Salicylic acid-driven association of LENRV and NIMIN1/NIMIN2 binding domain regions in the C-terminus of tobacco NPR1 transduces SAR signal

SummaryNONEXPRESSOR OF PATHOGENESIS-RELATED (PR) GENES1 (NPR1) is the central regulator of salicylic acid (SA)-induced PR-1 gene expression and systemic acquired resistance (SAR). The mechanism how SA is transduced through NPR1 is discussed controversially. Previously, we showed that Arabidopsis and tobacco (Nt) NPR1 contain two domains in their C-terminal thirds with relevance to SA signaling. SA sensitivity of NPR1 relies on the arginine residue in the LENRV motif, and SA-induced NIM1-INTERACTING (NIMIN, N) proteins bind to a highly conserved sequence termed N1/N2 binding domain (BD).We demonstrate that LENRV and N1/N2BD regions of tobacco NPR1, separated from each other, interact in yeast, in vitro, in plant and in animal cells. Physical association of LENRV and N1/N2BD parts is enhanced considerably by SA and functional analogs, but not by a non-functional analog. Furthermore, physical association requires R431 and is most effective with intact LENRV and N1/N2BD interfaces.Association of separated LENRV and N1/N2BD parts by SA reconstitutes a functional NtNPR1 C-terminus, displaying transcription activity and able to interact with TGA transcription factors at two distinct sites.Tobacco NIMIN proteins can assemble LENRV and N1/N2BD parts into ternary complexes suggesting that NIMINs shape the NPR1 C-terminus to modulate SA signaling.

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

Journal of Biological Chemistry ◽

10.1074/jbc.m009810200 ◽

2001 ◽

Vol 276 (15) ◽

pp. 11980-11987 ◽

Cited By ~ 88

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.

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Functional Dissection of Human and Mouse POT1 Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.01352-08 ◽

2008 ◽

Vol 29 (2) ◽

pp. 471-482 ◽

Cited By ~ 69

Author(s):

Wilhelm Palm ◽

Dirk Hockemeyer ◽

Tatsuya Kibe ◽

Titia de Lange

Keyword(s):

Dna Damage ◽

Dna Binding ◽

Dna Damage Response ◽

Binding Domain ◽

Dna Binding Domain ◽

Telomeric Dna ◽

C Terminus ◽

Damage Response ◽

End Resection

ABSTRACT The single-stranded telomeric DNA binding protein POT1 protects mammalian chromosome ends from the ATR-dependent DNA damage response, regulates telomerase-mediated telomere extension, and limits 5′-end resection at telomere termini. Whereas most mammals have a single POT1 gene, mice have two POT1 proteins that are functionally distinct. POT1a represses the DNA damage response, and POT1b controls 5′-end resection. In contrast, as we report here, POT1a and POT1b do not differ in their ability to repress telomere recombination. By swapping domains, we show that the DNA binding domain of POT1a specifies its ability to repress the DNA damage response. However, no differences were detected in the in vitro DNA binding features of POT1a and POT1b. In contrast to the repression of ATR signaling by POT1a, the ability of POT1b to control 5′-end resection was found to require two regions in the C terminus, one corresponding to the TPP1 binding domain and a second representing a new domain located between amino acids (aa) 300 and 350. Interestingly, the DNA binding domain of human POT1 can replace that of POT1a to repress ATR signaling, and the POT1b region from aa 300 to 350 required for the regulation of the telomere terminus is functionally conserved in human POT1. Thus, human POT1 combines the features of POT1a and POT1b.

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Phosphorylation of Connexin36 near the C-terminus switches binding affinities for PDZ-domain and 14–3–3 proteins in vitro

Scientific Reports ◽

10.1038/s41598-020-75375-0 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Stephan Tetenborg ◽

Helen Y. Wang ◽

Lena Nemitz ◽

Anne Depping ◽

Alexsandra B. Espejo ◽

...

Keyword(s):

Gap Junctions ◽

Binding Affinity ◽

Protein Microarray ◽

Pdz Domain ◽

Phosphorylation Site ◽

Binding Domain ◽

Dependent Manner ◽

C Terminus ◽

Activity Dependent

Abstract Connexin36 (Cx36) is the most abundant connexin in central nervous system neurons. It forms gap junction channels that act as electrical synapses. Similar to chemical synapses, Cx36-containing gap junctions undergo activity-dependent plasticity and complex regulation. Cx36 gap junctions represent multimolecular complexes and contain cytoskeletal, regulatory and scaffolding proteins, which regulate channel conductance, assembly and turnover. The amino acid sequence of mammalian Cx36 harbors a phosphorylation site for the Ca2+/calmodulin-dependent kinase II at serine 315. This regulatory site is homologous to the serine 298 in perch Cx35 and in close vicinity to a PDZ binding domain at the very C-terminal end of the protein. We hypothesized that this phosphorylation site may serve as a molecular switch, influencing the affinity of the PDZ binding domain for its binding partners. Protein microarray and pulldown experiments revealed that this is indeed the case: phosphorylation of serine 298 decreased the binding affinity for MUPP1, a known scaffolding partner of connexin36, and increased the binding affinity for two different 14–3–3 proteins. Although we did not find the same effect in cell culture experiments, our data suggest that phosphorylation of serine 315/298 may serve to recruit different proteins to connexin36/35-containing gap junctions in an activity-dependent manner.

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Competition between the Yops of Yersinia enterocolitica for Delivery into Eukaryotic Cells: Role of the SycE Chaperone Binding Domain of YopE

Journal of Bacteriology ◽

10.1128/jb.182.17.4811-4821.2000 ◽

2000 ◽

Vol 182 (17) ◽

pp. 4811-4821 ◽

Cited By ~ 106

Author(s):

Aoife P. Boyd ◽

Isabelle Lambermont ◽

Guy R. Cornelis

Keyword(s):

Binding Site ◽

In Vitro Release ◽

Inhibitory Effect ◽

Binding Domain ◽

Eukaryotic Cells ◽

Target Cells ◽

Wild Type ◽

Animal Cells

ABSTRACT A type III secretion-translocation system allowsYersinia adhering at the surface of animal cells to deliver a cocktail of effector Yops (YopH, -O, -P, -E, -M, and -T) into the cytosol of these cells. Residues or codons 1 to 77 contain all the information required for the complete delivery of YopE into the target cell (release from the bacterium and translocation across the eukaryotic cell membrane). Residues or codons 1 to 15 are sufficient for release from the wild-type bacterium under Ca2+-chelating conditions but not for delivery into target cells. Residues 15 to 50 comprise the binding domain for SycE, a chaperone specific for YopE that is necessary for release and translocation of full-length YopE. To understand the role of this chaperone, we studied the delivery of YopE-Cya reporter proteins and YopE deletants by polymutant Yersinia devoid of most of the Yop effectors (ΔHOPEM and ΔTHE strains). We first tested YopE-Cya hybrid proteins and YopE proteins deleted of the SycE-binding site. In contrast to wild-type strains, these mutants delivered YopE15-Cya as efficiently as YopE130-Cya. They were also able to deliver YopEΔ17–77. SycE was dispensable for these deliveries. These results show that residues or codons 1 to 15 are sufficient for delivery into eukaryotic cells and that there is no specific translocation signal in Yops. However, the fact that the SycE-binding site and SycE were necessary for delivery of YopE by wild-type Yersinia suggests that they could introduce hierarchy among the effectors to be delivered. We then tested a YopE-Cya hybrid and YopE proteins deleted of amino acids 2 to 15 but containing the SycE-binding domain. These constructs were neither released in vitro upon Ca2+ chelation nor delivered into cells by wild-type or polymutant bacteria, casting doubts on the hypothesis that SycE could be a secretion pilot. Finally, it appeared that residues 50 to 77 are inhibitory to YopE release and that binding of SycE overcomes this inhibitory effect. Removal of this domain allowed in vitro release and delivery in cells in the absence as well as in the presence of SycE.

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Effects of mutations within two hydrophilic regions of the bovine papillomavirus type 1 E1 DNA-binding domain on E1–E2 interaction

Journal of General Virology ◽

10.1099/0022-1317-82-10-2341 ◽

2001 ◽

Vol 82 (10) ◽

pp. 2341-2351 ◽

Cited By ~ 7

Author(s):

Kelly J. Woytek ◽

Dhandapani Rangasamy ◽

Cynthia Bazaldua-Hernandez ◽

Mike West ◽

Van G. Wilson

Keyword(s):

Dna Binding ◽

Binding Domain ◽

Genome Replication ◽

Bovine Papillomavirus ◽

Dna Binding Domain ◽

Transactivation Domain ◽

Recognition Element ◽

C Terminus

The interaction between papillomavirus E1 and E2 proteins is essential for viral genome replication. Using both in vivo and in vitro assays to evaluate the regions of the two proteins necessary for the E1–E2 interaction, three independent interactions were identified for bovine papillomavirus E1: the N terminus of E1 (E1N, residues 1–311) interacts with the E2 transactivation domain (E2TAD) and the E2 DNA-binding domain (E2DBD) and the C terminus of E1 (E1C, residues 315–605) interacts with E2. Nine mutations within E1N were evaluated for their effects on E2 interaction. Five mutations eliminated interaction with the E2TAD; four of these were located within two previously identified conserved, hydrophilic regions, HR1 and HR3. Since HR1 and HR3 residues appear to comprise the origin of replication recognition element for E1, simultaneous interaction with the E2TAD during initiation complex formation would seem unlikely. Consistent with this inference is the fact that three of the five mutants defective for E2TAD binding exhibited wild-type levels of replication. The replication-positive phenotype of these mutants suggests that the E1N–E2TAD interaction is not essential for replication function and is probably involved in some other E1–E2 function, such as regulating transcription. Only one of the five mutations defective for E2TAD binding also prevented E2DBD interaction, indicating that the regions of E1N that interact with the E2TAD and the E2DBD are not identical. The ability of E1N to cooperatively interact with E2 bound to E2-binding site (E2BS) 11 versus E2BS12 was also examined, and cooperative binding was only observed when E2 was bound to E2BS12.

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Ciboria carunculoides Suppresses Mulberry Immune Responses Through Regulation of Salicylic Acid Signaling

Frontiers in Plant Science ◽

10.3389/fpls.2021.658590 ◽

2021 ◽

Vol 12 ◽

Author(s):

Zhiyuan Lv ◽

Lijuan Hao ◽

Bi Ma ◽

Ziwen He ◽

Yiwei Luo ◽

...

Keyword(s):

Salicylic Acid ◽

Systemic Acquired Resistance ◽

Acquired Resistance ◽

Antimicrobial Properties ◽

Defense Responses ◽

Narrow Host Range ◽

Mulberry Fruit ◽

Pathogenesis Related Protein ◽

Ja Signaling ◽

Sa Signaling

Ciboria carunculoides is the dominant causal agent of mulberry sclerotial disease, and it is a necrotrophic fungal pathogen with a narrow host range that causes devastating diseases in mulberry fruit. However, little is known about the interaction between C. carunculoides and mulberry. Here, our transcriptome sequencing results showed that the transcription of genes in the secondary metabolism and defense-related hormone pathways were significantly altered in infected mulberry fruit. Due to the antimicrobial properties of proanthocyanidins (PAs), the activation of PA biosynthetic pathways contributes to defense against pathogens. Salicylic acid (SA) and jasmonic acid (JA) are major plant defense hormones. However, SA signaling and JA signaling are antagonistic to each other. Our results showed that SA signaling was activated, while JA signaling was inhibited, in mulberry fruit infected with C. carunculoides. Yet SA mediated responses are double-edged sword against necrotrophic pathogens, as SA not only activates systemic acquired resistance (SAR) but also suppresses JA signaling. We also show here that the small secreted protein CcSSP1 of C. carunculoides activates SA signaling by targeting pathogenesis-related protein 1 (PR1). These findings reveal that the infection strategy of C. carunculoides functions by regulating SA signaling to inhibit host defense responses.

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The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

Molecular Biology of the Cell ◽

10.1091/mbc.10.7.2119 ◽

1999 ◽

Vol 10 (7) ◽

pp. 2119-2129 ◽

Cited By ~ 43

Author(s):

Marcus P. Cooper ◽

Adayabalam S. Balajee ◽

Vilhelm A. Bohr

Keyword(s):

Dna Repair ◽

Nucleotide Excision Repair ◽

Excision Repair ◽

Binding Domain ◽

In Vivo Studies ◽

C Terminus ◽

Nucleotide Excision ◽

N Terminus

The protein p21Cip1, Waf1, Sdi1 is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase δ. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.

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PFA ependymoma-associated protein EZHIP inhibits PRC2 activity through a H3 K27M-like mechanism

10.1101/585729 ◽

2019 ◽

Author(s):

Siddhant U. Jain ◽

Truman J. Do ◽

Peder J. Lund ◽

Andrew Q. Rashoff ◽

Marcin Cieslik ◽

...

Keyword(s):

Cpg Islands ◽

Polycomb Group ◽

Inhibitory Protein ◽

Conserved Sequence ◽

H3k27 Methylation ◽

C Terminus ◽

Diffuse Intrinsic Pontine Gliomas ◽

Necessary And Sufficient

ABSTRACTPolycomb group (PcG) proteins are essential for development and are frequently misregulated in human cancers. Polycomb Repressive Complexes (PRC1, PRC2) function in a collaborative epigenetic cross-talk with H3K27me3 to initiate and maintain transcriptional silencing. Diffuse intrinsic pontine gliomas (DIPGs) have extremely low H3K27me3 levels mediated by H3 K27M oncohistone. Posterior fossa type A (PFA) ependymomas also exhibit very low H3K27 methylation but lack the K27M oncohistone. Instead, PFA tumors express high levels of EZHIP (Enhancer of Zeste Homologs Inhibitory Protein, also termed CXORF67). We find that a highly conserved sequence within the C-terminus of EZHIP is necessary and sufficient to inhibit the catalytic activity of PRC2 in vitro and in vivo. Our biochemical experiments indicate that EZHIP directly interacts with the active site of the EZH2 subunit in a mechanism that is remarkably similar to the K27M oncohistone. Furthermore, expression of H3 K27M or EZHIP in cells promote similar chromatin profiles: loss of broad H3K27me3 domains, but retention of H3K27me3 at the sites of PRC2 recruitment. Importantly, we find that H3K27me3-mediated allosteric activation of PRC2 substantially increases the inhibition potential of EZHIP and H3 K27M, providing a potential mechanism for loss of H3K27me3 spreading from CpG islands in vivo. Our data indicate that PFA ependymoma and DIPG are driven in part by the action of peptidyl PRC2 inhibitors– the K27M oncohistone and the EZHIP ‘oncohistone-mimic’– that dysregulate gene silencing to promote tumorigenesis.

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Regulation of Mdm2-Directed Degradation by the C Terminus of p53

Molecular and Cellular Biology ◽

10.1128/mcb.18.10.5690 ◽

1998 ◽

Vol 18 (10) ◽

pp. 5690-5698 ◽

Cited By ~ 113

Author(s):

Michael H. G. Kubbutat ◽

Robert L. Ludwig ◽

Margaret Ashcroft ◽

Karen H. Vousden

Keyword(s):

P53 Protein ◽

Regulatory Elements ◽

Binding Domain ◽

Tumor Suppressor Protein ◽

C Terminus ◽

The Stability ◽

P53 Tumor Suppressor Protein ◽

Inducible Gene ◽

Oligomerization Domain

ABSTRACT The stability of the p53 tumor suppressor protein is regulated by interaction with Mdm2, the product of a p53-inducible gene. Mdm2-targeted degradation of p53 depends on the interaction between the two proteins and is mediated by the proteasome. We show here that in addition to the N-terminal Mdm2 binding domain, the C terminus of p53 participates in the ability of p53 to be degraded by Mdm2. In contrast, alterations in the central DNA binding domain of p53, which change the conformation of the p53 protein, do not abrogate the sensitivity of the protein to Mdm2-mediated degradation. The importance of the C-terminal oligomerization domain to Mdm2-targeted degradation of p53 is likely to reflect the importance of oligomerization of the full-length p53 protein for interaction with Mdm2, as previously shown in vitro. Interestingly, the extreme C-terminal region of p53, outside the oligomerization domain, was also shown to be necessary for efficient degradation, and deletion of this region stabilized the protein without abrogating its ability to bind to Mdm2. Mdm2-resistant p53 mutants were not further stabilized following DNA damage, supporting a role for Mdm2 as the principal regulator of p53 stability in cells. The extreme C terminus of the p53 protein has previously been shown to contain several regulatory elements, raising the possibility that either allosteric regulation of p53 by this domain or interaction between this region and a third protein plays a role in determining the sensitivity of p53 to Mdm2-directed degradation.

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Endophytic Actinobacteria Induce Defense Pathways in Arabidopsis thaliana

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-2-0208 ◽

2008 ◽

Vol 21 (2) ◽

pp. 208-218 ◽

Cited By ~ 207

Author(s):

V. M. Conn ◽

A. R. Walker ◽

C. M. M. Franco

Keyword(s):

Gene Expression ◽

Arabidopsis Thaliana ◽

Salicylic Acid ◽

Systemic Acquired Resistance ◽

Quantitative Polymerase Chain Reaction ◽

In Planta ◽

Defense Gene Expression ◽

Streptomyces Sp ◽

Endophytic Actinobacteria

Endophytic actinobacteria, isolated from healthy wheat tissue, which are capable of suppressing a number wheat fungal pathogens both in vitro and in planta, were investigated for the ability to activate key genes in the systemic acquired resistance (SAR) or the jasmonate/ethylene (JA/ET) pathways in Arabidopsis thaliana. Inoculation of A. thaliana (Col-0) with selected endophytic strains induced a low level of SAR and JA/ET gene expression, measured using quantitative polymerase chain reaction. Upon pathogen challenge, endophyte-treated plants demonstrated a higher abundance of defense gene expression compared with the non-endophyte-treated controls. Resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora required the JA/ET pathway. On the other hand, resistance to the fungal pathogen Fusarium oxysporum involved primarily the SAR pathway. The endophytic actinobacteria appear to be able to “prime” both the SAR and JA/ET pathways, upregulating genes in either pathway depending on the infecting pathogen. Culture filtrates of the endophytic actinobacteria were investigated for the ability to also activate defense pathways. The culture filtrate of Micromonospora sp. strain EN43 grown in a minimal medium resulted in the induction of the SAR pathway; however, when grown in a complex medium, the JA/ET pathway was activated. Further analysis using Streptomyces sp. strain EN27 and defense-compromised mutants of A. thaliana indicated that resistance to E. carotovora subsp. carotovora occurred via an NPR1-independent pathway and required salicylic acid whereas the JA/ET signaling molecules were not essential. In contrast, resistance to F. oxysporum mediated by Streptomyces sp. strain EN27 occurred via an NPR1-dependent pathway but also required salicylic acid and was JA/ET independent.

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