels of erlotinib were added to reduce duration of EGFR kinase activation. Our results argue that RTK activation kinetics play a crucial role in determining MAP kinase cascade signaling dynamics and cell fate decisions, and that signaling outcome can be modified by activating a given RTK in different ways.

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49

Lim, Chunghun, Hekwang Sohn, Daeyoup Lee, Yousang Gwack, and Joonho Choe. "Functional Dissection of Latency-Associated Nuclear Antigen 1 of Kaposi's Sarcoma-Associated Herpesvirus Involved in Latent DNA Replication and Transcription of Terminal Repeats of the Viral Genome." Journal of Virology 76, no. 20 (October 15, 2002): 10320–31. http://dx.doi.org/10.1128/jvi.76.20.10320-10331.2002.

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ABSTRACT Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV) is implicated in the maintenance of the viral genome during latent infection. LANA1 colocalizes with KSHV episomes on the host chromosome and mediates their maintenance by attaching these viral structures to host chromosomes. Data from long-term selection of drug resistance in cells conferred by plasmids containing the terminal repeat (TR) sequence of KSHV revealed that KSHV TRs and LANA1 act as cis and trans elements of viral latent replication, respectively. In this study, we further characterized the cis- and trans-acting elements of KSHV latent replication by using a transient replication assay with a methylation-sensitive restriction enzyme, DpnI. Transient reporter and replication assays disclosed that the orientation and basal transcriptional activity of TR constructs did not significantly affect the efficiency of replication. However, at least two TR units were necessary for efficient replication. The N-terminal 90 amino acids comprising the chromosome-binding domain of LANA1 were required for the mediation of LANA1 C-terminal DNA-binding and dimerization domains to support the transient replication of KSHV TRs. LANA1 interacted with components of the origin recognition complexes (ORCs), similar to Epstein-Barr virus nuclear antigen 1. Our data suggest that LANA1 recruits ORCs to KSHV TRs for latent replication of the viral genome.

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50

Hunter, J. J., B. L. Bond, and T. G. Parslow. "Functional dissection of the human Bc12 protein: sequence requirements for inhibition of apoptosis." Molecular and Cellular Biology 16, no. 3 (March 1996): 877–83. http://dx.doi.org/10.1128/mcb.16.3.877.

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Overexpression of the cytoplasmic oncoprotein Bc12 blocks programmed cell death (apoptosis) in many cellular systems. To map the sequences in Bc12 that are necessary for its activity, we created a library of deletion-scanning mutants of this 239-amino-acid protein and tested their abilities to block staurosporine-induced fibroblast apoptosis, using a novel transient-transfection assay. Phenotypes of informative mutants were then confirmed by assaying for inhibition of steroid-induced apoptosis in stably transfected T-lymphoid cells. In accordance with earlier results, we found that Bc12 activity was only partially reduced after deletion of the hydrophobic tail that normally anchors it in cytoplasmic membranes. Essential sequences were found in the remainder of the protein and appeared to be organized in at least two discrete functional domains. The larger, more C-terminal region (within residues 90 to 203) encompassed, but extended beyond, two oligopeptide motifs called BH1 and BH2, which are known to mediate dimerization of Bc12 and related proteins. The second, more N-terminal regions (within residues 6 to 31) was not required for protein dimerization in vivo, but its deletion imparted a dominant negative phenotype, yielding mutants that promoted rather than inhibited apoptotic death. Residues 30 to 91 were not absolutely required for function; by deleting most of this region along with the hydrophobic tail, we derived a 155-residue mini-Bc12 that retains significant ability to inhibit apoptosis.

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