scholarly journals The Tomato Metallocarboxypeptidase Inhibitor I, which Interacts with a Heavy Metal-Associated Isoprenylated Protein, Is Implicated in Plant Response to Cadmium

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 700 ◽  
Author(s):  
Anna Manara ◽  
Elisa Fasani ◽  
Barbara Molesini ◽  
Giovanni DalCorso ◽  
Federica Pennisi ◽  
...  
Keyword(s):  
Heavy Metal ◽  
Active Site ◽  
Abiotic Stresses ◽  
Lower Amount ◽  
Interacting Proteins ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Model Species ◽  
Two Hybrid

Metallocarboxypeptidases are metal-dependent enzymes, whose biological activity is regulated by inhibitors directed on the metal-containing active site. Some metallocarboxypeptidase inhibitors are induced under stress conditions and have a role in defense against pests. This paper is aimed at investigating the response of the tomato metallocarboxypeptidase inhibitor (TCMP)-1 to Cd and other abiotic stresses. To this aim, the tomato TCMP-1 was ectopically expressed in the model species Arabidopsis thaliana, and a yeast two-hybrid analysis was performed to identify interacting proteins. We demonstrate that TCMP-1 is responsive to Cd, NaCl, and abscisic acid (ABA) and interacts with the tomato heavy metal-associated isoprenylated plant protein (HIPP)26. A. thaliana plants overexpressing TCMP-1 accumulate lower amount of Cd in shoots, display an increased expression of AtHIPP26 in comparison with wild-type plants, and are characterized by a modulation in the expression of antioxidant enzymes. Overall, these results suggest a possible role for the TCMP-1/HIPP26 complex in Cd response and compartmentalization.

scholarly journals ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery

2008 ◽  
Vol 183 (2) ◽  
pp. 313-322 ◽  
Author(s):  
Noveera T. Ahmed ◽  
Chunlei Gao ◽  
Ben F. Lucker ◽  
Douglas G. Cole ◽  
David R. Mitchell
Keyword(s):  
Binding Site ◽  
Intraflagellar Transport ◽  
Site Formation ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
B Subunit ◽  
Dynein Arms ◽  
Two Hybrid

Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.

scholarly journals Isolation and identification of l-dopa decarboxylase as a protein that binds to and enhances transcriptional activity of the androgen receptor using the repressed transactivator yeast two-hybrid system

2003 ◽  
Vol 375 (2) ◽  
pp. 373-383 ◽  
Author(s):  
Latif A. WAFA ◽  
Helen CHENG ◽  
Mira A. RAO ◽  
Colleen C. NELSON ◽  
Michael COX ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Androgen Receptor ◽  
Transcriptional Activity ◽  
Steroid Receptor ◽  
Receptor Activity ◽  
Dopa Decarboxylase ◽  
Interacting Proteins ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Two Hybrid

The AR (androgen receptor) is a ligand-regulated transcription factor, which belongs to the steroid receptor family and plays an essential role in growth and development of the prostate. Transcriptional activity of steroid receptors is modulated by interaction with co-regulator proteins and yeast two-hybrid analysis is commonly used to identify these steroid receptor-interacting proteins. However, a limitation of conventional two-hybrid systems for detecting AR protein partners has been that they only allow for analysis of the ligand- and DNA-binding domains of the receptor, as its NTD (N-terminal domain) possesses intrinsic transactivation activity. To identify AR N-terminus-interacting proteins, its NTD was used in the RTA (repressed transactivator) system, which is specifically designed for transactivator bait proteins and was shown to be suitable for two-hybrid analysis with the AR NTD. DDC (l-dopa decarboxylase) was detected multiple times as a novel AR-interacting protein, which was subsequently confirmed in vitro and in vivo. Furthermore, transient transfection of DDC in prostate cancer cells strongly enhanced ligand-dependent AR transcriptional activity, an effect that was antagonized using high concentrations of the anti-androgen bicalutamide. Glucocorticoid receptor activity was also strongly enhanced with DDC co-transfection, while oestrogen receptor activity was only mildly affected. Together, our data demonstrate that DDC interacts with AR to enhance steroid receptor transactivation, which may have important implications in prostate cancer progression.

scholarly journals Subunit Interactions in the mariner Transposase

Genetics ◽  
1996 ◽  
Vol 144 (3) ◽  
pp. 1087-1095 ◽  
Author(s):  
Allan R Lohe ◽  
David T Sullivan ◽  
Daniel L Hartl
Keyword(s):  
Hybrid System ◽  
Catalytic Domain ◽  
Wild Type ◽  
Target Element ◽  
Yeast Two Hybrid ◽  
Subunit Interactions ◽  
Hsp70 Promoter ◽  
Yeast Two Hybrid System ◽  
Transposition Reaction ◽  
Two Hybrid

Abstract We have studied the Mos1 transposase encoded by the transposable element mariner. This transposase is a member of the “D,D(35)E” superfamily of proteins exhibiting the motif D,D(34)D. It is not known whether this transposase, or other eukaryote transposases manifesting the D,D(35)E domain, functions in a multimeric form. Evidence for oligomerization was found in the negative complementation of Mos1 by an EMS-induced transposase mutation in the catalytic domain. The transposase produced by this mutation has a glycine-to-arginine replacement at position 292. The G292R mutation strongly interferes with the ability of wild-type transposase to catalyze excision of a target element. Negative complementation was also observed for two other EMS mutations, although the effect was weaker than observed with G292R. Results from the yeast two-hybrid system also imply that Mos1 subunits interact, suggesting the possibility of subunit oligomerization in the transposition reaction. Overproduction of Mos1 subunits through an hsp70 promoter also inhibits excision of the target element, possibly through autoregulatory feedback on transcription or through formation of inactive or less active oligomers. The effects of both negative complementation and overproduction may contribute to the regulation of mariner transposition.

scholarly journals Genetic Analysis of theEscherichia coliFtsZ·ZipA Interaction in the Yeast Two-hybrid System

2001 ◽  
Vol 276 (15) ◽  
pp. 11980-11987 ◽  
Author(s):  
Steven A. Haney ◽  
Elizabeth Glasfeld ◽  
Cynthia Hale ◽  
David Keeney ◽  
Zhizhen He ◽  
...  
Keyword(s):  
Hybrid System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Conserved Sequence ◽  
Yeast Two Hybrid System ◽  
C Terminus ◽  
Two Hybrid

The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division inEscherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZD373G) identified eight different changes at two residues within this sequence.In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZD373Gfailed to interact. Two mutant proteins examined restored this interaction significantly.In vivo, the alleles tested are significantly more toxic than the wild typeftsZand cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZD373G, no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.

scholarly journals Mechanism of Signalling and Adaptation through the Rhodobacter sphaeroides Cytoplasmic Chemoreceptor Cluster

2019 ◽  
Vol 20 (20) ◽  
pp. 5095
Author(s):  
Beyer ◽  
Szöllössi ◽  
Byles ◽  
Fischer ◽  
Armitage
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Tandem Mass ◽  
Wild Type ◽  
Hybrid Analysis ◽  
Phosphatase Domain ◽  
E Coli ◽  
Tandem Mass Spectroscopy ◽  
Membrane Spanning ◽  
State Concentration ◽  
Two Hybrid

Rhodobacter sphaeroides has two chemotaxis clusters, an Escherichia coli-like cluster with membrane-spanning chemoreceptors and a less-understood cytoplasmic cluster. The cytoplasmic CheA is split into CheA4, a kinase, and CheA3, a His-domain phosphorylated by CheA4 and a phosphatase domain, which together phosphorylate and dephosphorylate motor-stopping CheY6. In bacterial two-hybrid analysis, one major cytoplasmic chemoreceptor, TlpT, interacted with CheA4, while the other, TlpC, interacted with CheA3. Both clusters have associated adaptation proteins. Deleting their methyltransferases and methylesterases singly and together removed chemotaxis, but with opposite effects. The cytoplasmic cluster signal overrode the membrane cluster signal. Methylation and demethylation of specific chemoreceptor glutamates controls adaptation. Tandem mass spectroscopy and bioinformatics identified four putative sites on TlpT, three glutamates and a glutamine. Mutating each glutamate to alanine resulted in smooth swimming and loss of chemotaxis, unlike similar mutations in E. coli chemoreceptors. Cells with two mutated glutamates were more stoppy than wild-type and responded and adapted to attractant addition, not removal. Mutating all four sites amplified the effect. Cells were non-motile, began smooth swimming on attractant addition, and rapidly adapted back to non-motile before attractant removal. We propose that TlpT responds and adapts to the cell’s metabolic state, generating the steady-state concentration of motor-stopping CheY6~P. Membrane-cluster signalling produces a pulse of CheY3/CheY4~P that displaces CheY6~P and allows flagellar rotation and smooth swimming before both clusters adapt.

scholarly journals Evaluation of Interactions of Human Cytomegalovirus Immediate-Early IE2 Regulatory Protein with Small Ubiquitin-Like Modifiers and Their Conjugation Enzyme Ubc9

2001 ◽  
Vol 75 (8) ◽  
pp. 3859-3872 ◽  
Author(s):  
Jin-Hyun Ahn ◽  
Yixun Xu ◽  
Won-Jong Jang ◽  
Michael J. Matunis ◽  
Gary S. Hayward
Keyword(s):  
Hcmv Infection ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
Binding Assays ◽  
Yeast Two Hybrid ◽  
Vitro Binding ◽  
Lysine Residues ◽  
Infected Cells ◽  
Two Hybrid

ABSTRACT The human cytomegalovirus (HCMV) major immediate-early protein IE2 is a nuclear phosphoprotein that is believed to be a key regulator in both lytic and latent infections. Using yeast two-hybrid screening, small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) and a SUMO-conjugating enzyme (Ubc9) were isolated as IE2-interacting proteins. In vitro binding assays with glutathioneS-transferase (GST) fusion proteins provided evidence for direct protein-protein interaction. Mapping data showed that the C-terminal end of SUMO-1 is critical for interaction with IE2 in both yeast and in vitro binding assays. IE2 was efficiently modified by SUMO-1 or SUMO-2 in cotransfected cells and in cells infected with a recombinant adenovirus expressing HCMV IE2, although the level of modification was much lower in HCMV-infected cells. Two lysine residues at positions 175 and 180 were mapped as major alternative SUMO-1 conjugation sites in both cotransfected cells and an in vitro sumoylation assay and could be conjugated by SUMO-1 simultaneously. Although mutations of these lysine residues did not interfere with the POD (or ND10) targeting of IE2, overexpression of SUMO-1 enhanced IE2-mediated transactivation in a promoter-dependent manner in reporter assays. Interestingly, many other cellular proteins identified as IE2 interaction partners in yeast two-hybrid assays also interact with SUMO-1, suggesting that either directly bound or covalently conjugated SUMO moieties may act as a bridge for interactions between IE2 and other SUMO-1-modified or SUMO-1-interacting proteins. When we investigated the intracellular localization of SUMO-1 in HCMV-infected cells, the pattern changed from nuclear punctate to predominantly nuclear diffuse in an IE1-dependent manner at very early times after infection, but with some SUMO-1 protein now associated with IE2 punctate domains. However, at late times after infection, SUMO-1 was predominantly detected within viral DNA replication compartments containing IE2. Taken together, these results show that HCMV infection causes the redistribution of SUMO-1 and that IE2 both physically binds to and is covalently modified by SUMO moieties, suggesting possible modulation of both the function of SUMO-1 and protein-protein interactions of IE2 during HCMV infection.

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