scholarly journals Phylogenetic Analysis and In Vitro Bifunctional Nuclease Assay of Arabidopsis BBD1 and BBD2

Molecules ◽  
2020 ◽  
Vol 25 (9) ◽  
pp. 2169
Author(s):  
A. K. M. Mahmudul Huque ◽  
Won Mi So ◽  
Min Kyoung You ◽  
Jeong Sheop Shin
Keyword(s):  
Phylogenetic Analysis ◽  
Diverse Group ◽  
Yeast Two Hybrid ◽  
Sequence Identity ◽  
Physiological Processes ◽  
Metal Cofactor ◽  
Domain Of Unknown Function ◽  
Two Hybrid ◽  
Nuclease Assay

Nucleases are a very diverse group of enzymes that play important roles in many crucial physiological processes in plants. We previously reported that the highly conserved region (HCR), domain of unknown function 151 (DUF151) and UV responsive (UVR) domain-containing OmBBD is a novel nuclease that does not share homology with other well-studied plant nucleases. Here, we report that DUF151 domain-containing proteins are present in bacteria, archaea and only Viridiplantae kingdom of eukarya, but not in any other eukaryotes. Two Arabidopsis homologs of OmBBD, AtBBD1 and AtBBD2, shared 43.69% and 44.38% sequence identity and contained all three distinct domains of OmBBD. We confirmed that the recombinant MBP-AtBBD1 and MBP-AtBBD2 exhibited non-substrate-specific DNase and RNase activity, like OmBBD. We also found that a metal cofactor is not necessarily required for DNase activity of AtBBD1 and AtBBD2, but their activities were much enhanced in the presence of Mg2+ or Mn2+. Using a yeast two-hybrid assay, we found that AtBBD1 and AtBBD2 each form a homodimer but not a heterodimer and that the HCR domain is possibly crucial for dimerization.

scholarly journals Two LIM Domain Proteins and UNC-96 Link UNC-97/PINCH to Myosin Thick Filaments in Caenorhabditis elegans Muscle

2007 ◽  
Vol 18 (11) ◽  
pp. 4317-4326 ◽  
Author(s):  
Hiroshi Qadota ◽  
Kristina B. Mercer ◽  
Rachel K. Miller ◽  
Kozo Kaibuchi ◽  
Guy M. Benian
Keyword(s):  
Caenorhabditis Elegans ◽  
Binding Assays ◽  
Lim Domain ◽  
Yeast Two Hybrid ◽  
Lim Domains ◽  
Thick Filaments ◽  
Vitro Binding ◽  
Two Hybrid ◽  
Hybrid Screening

By yeast two-hybrid screening, we found three novel interactors (UNC-95, LIM-8, and LIM-9) for UNC-97/PINCH in Caenorhabditis elegans. All three proteins contain LIM domains that are required for binding. Among the three interactors, LIM-8 and LIM-9 also bind to UNC-96, a component of sarcomeric M-lines. UNC-96 and LIM-8 also bind to the C-terminal portion of a myosin heavy chain (MHC), MHC A, which resides in the middle of thick filaments in the proximity of M-lines. All interactions identified by yeast two-hybrid assays were confirmed by in vitro binding assays using purified proteins. All three novel UNC-97 interactors are expressed in body wall muscle and by antibodies localize to M-lines. Either a decreased or an increased dosage of UNC-96 results in disorganization of thick filaments. Our previous studies showed that UNC-98, a C2H2 Zn finger protein, acts as a linkage between UNC-97, an integrin-associated protein, and MHC A in myosin thick filaments. In this study, we demonstrate another mechanism by which this linkage occurs: from UNC-97 through LIM-8 or LIM-9/UNC-96 to myosin.

scholarly journals A Novel Human Ada2 Homologue Functions with Gcn5 or Brg1 To Coactivate Transcription

2003 ◽  
Vol 23 (19) ◽  
pp. 6944-6957 ◽  
Author(s):  
Nickolai A. Barlev ◽  
Alexander V. Emelyanov ◽  
Paola Castagnino ◽  
Philip Zegerman ◽  
Andrew J. Bannister ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Chromatin Remodeling ◽  
Adaptor Protein ◽  
Saga Complex ◽  
Yeast Two Hybrid ◽  
Functional Assays ◽  
Two Hybrid ◽  
Histone Acetyltransferase Activity

ABSTRACT In yeast, the transcriptional adaptor yeast Ada2 (yAda2) is a part of the multicomponent SAGA complex, which possesses histone acetyltransferase activity through action of the yGcn5 catalytic enzyme. yAda2, among several SAGA proteins, serves to recruit SAGA to genes via interactions with promoter-bound transcription factors. Here we report identification of a new human Ada2 homologue, hAda2β. Ada2β differs both biochemically and functionally from the previously characterized hAda2α, which is a stable component of the human PCAF (human Gcn5 homologue) acetylase complex. Ada2β, relative to Ada2α, interacted selectively, although not stably, with the Gcn5-containing histone acetylation complex TFTC/STAGA. In addition, Ada2β interacted with Baf57 (a component of the human Swi/Snf complex) in a yeast two-hybrid screen and associated with human Swi/Snf in vitro. In functional assays, hAda2β (but not Ada2α), working in concert with Gcn5 (but not PCAF) or Brg1 (the catalytic component of hSwi/Snf complex), increased transcription via the B-cell-specific transcription factor Pax5/BSAP. These findings support the view that Gcn5 and PCAF have distinct roles in vivo and suggest a new mechanism of coactivator function, in which a single adaptor protein (Ada2β) can coordinate targeting of both histone acetylation and chromatin remodeling activities.

scholarly journals Interaction of Proteus mirabilis Urease Apoenzyme and Accessory Proteins Identified with Yeast Two-Hybrid Technology

2001 ◽  
Vol 183 (4) ◽  
pp. 1423-1433 ◽  
Author(s):  
Susan R. Heimer ◽  
Harry L. T. Mobley
Keyword(s):  
Proteus Mirabilis ◽  
Accessory Proteins ◽  
Structural Protein ◽  
Yeast Two Hybrid ◽  
Nickel Ions ◽  
Catalytically Active ◽  
Tract Infections ◽  
Two Hybrid

ABSTRACT Proteus mirabilis, a gram-negative bacterium associated with complicated urinary tract infections, produces a metalloenzyme urease which hydrolyzes urea to ammonia and carbon dioxide. The apourease is comprised of three structural subunits, UreA, UreB, and UreC, assembled as a homotrimer of individual UreABC heterotrimers (UreABC)3. To become catalytically active, apourease acquires divalent nickel ions through a poorly understood process involving four accessory proteins, UreD, UreE, UreF, and UreG. While homologues of UreD, UreF, and UreG have been copurified with apourease, it remains unclear specifically how these polypeptides associate with the apourease or each other. To identify interactions among P. mirabilis accessory proteins, in vitro immunoprecipitation and in vivo yeast two-hybrid assays were employed. A complex containing accessory protein UreD and structural protein UreC was isolated by immunoprecipitation and characterized with immunoblots. This association occurs independently of coaccessory proteins UreE, UreF, and UreG and structural protein UreA. In a yeast two-hybrid screen, UreD was found to directly interact in vivo with coaccessory protein UreF. Unique homomultimeric interactions of UreD and UreF were also detected in vivo. To substantiate the study of urease proteins with a yeast two-hybrid assay, previously described UreE dimers and homomultimeric UreA interactions among apourease trimers were confirmed in vivo. Similarly, a known structural interaction involving UreA and UreC was also verified. This report suggests that in vivo, P. mirabilis UreD may be important for recruitment of UreF to the apourease and that crucial homomultimeric associations occur among these accessory proteins.

ralGDS family members interact with the effector loop of ras p21

1994 ◽  
Vol 14 (11) ◽  
pp. 7483-7491
Author(s):  
A Kikuchi ◽  
S D Demo ◽  
Z H Ye ◽  
Y W Chen ◽  
L T Williams
Keyword(s):  
Hybrid System ◽  
Family Members ◽  
Dominant Negative Mutant ◽  
Yeast Two Hybrid ◽  
Amino Acid Homology ◽  
Yeast Two Hybrid System ◽  
Ras P21 ◽  
Two Hybrid ◽  
Interacting Domain

Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21. In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.

scholarly journals Inhibition of Ubiquitination and Stabilization of Human Ubiquitin E3 Ligase PIRH2 by Measles Virus Phosphoprotein

2005 ◽  
Vol 79 (18) ◽  
pp. 11824-11836 ◽  
Author(s):  
Mingzhou Chen ◽  
Jean-Claude Cortay ◽  
Ian R. Logan ◽  
Vasileia Sapountzi ◽  
Craig N. Robson ◽  
...  
Keyword(s):  
Binding Site ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
E3 Ligase ◽  
Yeast Two Hybrid ◽  
Ubiquitin E3 Ligase ◽  
P Protein ◽  
Two Hybrid

ABSTRACT Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally coimmunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by coimmunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.

scholarly journals Definition of a minimal munc18c domain that interacts with syntaxin 4

2000 ◽  
Vol 350 (3) ◽  
pp. 741-746 ◽  
Author(s):  
Julian GRUSOVIN ◽  
Violet STOICHEVSKA ◽  
Keith H. GOUGH ◽  
Katrina NUNAN ◽  
Colin W. WARD ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Full Length ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Interaction Studies ◽  
Syntaxin 4 ◽  
Definition Of ◽  
Two Hybrid

munc18c is a critical protein involved in trafficking events associated with syntaxin 4 and which also mediates inhibitory effects on vesicle docking and/or fusion. To investigate the domains of munc18c responsible for its interaction with syntaxin 4, fragments of munc18c were generated and their interaction with syntaxin 4 examined in vivo by the yeast two-hybrid assay. In vitro protein–protein interaction studies were then used to confirm that the interaction between the proteins was direct. Full-length munc18c1–592, munc18c1–139 and munc18c1–225, but not munc18c226–592, munc18c1–100, munc18c43–139 or munc18c66–139, interacted with the cytoplasmic portion of syntaxin 4, Stx42–273, as assessed by yeast two-hybrid assay of growth on nutritionally deficient media and by β-galactosidase reporter induction. The N-terminal predicted helix-a-helix-b-helix-c region of syntaxin 4, Stx429–157, failed to interact with full-length munc18c1–592, indicating that a larger portion of syntaxin 4 is necessary for the interaction. The yeast two-hybrid results were confirmed by protein–protein interaction studies between Stx42–273 and glutathione S-transferase fusion proteins of munc18c. Full-length munc18c1–592, munc18c1–139 and munc18c1–225 interacted with Stx42–273 whereas munc18c1–100 did not, consistent with the yeast two-hybrid data. These data thus identify a region of munc18c between residues 1 and 139 as a minimal domain for its interaction with syntaxin 4.

scholarly journals Dominant Negative Dimerization of a Mutant Homeodomain Protein in Axenfeld-Rieger Syndrome

2003 ◽  
Vol 23 (6) ◽  
pp. 1968-1982 ◽  
Author(s):  
Irfan Saadi ◽  
Adisa Kuburas ◽  
Jamison J. Engle ◽  
Andrew F. Russo
Keyword(s):  
Dominant Negative ◽  
Hill Coefficient ◽  
Homeodomain Protein ◽  
Yeast Two Hybrid ◽  
Autosomal Dominant Disorder ◽  
Rieger Syndrome ◽  
A Charge ◽  
Two Hybrid

ABSTRACT Axenfeld-Rieger syndrome is an autosomal-dominant disorder caused by mutations in the PITX2 homeodomain protein. We have studied the mechanism underlying the dominant negative K88E mutation, which occurs at position 50 of the homeodomain. By using yeast two-hybrid and in vitro pulldown assays, we have documented that PITX2a can form homodimers in the absence of DNA. Moreover, the K88E mutant had even stronger dimerization ability, primarily due to interactions involving the C-terminal region. Dimerization allowed cooperative binding of wild-type (WT) PITX2a to DNA containing tandem bicoid sites in a head-to-tail orientation (Hill coefficient, 1.73). In contrast, the WT-K88E heterodimer bound the tandem sites with greatly reduced cooperativity and decreased transactivation activity. To further explore the role of position 50 in PITX2a dimerization, we introduced a charge-conservative mutation of lysine to arginine (K88R). The K88R protein had greatly reduced binding to a TAATCC element and did not specifically bind any other TAATNN motif. Like K88E, K88R formed relatively stronger dimers with WT. As predicted by our model, the K88R protein acted in a dominant negative manner to suppress WT PITX2a activity. These results suggest that the position 50 residue in the PITX2 homeodomain plays an important role in both DNA binding and dimerization activities.

Abstract 3600: HspA12B Promotes Angiogenesis through Suppressing AKAP12 and Up-Regulating VEGF Pathway

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Rebecca J Steagall ◽  
Fang Hua ◽  
Mahesh Thirunazukarasu ◽  
Lijun Zhan ◽  
Chuanfu Li ◽  
...  
Keyword(s):  
Cancer Metastasis ◽  
Yeast Two Hybrid ◽  
Vegf Expression ◽  
Tubule Formation ◽  
Interacting Protein ◽  
Over Expression ◽  
Vegf Pathway ◽  
Two Hybrid ◽  
Hybrid Screening

We have previously shown that HspA12B, a member of HspA70 family subfamily 12, is a novel angiogenesis regulator that is preferentially expressed in endothelial cells (ECs) and required for angiogenesis in vitro . The mechanism by which HspA12B regulates angiogenesis, however, is unknown. In this study we identified AKAP12/SSeCKS as a HSPA12B-interacting protein through a yeast two-hybrid screening and confirmed the interaction by co-immunoprecipitation and co-localization. We observed that HspA12B negatively regulated the expression of AKAP12/SSeCKS, a cancer metastasis repressor that inhibits VEGF expression and angiogen-esis. In HUVEC, HspA12B knockdown increased AKAP12 levels, decreased VEGF by more than 75%, and down-regulated Akt and pAkt; whereas HspA12B over expression decreased AKAP12 and more than doubled VEGF levels. We further identified a 32-AA domain in AKAP12 that was capable of interacting with HspA12B. Overexpression of this 32-AA domain in HUVEC disrupted the HspA12B-AKAP12 interaction and decreased VEGF expression by more than 70%, suggesting the importance of HspA12B-AKAP12 interaction in regulating VEGF. We also observed that HspA12B expression was increased more than 2 folds in ECs by hypoxia or shearing stress, and induced in ischemic rat heart. Inhibition of HspA12B abolished hypoxia-induced tubule formation. Adeno-HspA12B promoted angiogenesis in DIVAA assay. We concluded that this is the first evidence that HspA12B promotes angiogenesis through regulating VEGF by way of suppressing AKAP12. Our finding is the first example of an EC-specific molecular chaperone acting as the regulator of angiogenesis.

PSD-93 mediate the dialogue between neuron and microglia and facilitate acute ischemic stroke by binding 357-395 amino acid sequence of CX3CL1

2020 ◽  
Author(s):  
Qingxiu Zhang ◽  
Lei He ◽  
Mo Chen ◽  
Hui Yang ◽  
Xiaowei Cao ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Ischemia Reperfusion ◽  
Yeast Two Hybrid ◽  
Neuron Death ◽  
Specific Amino Acid ◽  
Ischemic Brain ◽  
Two Hybrid

Abstract Background: Our previous experiments demonstrated that PSD-93 mediates glutamate excitotoxicity induced by ischemic brain injury, which promotes the release of inflammatory cytokines in early ischemic brain injury by activating the NMDA receptor. Glutamate activity is the key to neuronal excitatory toxicity and microglial cell inflammatory response in the joints. However, the underlying mechanisms of how does PSD-93 mediate the dialogue between neurons and microglia in the postsynaptic dense region remain elusive. And CX3 chemokine ligand 1 (CX3CL1) is a chemokine that is specifically expressed in neurons. Its only receptor CX3CR1 is highly expressed in microglia and its main forms are membrane binding and soluble. In this study, we aim to clarify the specific amino acid sequence of the binding of psd-93 and CX3CL1 and investigate role of PSD-93 on regulating the crosstalk between neuron and microglia in acute ischemic stroke. Methods: In this study, male C57BL/6 mice aged 8-12 weeks and weighted 22-26g were applied with Middle Cerebral Artery Occlusion (MCAO) model and randomly divided into different groups. Firstly, co-immunoprecipitation and immunoblotting were used to detect the binding of PSD-93 and CX3CL1 at different time points 3h, 6h, 12h 24h, 48h and 72h following cerebral ischemic/reperfusion. Meanwhile, ELISA was used to investigate the expression of soluble CX3CL1 at the same time points to confirm the relationship between of the expression of soluble CX3CL1 and the combination of PSD-93 and CX3CL1. Secondly, two bait plasmids pSos-PSD-93-full length, pSos-CX3CL1-full length and five mutant plasmids: pMyr-PSD-93-mut1, pMyr-PSD-93-mut2, pMyr-PSD-93-mut3, pMyr-PSD-93-mut4, and pMyr-CX3CL1-mut, were constructed and used a yeast two-hybrid system to screen and identify positive clones and to determine the sequence in which the two proteins bind to each other. Thirdly, the proteins corresponding to the three positive clones obtained in the yeast two-hybrid experiment were used to construct plasmids for transfection of eukaryotic cells and the protein expression binding was verified again by in vitro co-immunoprecipitation. Finally, a specific fusion small peptide Tat-CX3CL1 were designed according to above experiment to inhibit the integration of PSD-93 and CX3CL1 and to explore their role on neuron death following reperfusion. Results: We found that the binding capacity of PSD-93 and CX3CL1 proteins peaked at 6h after ischemia/reperfusion and then decreased gradually. The specific amino acid sequence of PSD-93 and CX3CL1 binding was obtained by yeast double hybridization and in vitro immunoprecipitation. We identified that their binding sites are located in the 420-535 amino acid sequence of PSD-93 and 357-395 amino acid sequence of CX3CL1. And a specific fusion small peptide Tat-CX3CL1 (357-395aa) were designed to inhibit the integration of PSD-93 and CX3CL1 and perform neuroprotection on neuron death following reperfusion. Conclusions: Our results suggest that PSD-93 promotes the formation of its soluble form by binding to CX3CL1, which is recruited to the surface of microglia to bind to CX3CR1, thereby activating microglia to initiate inflammation. Thus, specific blockade of PSD-93-CX3CL1 coupling can reduce ischemia-reperfusion induced neuronal cell death, which provide a new target to treat ischemic stroke.

scholarly journals Induction of apoptosis by human Nbk/Bik, a BH3-containing protein that interacts with E1B 19K.

1996 ◽  
Vol 16 (10) ◽  
pp. 5857-5864 ◽  
Author(s):  
J Han ◽  
P Sabbatini ◽  
E White
Keyword(s):  
Apoptotic Cell ◽  
Yeast Two Hybrid ◽  
Nuclear Membranes ◽  
Induction Of Apoptosis ◽  
Low Levels ◽  
Apoptosis Inhibitor ◽  
Apoptosis Proteins ◽  
Two Hybrid

The E1B 19-kilodalton protein (19K protein) is a potent apoptosis inhibitor and the adenovirus homolog of Bcl-2 (E. White, Genes Dev. 10:1-15, 1996). To obtain a better understanding of the biochemical mechanism by which the E1B 19K protein regulates apoptosis, proteins that interact with 19K have been identified; one of these is Bax (J. Han, P. Sabbatini, D. Perez, L. Rao, D. Mohda, and E. White, Genes Dev. 10:461-477, 1996), and another is Bak (S. N. Farrow, J. H. M. White, I. Martinou, T. Raven, K.-T. Pun, C. J. Grinham, J.-C. Martinou, and R. Brown, Nature (London) 374:731-733, 1995). Bax and Bak are Bcl-2 family members which contain Bcl-2 homology regions 1, 2, and 3 (BH1, BH2, and BH3), which interact with E1B 19K and Bcl-2 and promote apoptosis. Like Bax and Bak, Nbk was cloned from a yeast two-hybrid screen for proteins that interact with E1B 19K. Nbk contained BH3 but not BH1 or BH2. It also interacted with Bcl-2 but not with Bax. Both Bcl-2 and E1B 19K interacted with Nbk in vitro, and this interaction was highly specific. In vivo, the Nbk and E1B 19K proteins may colocalize with cytoplasmic and nuclear membranes. Nbk expression functionally antagonized 19K-mediated inhibition of apoptotic cell death and completely prevented transformation by E1A and E1B 19K. Nbk was sufficient for induction of apoptosis in the presence of mutant p53 and thus low levels of Bax, suggesting that Nbk functions independently of Bax to induce apoptosis. Nbk may therefore represent a novel death regulator which contains only a BH3 that interacts with and antagonizes apoptosis inhibitors such as the E1B 19K protein.

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