scholarly journals Divergence in Thermostability of Arabidopsis Mitochondrial Nucleotide Exchange Factors Encoded by Duplicate Genes, MGE1 and MGE2

2019 ◽  
Author(s):  
Zih-teng Chen ◽  
Meng-Ju Hung ◽  
Shih-Jiun Yu ◽  
Tai-Yan Liao ◽  
Yao-Pin Lin ◽  
...  
Keyword(s):  
Elevated Temperatures ◽  
Duplication Event ◽  
Duplicate Genes ◽  
Nucleotide Exchange ◽  
Homologous Proteins ◽  
Genome Duplication Event ◽  
E Coli ◽  
Conserved Sequence ◽  
Nucleotide Exchange Factors

ABSTRACTThe divergence of duplicate genes links to organismic adaptation. In Arabidopsis thaliana two nuclear genes encode mitochondrial GrpEs, MGE1 and MGE2, the nucleotide exchange factors of DnaK/HSP70 chaperone. MGE1 and MGE2 are duplicate genes originated from a whole genome duplication event. They respond differentially to high temperature; MGE2 is heat-inducible and is required for Arabidopsis seedlings to tolerate prolonged heat stress, while MGE1 is constitutively expressed. Heterologous expression of MGE2 but not MGE1 restored the growth of E. coli grpE mutant cells at elevated temperatures, suggesting that MGE2 is more thermostable than MGE1. In this study, we directly compared the thermostability of the purified recombinant MGE1 and MGE2 by circular dichroism spectroscopy. The temperature midpoints of the unfolding transition (Tm) of MGE1 and MGE2 were about 38 and 46 °C, respectively, indicating that MGE2 is remarkably more stable than MGE1 at higher temperature. Domain swapping between the two homologous proteins showed that the N-terminal region, including an unstructured sequence and a long α-helix domain, is the major determinant of the thermostability. Although MGE2 contains a conserved sequence derived from an exonized intron within the N-terminus unstructured region, deletion of this sequence did not substantially affect protein thermostability in vitro and complementation of E. coli and Arabidopsis heat sensitive mutants. Taken together, our results suggest that Arabidopsis MGE1 and MGE2 had diverged not only in transcriptional response but also in the thermostability of the encoded proteins, which may contribute to adaptation of plants to higher temperatures.

scholarly journals Essential Role of Vav Family Guanine Nucleotide Exchange Factors in EphA Receptor-Mediated Angiogenesis

2006 ◽  
Vol 26 (13) ◽  
pp. 4830-4842 ◽  
Author(s):  
Sonja G. Hunter ◽  
Guanglei Zhuang ◽  
Dana Brantley-Sieders ◽  
Wojciech Swat ◽  
Christopher W. Cowan ◽  
...  
Keyword(s):  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Rac1 Gtpase ◽  
Rho Family ◽  
Epha Receptor

ABSTRACT Angiogenesis, the process by which new blood vessels are formed from preexisting vasculature, is critical for vascular remodeling during development and contributes to the pathogenesis of diseases such as cancer. Prior studies from our laboratory demonstrate that the EphA2 receptor tyrosine kinase is a key regulator of angiogenesis in vivo. The EphA receptor-mediated angiogenic response is dependent on activation of Rho family GTPase Rac1 and is regulated by phosphatidylinositol 3-kinase. Here we report the identification of Vav2 and Vav3 as guanine nucleotide exchange factors (GEFs) that link the EphA2 receptor to Rho family GTPase activation and angiogenesis. Ephrin-A1 stimulation recruits the binding of Vav proteins to the activated EphA2 receptor. The induced association of EphA receptor and Vav proteins modulates the activity of Vav GEFs, leading to activation of Rac1 GTPase. Overexpression of either Vav2 or Vav3 in primary microvascular endothelial cells promotes Rac1 activation, cell migration, and assembly in response to ephrin-A1 stimulation. Conversely, loss of Vav2 and Vav3 GEFs inhibits Rac1 activation and ephrin-A1-induced angiogenic responses both in vitro and in vivo. In addition, embryonic fibroblasts derived from Vav2−/− Vav3−/− mice fail to spread on an ephrin-A1-coated surface and exhibit a significant decrease in the formation of ephrin-A1-induced lamellipodia and filopodia. These findings suggest that Vav GEFs serve as a molecular link between EphA2 receptors and the actin cytoskeleton and provide an important mechanism for EphA2-mediated angiogenesis.

scholarly journals Conserved Stem II of the Box C/D Motif Is Essential for Nucleolar Localization and Is Required, Along with the 15.5K Protein, for the Hierarchical Assembly of the Box C/D snoRNP

2002 ◽  
Vol 22 (23) ◽  
pp. 8342-8352 ◽  
Author(s):  
Nicholas J. Watkins ◽  
Achim Dickmanns ◽  
Reinhard Lührmann
Keyword(s):  
Specific Binding ◽  
Nucleolar Localization ◽  
Homologous Proteins ◽  
Stem Loop ◽  
Hierarchical Assembly ◽  
Conserved Sequence ◽  
Associated Proteins ◽  
The Individual ◽  
Stem Structure

ABSTRACT The 5′ stem-loop of the U4 snRNA and the box C/D motif of the box C/D snoRNAs can both be folded into a similar stem-internal loop-stem structure that binds the 15.5K protein. The homologous proteins NOP56 and NOP58 and 61K (hPrp31) associate with the box C/D snoRNPs and the U4/U6 snRNP, respectively. This raises the intriguing question of how the two homologous RNP complexes specifically assemble onto similar RNAs. Here we investigate the requirements for the specific binding of the individual snoRNP proteins to the U14 box C/D snoRNPs in vitro. This revealed that the binding of 15.5K to the box C/D motif is essential for the association of the remaining snoRNP-associated proteins, namely, NOP56, NOP58, fibrillarin, and the nucleoplasmic proteins TIP48 and TIP49. Stem II of the box C/D motif, in contrast to the U4 5′ stem-loop, is highly conserved, and we show that this sequence is responsible for the binding of NOP56, NOP58, fibrillarin, TIP48, and TIP49, but not of 15.5K, to the snoRNA. Indeed, the sequence of stem II was essential for nucleolar localization of U14 snoRNA microinjected into HeLa cells. Thus, the conserved sequence of stem II determines the specific assembly of the box C/D snoRNP.

scholarly journals Distinct Subclasses of Small GTPases Interact with Guanine Nucleotide Exchange Factors in a Similar Manner

1998 ◽  
Vol 18 (12) ◽  
pp. 7444-7454 ◽  
Author(s):  
Gwo-Jen Day ◽  
Raymond D. Mosteller ◽  
Daniel Broek
Keyword(s):  
Alpha Helix ◽  
Small Gtpases ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Structural Determinants ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Ras Superfamily

ABSTRACT The Ras-related GTPases are small, 20- to 25-kDa proteins which cycle between an inactive GDP-bound form and an active GTP-bound state. The Ras superfamily includes the Ras, Rho, Ran, Arf, and Rab/YPT1 families, each of which controls distinct cellular functions. The crystal structures of Ras, Rac, Arf, and Ran reveal a nearly superimposible structure surrounding the GTP-binding pocket, and it is generally presumed that the Rab/YPT1 family shares this core structure. The Ras, Rac, Ran, Arf, and Rab/YPT1 families are activated by interaction with family-specific guanine nucleotide exchange factors (GEFs). The structural determinants of GTPases required for interaction with family-specific GEFs have begun to emerge. We sought to determine the sites on YPT1 which interact with GEFs. We found that mutations of YPT1 at position 42, 43, or 49 (effector loop; switch I), position 69, 71, 73, or 75 (switch II), and position 107, 109, or 115 (alpha-helix 3–loop 7 [α3-L7]) are intragenic suppressors of dominant interfering YPT1 mutant N22 (YPT1-N22), suggesting these mutations prevent YPT1-N22 from binding to and sequestering an endogenous GEF. Mutations at these positions prevent interaction with the DSS4 GEF in vitro. Mutations in the switch II and α3-L7 regions do not prevent downstream signaling in yeast when combined with a GTPase-defective (activating) mutation. Together, these results show that the YPT1 GTPase interacts with GEFs in a manner reminiscent of that for Ras and Arf in that these GTPases use divergent sequences corresponding to the switch I and II regions and α3-L7 of Ras to interact with family-specific GEFs. This finding suggests that GTPases of the Ras superfamily each may share common features of GEF-mediated guanine nucleotide exchange even though the GEFs for each of the Ras subfamilies appear evolutionarily unrelated.

scholarly journals RapGEF2 is essential for embryonic hematopoiesis but dispensable for adult hematopoiesis

Blood ◽  
2010 ◽  
Vol 116 (16) ◽  
pp. 2921-2931 ◽  
Author(s):  
Ande Satyanarayana ◽  
Kristbjorn Orri Gudmundsson ◽  
Xiu Chen ◽  
Vincenzo Coppola ◽  
Lino Tessarollo ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Conditional Knockout ◽  
Nucleotide Exchange ◽  
Hematopoietic Stem ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
And Migration ◽  
Rap1 Activation ◽  
Transcription Factor Expression

Abstract RapGEF2 is one of many guanine nucleotide exchange factors (GEFs) that specifically activate Rap1. Here, we generated RapGEF2 conditional knockout mice and studied its role in embryogenesis and fetal as well as adult hematopoietic stem cell (HSC) regulation. RapGEF2 deficiency led to embryonic lethality at ∼ E11.5 due to severe yolk sac vascular defects. However, a similar number of Flk1+ cells were present in RapGEF2+/+ and RapGEF2−/− yolk sacs indicating that the bipotential early progenitors were in fact generated in the absence of RapGEF2. Further analysis of yolk sacs and embryos revealed a significant reduction of CD41 expressing cells in RapGEF2−/− genotype, suggesting a defect in the maintenance of definitive hematopoiesis. RapGEF2−/− cells displayed defects in proliferation and migration, and the in vitro colony formation ability of hematopoietic progenitors was also impaired. At the molecular level, Rap1 activation was impaired in RapGEF2−/− cells that in turn lead to defective B-raf/ERK signaling. Scl/Gata transcription factor expression was significantly reduced, indicating that the defects observed in RapGEF2−/− cells could be mediated through Scl/Gata deregulation. Inducible deletion of RapGEF2 during late embryogenesis in RapGEF2cko/ckoERcre mice leads to defective fetal liver erythropoiesis. Conversely, inducible deletion in the adult bone marrow, or specific deletion in B cells, T cells, HSCs, and endothelial cells has no impact on hematopoiesis.

scholarly journals A stutter in the coiled coil domain of E.coli co-chaperone GrpE connects structure with function

2020 ◽  
Author(s):  
Tulsi Upadhyay ◽  
Vaibhav V Karekar ◽  
Ishu Saraogi
Keyword(s):  
Heat Stress ◽  
Coiled Coil ◽  
Bacterial Survival ◽  
Nucleotide Exchange ◽  
E Coli ◽  
Coiled Coil Domain ◽  
Nucleotide Exchange Factor ◽  
First Time

AbstractIn bacteria, the co-chaperone GrpE acts as a nucleotide exchange factor and plays an important role in controlling the chaperone cycle of DnaK. The functional form of GrpE is an asymmetric dimer, consisting of a long non-ideal coiled-coil. During heat stress, this region partially unfolds and prevents DnaK nucleotide exchange, ultimately ceasing the chaperone cycle. In this study, we elucidate the role of thermal unfolding of the coiled-coil domain of E. coli GrpE in regulating its co-chaperonic activity. The presence of a stutter disrupts the regular heptad arrangement typically found in an ideal coiled coil resulting in structural distortion. Introduction of hydrophobic residues at the stutter altered the structural stability of the coiled-coil. Using an in vitro FRET assay, we show for the first time that the enhanced stability of GrpE resulted in an increased affinity for DnaK. However, the mutants were defective in in vitro functional assays, and were unable to support bacterial growth at heat shock temperature in a grpE-deleted E. coli strain. This work provides valuable insights into the functional role of a stutter in the GrpE coiled-coil, and its role in regulating the DnaK-chaperone cycle for bacterial survival during heat stress. More generally, our findings illustrate how a sequence specific stutter in a coiled-coil domain regulates the structure function trade-off in proteins.

scholarly journals A biochemical and genetic discovery pipeline identifies PLCδ4b as a nonreceptor activator of heterotrimeric G-proteins

2018 ◽  
Vol 293 (44) ◽  
pp. 16964-16983 ◽  
Author(s):  
Marcin Maziarz ◽  
Stefan Broselid ◽  
Vincent DiGiacomo ◽  
Jong-Chan Park ◽  
Alex Luebbers ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Cell Function ◽  
Sequence Similarity ◽  
Heterotrimeric G Proteins ◽  
Nucleotide Exchange ◽  
Protein Activity ◽  
Conserved Sequence ◽  
Discovery Pipeline

Recent evidence has revealed that heterotrimeric G-proteins can be activated by cytoplasmic proteins that share an evolutionarily conserved sequence called the Gα-binding-and-activating (GBA) motif. This mechanism provides an alternative to canonical activation by G-protein–coupled receptors (GPCRs) and plays important roles in cell function, and its dysregulation is linked to diseases such as cancer. Here, we describe a discovery pipeline that uses biochemical and genetic approaches to validate GBA candidates identified by sequence similarity. First, putative GBA motifs discovered in bioinformatics searches were synthesized on peptide arrays and probed in batch for Gαi3 binding. Then, cDNAs encoding proteins with Gαi3-binding sequences were expressed in a genetically-modified yeast strain that reports mammalian G-protein activity in the absence of GPCRs. The resulting GBA motif candidates were characterized by comparison of their biochemical, structural, and signaling properties with those of all previously described GBA motifs in mammals (GIV/Girdin, DAPLE, Calnuc, and NUCB2). We found that the phospholipase Cδ4 (PLCδ4) GBA motif binds G-proteins with high affinity, has guanine nucleotide exchange factor activity in vitro, and activates G-protein signaling in cells, as indicated by bioluminescence resonance energy transfer (BRET)-based biosensors of G-protein activity. Interestingly, the PLCδ4 isoform b (PLCδ4b), which lacks the domains required for PLC activity, bound and activated G-proteins more efficiently than the full-length isoform a, suggesting that PLCδ4b functions as a G-protein regulator rather than as a PLC. In summary, we have identified PLCδ4 as a nonreceptor activator of G-proteins and established an experimental pipeline to discover and characterize GBA motif–containing proteins.

scholarly journals Activation of Cellular Arf GTPases by Poliovirus Protein 3CD Correlates with Virus Replication

2007 ◽  
Vol 81 (17) ◽  
pp. 9259-9267 ◽  
Author(s):  
George A. Belov ◽  
Courtney Habbersett ◽  
David Franco ◽  
Ellie Ehrenfeld
Keyword(s):  
Cellular Membrane ◽  
Viral Rna ◽  
Nucleotide Exchange ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Cell Extracts ◽  
Infected Cells ◽  
Arf Gtpases ◽  
Poliovirus Protein

ABSTRACT We have previously shown that synthesis of poliovirus protein 3CD in uninfected HeLa cell extracts induces an increased association with membranes of the cellular Arf GTPases, which are key players in cellular membrane traffic. Arfs cycle between an inactive, cytoplasmic, GDP-bound form and an active, membrane-associated, GTP-bound form. 3CD promotes binding of Arf to membranes by initiating recruitment to membranes of guanine nucleotide exchange factors (GEFs), BIG1 and BIG2. GEFs activate Arf by replacing GDP with GTP. In poliovirus-infected cells, there is a dramatic redistribution of cellular Arf pools that coincides with the reorganization of membranes used to form viral RNA replication complexes. Here we demonstrate that Arf translocation in vitro can be induced by purified recombinant 3CD protein; thus, concurrent translation of viral RNA is not required. Coexpression of 3C and 3D proteins was not sufficient to target Arf to membranes. 3CD expressed in HeLa cells was retained after treatment of the cells with digitonin, indicating that it may interact with a membrane-bound host factor. A F441S mutant of 3CD was shown previously to have lost Arf translocation activity and was also defective in attracting the corresponding GEFs to membranes. A series of other mutations were introduced at 3CD residue F441. Mutations that retained Arf translocation activity of 3CD also supported efficient growth of virus, regardless of their effects on 3D polymerase elongation activity. Those that abrogated Arf activation by 3CD generated quasi-infectious RNAs that produced some plaques from which revertants that always restored the Arf activation property of 3CD were rescued.

scholarly journals Vav guanine nucleotide exchange factors link hyperlipidemia and a prothrombotic state

Blood ◽  
2011 ◽  
Vol 117 (21) ◽  
pp. 5744-5750 ◽  
Author(s):  
Kan Chen ◽  
Wei Li ◽  
Jennifer Major ◽  
Shaik Ohidar Rahaman ◽  
Maria Febbraio ◽  
...  
Keyword(s):  
Oxidized Ldl ◽  
Flow Cytometric Analysis ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Prothrombotic State ◽  
Aggregation Assay ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors

AbstractPlatelet hyperactivity associated with hyperlipidemia contributes to development of a pro-thrombotic state. We previously showed that oxidized LDL (oxLDL) formed in the setting of hyperlipidemia and atherosclerosis initiated a CD36-mediated signaling cascade leading to platelet hyperactivity. We now show that the guanine nucleotide exchange factors Vav1 and Vav3 were tyrosine phosphorylated in platelets exposed to oxLDL. Pharmacologic inhibition of src family kinases abolished Vav1 phosphorylation by oxLDL in vitro. Coimmunoprecipitations revealed the tyrosine phosphorylated form of src kinase Fyn was associated with Vav1 in platelets exposed to oxLDL. Using a platelet aggregation assay, we demonstrated that Vav1 deficiency, Fyn deficiency, or Vav1/Vav3 deficiency protected mice from diet-induced platelet hyperactivity. Furthermore, flow cytometric analysis revealed that Vav1/Vav3 deficiency significantly inhibited oxLDL-mediated integrin αIIbβIII activation of platelets costimulated with ADP. Finally, we showed with an in vivo carotid artery thrombosis model that genetic deletion of Vav1 and Vav3 together may prevent the development of occlusive thrombi in mice fed a high-fat diet. These findings implicate Vav proteins in oxLDL-mediated platelet activation and suggest that Vav family member(s) may act as critical modulators linking a prothrombotic state and hyperlipidemia.

scholarly journals Erythrocyte Sensitization by Blood Group-Specific Bacterial Antigens

1964 ◽  
Vol 47 (6) ◽  
pp. 1229-1250 ◽  
Author(s):  
Georg F. Springer ◽  
Richard E. Horton
Keyword(s):  
Blood Group ◽  
Elevated Temperatures ◽  
E Coli ◽  
Tourniquet Shock ◽  
Group B ◽  
Chicken Erythrocytes ◽  
Plasma Factors ◽  
Blood Group H

Human and chicken erythrocytes are readily coated in vitro by blood group active protein-lipopolysaccharides and lipopolysaccharides from E. coli O86 and E. coli O128. Serum albumin, α2- and ß-lipoproteins inhibit this sensitization. Blood group B specific agglutination of erythrocytes with B or B-like antigens was obtained with antibodies purified by adsorption on and elution from B erythrocytes. Anti-blood group B and E. coli O86-specific antibodies could be eluted from E. coli O86-coated O erythrocytes. Eel anti-H(O) serum agglutinated O erythrocytes and only those A1B red cells which were coated with blood group H(O) active E. coli products. Blood group active substances specifically inhibited agglutination of lipopolysaccharide-coated erythrocytes by anti-B and anti-H(O) agglutinins. Demonstrable amounts of lipopolysaccharide could only be removed from coated erythrocytes by washing them at elevated temperatures (58°C) in physiological solutions. Red cell sensitization with B active E. coli O86 substances was achieved in vivo in a minority of severely diseased infants and in germ-free and ordinary chicks which were in tourniquet shock after treatment with cathartics. Therefore, a possible mode by which erythrocytes of patients with severe intestinal disorders acquire antigens is the fixation of bacterial substances to their surfaces, if there are not enough of the normally interfering plasma factors present.

scholarly journals The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis

2015 ◽  
Vol 26 (2) ◽  
pp. 238-255 ◽  
Author(s):  
Ning Wang ◽  
Mo Wang ◽  
Yi-Hua Zhu ◽  
Timothy W. Grosel ◽  
Daokun Sun ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Rho Gtpases ◽  
Cell Polarization ◽  
Nucleotide Exchange ◽  
Cellular Processes ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
Rho Gef

Rho GTPases, activated by Rho guanine nucleotide exchange factors (GEFs), are conserved molecular switches for signal transductions that regulate diverse cellular processes, including cell polarization and cytokinesis. The fission yeast Schizosaccharomyces pombe has six Rho GTPases (Cdc42 and Rho1–Rho5) and seven Rho GEFs (Scd1, Rgf1–Rgf3, and Gef1–Gef3). The GEFs for Rho2–Rho5 have not been unequivocally assigned. In particular, Gef3, the smallest Rho GEF, was barely studied. Here we show that Gef3 colocalizes with septins at the cell equator. Gef3 physically interacts with septins and anillin Mid2 and depends on them to localize. Gef3 coprecipitates with GDP-bound Rho4 in vitro and accelerates nucleotide exchange of Rho4, suggesting that Gef3 is a GEF for Rho4. Consistently, Gef3 and Rho4 are in the same genetic pathways to regulate septum formation and/or cell separation. In gef3∆ cells, the localizations of two potential Rho4 effectors—glucanases Eng1 and Agn1—are abnormal, and active Rho4 level is reduced, indicating that Gef3 is involved in Rho4 activation in vivo. Moreover, overexpression of active Rho4 or Eng1 rescues the septation defects of mutants containing gef3∆. Together our data support that Gef3 interacts with the septin complex and activates Rho4 GTPase as a Rho GEF for septation in fission yeast.

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