scholarly journals Novel Insights Regarding the Operational Characteristics and Teleological Purpose of the Renal Na+-K+-Cl2 Cotransporter (NKCC2s) Splice Variants

2005 ◽  
Vol 126 (4) ◽  
pp. 325-337 ◽  
Author(s):  
Geneviève M. Brunet ◽  
Edith Gagnon ◽  
Charles F. Simard ◽  
Nikolas D. Daigle ◽  
Luc Caron ◽  
...  
Keyword(s):  
Structural Organization ◽  
Apical Membrane ◽  
Splice Variants ◽  
Mrna Levels ◽  
Hybrid Mapping ◽  
Yeast Two Hybrid ◽  
Additional Insight ◽  
Operational Characteristics ◽  
Mapping Analysis ◽  
Two Hybrid

The absorptive Na+-K+-Cl− cotransporter (NKCC2) is a polytopic protein that forms homooligomeric complexes in the apical membrane of the thick ascending loop of Henle (TAL). It occurs in at least four splice variants (called B, A, F, and AF) that are identical to one another except for a short region in the membrane-associated domain. Although each of these variants exhibits unique functional properties and distributions along the TAL, their teleological purpose and structural organization remain poorly defined. In the current work, we provide additional insight in these regards by showing in mouse that the administration of either furosemide or an H2O-rich diet, which are predicted to alter NKCC2 expression in the TAL, exerts differential effects on mRNA levels for the variants, increasing those of A (furosemide) but decreasing those of F and AF (furosemide or H2O). Based on a yeast two-hybrid mapping analysis, we also show that the formation of homooligomeric complexes is mediated by two self-interacting domains in the COOH terminus (residues 671 to 816 and 910 to 1098), and that these complexes could probably include more than one type of variant. Taken together, the data reported here suggest that A, F, and AF each play unique roles that are adapted to specific physiological needs, and that the accomplishment of such roles is coordinated through the splicing machinery as well as complex NKCC2–NKCC2 interactions.

scholarly journals The Multivalent PDZ Domain-containing Protein PDZK1 Regulates Transport Activity of Renal Urate-Anion Exchanger URAT1 via Its C Terminus

2004 ◽  
Vol 279 (44) ◽  
pp. 45942-45950 ◽  
Author(s):  
Naohiko Anzai ◽  
Hiroki Miyazaki ◽  
Rie Noshiro ◽  
Suparat Khamdang ◽  
Arthit Chairoungdua ◽  
...  
Keyword(s):  
Anion Exchanger ◽  
Apical Membrane ◽  
Organic Anion ◽  
Pdz Domain ◽  
Hek293 Cells ◽  
Transport Activity ◽  
Yeast Two Hybrid ◽  
Urate Transport ◽  
Anion Transporter ◽  
Two Hybrid

The urate-anion exchanger URAT1 is a member of the organic anion transporter (OAT) family that regulates blood urate level in humans and is targeted by uricosuric and antiuricosuric agents (Enomoto, A., Kimura, H., Chairoungdua, A., Shigeta, Y., Jutabha, P., Cha, S. H., Hosoyamada, M., Takeda, M., Sekine, T., Igarashi, T., Matsuo, H., Kikuchi, Y., Oda, T., Ichida, K., Hosoya, T., Shimotaka, K., Niwa, T., Kanai, Y., and Endou, H. (2002)Nature417, 447–452). URAT1 is expressed only in the kidney, where it is thought to participate in tubular urate reabsorption. We found that the multivalent PDZ (PSD-95,Drosophiladiscs-large protein,Zonula occludensprotein 1) domain-containing protein, PDZK1 interacts with URAT1 in a yeast two-hybrid screen. Such an interaction requires the PDZ motif of URAT1 in its extreme intracellular C-terminal region and the first, second, and fourth PDZ domains of PDZK1 as identified by yeast two-hybrid assay,in vitrobinding assay and surface plasmon resonance analysis (KD= 1.97–514 nm). Coimmunoprecipitation studies revealed that the wild-type URAT1, but not its mutant lacking the PDZ-motif, directly interacts with PDZK1. Colocalization of URAT1 and PDZK1 was observed at the apical membrane of renal proximal tubular cells. The association of URAT1 with PDZK1 enhanced urate transport activities in HEK293 cells (1.4-fold), and the deletion of the URAT1 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by a significant increase in theVmaxof urate transport via URAT1 and was associated with the increased surface expression level of URAT1 protein from HEK293 cells stably expressing URAT1 transfected with PDZK1. Taken together, the present study indicates the novel role of PDZK1 in regulating the functional activity of URAT1-mediated urate transport in the apical membrane of renal proximal tubules.

scholarly journals Ebolavirus VP35 Interacts with the Cytoplasmic Dynein Light Chain 8

2009 ◽  
Vol 83 (13) ◽  
pp. 6952-6956 ◽  
Author(s):  
Toru Kubota ◽  
Mayumi Matsuoka ◽  
Tsung-Hsien Chang ◽  
Mike Bray ◽  
Steven Jones ◽  
...  
Keyword(s):  
Life Cycle ◽  
Light Chain ◽  
Viral Protein ◽  
Cytoplasmic Dynein ◽  
Type I ◽  
Dynein Light Chain ◽  
Yeast Two Hybrid ◽  
Binding Partners ◽  
Mapping Analysis ◽  
Two Hybrid

ABSTRACT The viral protein VP35 of ebolavirus (EBOV) is implicated to have diverse roles in the viral life cycle. We employed a yeast two-hybrid screen to search for VP35 binding partners and identified the cytoplasmic dynein light chain (DLC8) as a protein that interacts with VP35. Mapping analysis unraveled a consensus motif, SQTQT, within VP35 through which VP35 binds to DLC8. The disruption of DLC8 binding does not affect the ability of VP35 to inhibit type I IFN production. Given that VP35 from various EBOV species interacts with DLC8, this interaction may have a role in regulating the EBOV life cycle.

scholarly journals Silencing Suppressor Protein VPg of a Potyvirus Interacts With the Plant Silencing-Related Protein SGS3

2014 ◽  
Vol 27 (11) ◽  
pp. 1199-1210 ◽  
Author(s):  
Minna-Liisa Rajamäki ◽  
Janne Streng ◽  
Jari P. T. Valkonen
Keyword(s):  
Rna Silencing ◽  
Bimolecular Fluorescence Complementation ◽  
Mrna Levels ◽  
Yeast Two Hybrid ◽  
Main Determinant ◽  
Potato Virus A ◽  
Cytoplasmic Bodies ◽  
Multiple Protein ◽  
Suppressor Of Rna Silencing ◽  
Two Hybrid

Viral genome-linked protein (VPg) of potyviruses is involved in multiple steps of the potyvirus infection cycle, including viral multiplication and movement in plants. Recently, we showed that VPg of Potato virus A (PVA; genus Potyvirus) suppresses sense-mediated RNA silencing, which is linked to one or both nuclear or nucleolar localization. Here, we studied interactions between VPg and components of the plant RNA silencing pathway. Results showed that VPg interacts with the SGS3 protein of Solanum tuberosum and Arabidopsis thaliana, as shown by yeast two-hybrid analysis and bimolecular fluorescence complementation assays. VPg–SGS3 interactions co-localized with small cytoplasmic bodies that contained plant RNA-dependent RNA polymerase 6 (RDR6) (likely SGS3/RDR6 bodies). The N-terminal zinc finger (ZF) domain of SGS3 was the main determinant of the VPg interaction. Our data also suggest that the ZF domain controls SGS3 localization. SGS3 homodimerization was controlled by multiple protein regions. The VPg–SGS3 interaction appeared beneficial for PVA, as viral RNA levels correlated positively with sgs3 mRNA levels in the SGS3-silenced and SGS3-overexpressing leaves of Nicotiana benthamiana. The data support the idea that VPg acts as a suppressor of RNA silencing and suggest that an interaction with SGS3 may be important, especially in suppression of sense-mediated RNA silencing.

Gfi1 Protein Turnover Is Regulated by the Ubiquitin Ligase Triad1.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1173-1173
Author(s):  
Laurens T. van der Meer ◽  
Jurgen A.F. Marteijn ◽  
Theo M. de Witte ◽  
Joop H. Jansen ◽  
Bert A. van der Reijden
Keyword(s):  
Ubiquitin Ligase ◽  
Half Life ◽  
Proteasome Inhibitors ◽  
Ring Finger ◽  
Ubiquitin Ligases ◽  
Proteasomal Degradation ◽  
Mrna Levels ◽  
Yeast Two Hybrid ◽  
Protein Levels ◽  
Two Hybrid

Abstract The transcriptional repressor Growth factor independence-1 (Gfi1) plays an essential role during various stages of hematopoiesis. It is crucial for the self-renewal and long-term reconstituting potential of stem cells, essential for neutrophilic differentiation, and it plays an important role in T-cell and dendritic cell development. Gfi1 has also been implicated in malignant hematopoeisis because the Gfi1 gene is a common proviral integration site in murine leukemia models. We recently found that Gfi1 protein levels are mainly regulated by the ubiquitin-proteasome system. Although Gfi1 mRNA levels are low in primary human monocytes, the protein levels are high due to low proteasomal degradation. Conversely, in mature granulocytes Gfi1 mRNA levels are high but protein levels are low due to strong proteasome-mediated turnover. Because Gfi1 plays an important role in normal and malignant hematopoiesis it will be of great interest to identify the ubiquitin ligases that regulate its turnover. Previously, we showed that the RING finger ubiquitin ligase Triad1 regulates myeloid cell proliferation. Using yeast-two-hybrid assays we found that Triad1 binds the zinc finger region of Gfi1. This interaction was confirmed in co-immunoprecipitation experiments. To study whether the turnover of Gfi1 is regulated by Triad1 we performed ubiquitination assays. To our suprise we found that instead of promoting ubiquitination, Triad1 inhibited Gfi1 protein ubiquitination, also in the presence of proteasome inhibitors. RNAi mediated down regulation of Triad1 protein levels stimulated Gfi1 ubiquitination. Importantly, expression of a Triad1 point mutant (H158A) that fails to bind the ubiquitin conjugating enzyme UbcH7 also inhibited Gfi1 ubiquitination. To study whether the observed diminished ubiquitination by Triad1 affected the turnover of Gfi1 we analyzed Gfi1 protein half-life using the protein synthesis inhibitor cycloheximide. This showed that Triad1 co-expression prolonged the half-life of Gfi1 significantly. We conclude that Triad1 inhibits Gfi1 ubiquitination, resulting in decreased turnover of the protein. As this inhibition also occurs in the presence of proteasome inhibitors and is independent of the ubiquitin ligase activity of Triad1, these data support a model in which Triad1 competes for Gfi1 binding with other ubiquitin ligases that do mark Gfi1 for proteasomal degradation. Currently, we are testing candidate ubiquitin ligases (RING finger and HECT proteins) that were found to associate with Gfi1 in yeast-two-hybrid assays to gain more insight in how the activity of this important transcription factor is regulated.

scholarly journals The Peptidyl Prolyl cis/trans Isomerase FKBP38 Determines Hypoxia-Inducible Transcription Factor Prolyl-4-Hydroxylase PHD2 Protein Stability

2007 ◽  
Vol 27 (10) ◽  
pp. 3758-3768 ◽  
Author(s):  
Sandra Barth ◽  
Jutta Nesper ◽  
Philippe A. Hasgall ◽  
Renato Wirthner ◽  
Katarzyna J. Nytko ◽  
...  
Keyword(s):  
Protein Stability ◽  
Transcriptional Activity ◽  
Mrna Levels ◽  
Yeast Two Hybrid ◽  
Isomerase Activity ◽  
Fk506 Binding Protein ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Α Subunits ◽  
Two Hybrid

ABSTRACT The heterodimeric hypoxia-inducible transcription factors (HIFs) are central regulators of the response to low oxygenation. HIF-α subunits are constitutively expressed but rapidly degraded under normoxic conditions. Oxygen-dependent hydroxylation of two conserved prolyl residues by prolyl-4-hydroxylase domain-containing enzymes (PHDs) targets HIF-α for proteasomal destruction. We identified the peptidyl prolyl cis/trans isomerase FK506-binding protein 38 (FKBP38) as a novel interactor of PHD2. Yeast two-hybrid, glutathione S-transferase pull-down, coimmunoprecipitation, colocalization, and mammalian two-hybrid studies confirmed specific FKBP38 interaction with PHD2, but not with PHD1 or PHD3. PHD2 and FKBP38 associated with their N-terminal regions, which contain no known interaction motifs. Neither FKBP38 mRNA nor protein levels were regulated under hypoxic conditions or after PHD inhibition, suggesting that FKBP38 is not a HIF/PHD target. Stable RNA interference-mediated depletion of FKBP38 resulted in increased PHD hydroxylation activity and decreased HIF protein levels and transcriptional activity. Reconstitution of FKBP38 expression abolished these effects, which were independent of the peptidyl prolyl cis/trans isomerase activity. Downregulation of FKBP38 did not affect PHD2 mRNA levels but prolonged PHD2 protein stability, suggesting that FKBP38 is involved in PHD2 protein regulation.

Identifications of DNA Sequences Encoding Key Region of SCR Interacting with SRK Extracellular Domain by Using Yeast Two-Hybrid System

2013 ◽  
Vol 38 (9) ◽  
pp. 1583-1591
Author(s):  
Li-Yan XUE ◽  
Bing LUO ◽  
Li-Quan ZHU ◽  
Yong-Jun YANG ◽  
He-Cui ZHANG ◽  
...  
Keyword(s):  
Hybrid System ◽  
Dna Sequences ◽  
Extracellular Domain ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

scholarly journals Structural Glycoprotein E2 of Classical Swine Fever Virus Interacts with Host Protein Dynactin Subunit 6 (DCTN6) during the Virus Infectious Cycle

2019 ◽  
Vol 94 (1) ◽  
Author(s):  
M. V. Borca ◽  
E. A. Vuono ◽  
E. Ramirez-Medina ◽  
P. Azzinaro ◽  
K. A. Berggren ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Interaction ◽  
Hybrid Approach ◽  
Classical Swine Fever ◽  
Host Protein ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Viral Virulence ◽  
Glycoprotein E2 ◽  
Two Hybrid

ABSTRACT The E2 protein in classical swine fever (CSF) virus (CSFV) is the major virus structural glycoprotein and is an essential component of the viral particle. E2 has been shown to be involved in several functions, including virus adsorption, induction of protective immunity, and virulence in swine. Using the yeast two-hybrid system, we previously identified a swine host protein, dynactin subunit 6 (DCTN6) (a component of the cell dynactin complex), as a specific binding partner for E2. We confirmed the interaction between DCTN6 and E2 proteins in CSFV-infected swine cells by using two additional independent methodologies, i.e., coimmunoprecipitation and proximity ligation assays. E2 residues critical for mediating the protein-protein interaction with DCTN6 were mapped by a reverse yeast two-hybrid approach using a randomly mutated E2 library. A recombinant CSFV mutant, E2ΔDCTN6v, harboring specific substitutions in those critical residues was developed to assess the importance of the E2-DCTN6 protein-protein interaction for virus replication and virulence in swine. CSFV E2ΔDCTN6v showed reduced replication, compared with the parental virus, in an established swine cell line (SK6) and in primary swine macrophage cultures. Remarkably, animals infected with CSFV E2ΔDCTN6v remained clinically normal during the 21-day observation period, which suggests that the ability of CSFV E2 to bind host DCTN6 protein efficiently during infection may play a role in viral virulence. IMPORTANCE Structural glycoprotein E2 is an important component of CSFV due to its involvement in many virus activities, particularly virus-host interactions. Here, we present the description and characterization of the protein-protein interaction between E2 and the swine host protein DCTN6 during virus infection. The E2 amino acid residues mediating the interaction with DCTN6 were also identified. A recombinant CSFV harboring mutations disrupting the E2-DCTN6 interaction was created. The effect of disrupting the E2-DCTN6 protein-protein interaction was studied using reverse genetics. It was shown that the same amino acid substitutions that abrogated the E2-DCTN6 interaction in vitro constituted a critical factor in viral virulence in the natural host, domestic swine. This highlights the potential importance of the E2-DCTN6 protein-protein interaction in CSFV virulence and provides possible mechanisms of virus attenuation for the development of improved CSF vaccines.

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