Functional analyses of mammalian reovirus nonstructural protein μNS

Genome replication of reovirus occurs in cytoplasmic inclusion bodies called viral factories or viroplasms. The viral nonstructural protein μNS, encoded by genome segment M3, is not a component of mature virions, but is expressed to high levels in infected cells and is concentrated in the infected cell factory matrix. Recent studies have demonstrated that μNS plays a central role in forming the matrix of these structures, as well as in recruiting other components to them for putative roles in genome replication and particle assembly.     

Nuclear localization of Sindbis virus nonstructural protein nsP2

In early infection, approximately 10% of nonstructural protein nsP2 of Sindbis virus was transported into the nuclei of virus-infected BHK-21 cells. Nuclear asP2 was dominantly associated with nuclear matrix. During the course of infection, increasing amounts of nsP2 accumulated in the nuclear fraction. A prominent accumulation of nuclear nsP2 occurred early in infection, from 1 h to 3 h postinfection. Meanwhile. a weak NTPase activity was found to be associated with the immunocomplexed nsP2. Nuclear localization of nsP2 and its possible role were diseussed in relation to the inhibition of host macromolecular synthesis.     

Smurf1 protein negatively regulates interferon-γ signaling through promoting STAT1 protein ubiquitination and degradation

Interferons are important cytokines that mediate antiviral, antiproliferative, antitumor, and immunoregulatory activities. However, uncontrolled IFN signaling may lead to autoimmune diseases. Here we identified Smurf1 as a negative regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation. Smurf1 interacted with STAT1 through the WW domains of Smurf1 and the PY motif in STAT1 and catalyzed K48-linked polyubiquitination of STAT1. Interestingly, the Smurf1-mediated ubiquitination and degradation did not require STAT1 tyrosine and serine phosphorylation. Subsequently, overexpression of Smurf1 attenuated IFN-γ-mediated STAT1 activation and antiviral immune responses, whereas knockdown of Smurf1 enhanced IFN-γ-mediated STAT1 activation, expression of STAT1 target genes, and antiviral immune responses. Furthermore, IFN-γ stimulation led to enhanced expression of Smurf1. Therefore, our results demonstrate that Smurf1 is a negative feedback regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation.     

Coronavirus infection induces DNA replication stress partly through interaction of its nonstructural protein 13 with the p125 subunit of DNA polymerase δ

Perturbation of cell cycle regulation is a characteristic feature of infection by many DNA and RNA viruses, including Coronavirus infectious bronchitis virus (IBV). IBV infection was shown to induce cell cycle arrest at both S and G(2)/M phases for the enhancement of viral replication and progeny production. However, the underlying mechanisms are not well explored. In this study we show that activation of cellular DNA damage response is one of the mechanisms exploited by Coronavirus to induce cell cycle arrest. An ATR-dependent cellular DNA damage response was shown to be activated by IBV infection. Suppression of the ATR kinase activity by chemical inhibitors and siRNA-mediated knockdown of ATR reduced the IBV-induced ATR signaling and inhibited the replication of IBV. Furthermore, yeast two-hybrid screens and subsequent biochemical and functional studies demonstrated that interaction between Coronavirus nsp13 and DNA polymerase δ induced DNA replication stress in IBV-infected cells. These findings indicate that the ATR signaling activated by IBV replication contributes to the IBV-induced S-phase arrest and is required for efficient IBV replication and progeny production.     

Interactome of signaling networks in wheat: the protein–protein interaction between TaRAR1 and TaSGT1

RAR1 and SGT1 are required for development and disease resistance in plants. In many cases, RAR1 and SGT1 regulate the resistance (R)-gene-mediated defense signaling pathways. Lr21 is the first identified NBS-LRR-type R protein in wheat and is required for resistance to the leaf rust pathogen. The Lr21-mediated signaling pathways require the wheat homologs of RAR1, SGT1, and HSP90. However, the molecular mechanisms of the Lr21-mediated signaling networks remain unknown. Here I present the DNA and protein sequences of TaRAR1 and TaSGT1, and demonstrate for the first time a direct protein-protein interaction between them.     

Identification of a protein–protein interaction between KCNE1 and the activation gate machinery of KCNQ1

KCNQ1 channels assemble with KCNE1 transmembrane (TM) peptides to form voltage-gated K⁺ channel complexes with slow activation gate opening. The cytoplasmic C-terminal domain that abuts the KCNE1 TM segment has been implicated in regulating KCNQ1 gating, yet its interaction with KCNQ1 has not been described. Here, we identified a protein–protein interaction between the KCNE1 C-terminal domain and the KCNQ1 S6 activation gate and S4–S5 linker. Using cysteine cross-linking, we biochemically screened over 300 cysteine pairs in the KCNQ1–KCNE1 complex and identified three residues in KCNQ1 (H363C, P369C, and I257C) that formed disulfide bonds with cysteine residues in the KCNE1 C-terminal domain. Statistical analysis of cross-link efficiency showed that H363C preferentially reacted with KCNE1 residues H73C, S74C, and D76C, whereas P369C showed preference for only D76C. Electrophysiological investigation of the mutant K⁺ channel complexes revealed that the KCNQ1 residue, H363C, formed cross-links not only with KCNE1 subunits, but also with neighboring KCNQ1 subunits in the complex. Cross-link formation involving the H363C residue was state dependent, primarily occurring when the KCNQ1–KCNE1 complex was closed. Based on these biochemical and electrophysiological data, we generated a closed-state model of the KCNQ1–KCNE1 cytoplasmic region where these protein–protein interactions are poised to slow activation gate opening.     

N-terminal region of rice polycomb group protein OsEZ1 is required for OsEZ1–OsFIE2 protein interaction

Polycomb group (PcG) proteins form Polycomb Repressive Complex 2 (PRC2), which regulates seed development by the epigenetic control of gene expression. Interaction assay among Arabidopsis Fertilization-independent-seed2 (FIS) class PcG proteins showed that Fertilization-independent endosperm (FIE) interacts with Medea (MEA), a SET-domain polycomb protein, of which N-terminal region is crucial for the interaction. In this study, rice SET-domain PcG protein OsEZ1, also known as OsiEZ1 in indica rice, was analyzed to identify an interacting domain of OsEZ1 required for OsEZ1–OsFIE2 protein interaction. A series of OsEZ1 deletions were generated and used to determine an interacting domain of OsEZ1 with OsFIE2 using the yeast two-hybrid system. Among OsEZ1 deletions, only OsEZ1?2 and OsEZ1?3 interacted with OsFIE2, indicating that the 155K–169R or N-proximal region of OsEZ1 is crucial for OsFIE2–OsEZ1 interaction. To examine the physiological roles of OsEZ1, 35S:OsEZ1 Arabidopsis lines were generated. OsEZ1 overexpressors exhibited altered seedling growth and seed size, implying that OsEZ1 may play important roles in seedling and seed development.     

Chk1 modulates the interaction between myosin phosphatase targeting protein 1 (MYPT1) and protein phosphatase 1cβ (PP1cβ)

Polo-like kinase 1 (Plk1) is an instrumental kinase that modulates many aspects of the cell cycle. Previous investigations have indicated that Plk1 is a target of the DNA damage response, and Plk1 inhibition is dependent on ATM/ATR and Chk1. But the exact mechanism remains elusive. In a proteomic screen to identify Chk1-interacting proteins, we found that myosin phosphatase targeting protein 1 (MYPT1) was present in the immunocomplex. MYPT1 is phosphorylated by CDK1, thus recruiting protein phosphatase 1β (PP1cβ) to dephosphorylate and inactivate Plk1. Here we identified that Chk1 directly interacts with MYPT1 and preferentially phosphorylates MYPT1 at Ser20, which is essential for MYPT1-PP1cβ interaction and subsequent Plk1 dephosphorylation. Phosphorylation of Ser20 is abolished during mitotic damage when Chk1 is inhibited. The degradation of MYPT1 is also regulated by Chk1 phosphorylation. Our results thus unveil the underlying machinery that attenuates Plk1 activity during mitotic damage through Chk1-induced phosphorylation of MYPT1.     

Inositol-requiring protein 1 – X-box-binding protein 1 pathway promotes epithelial–mesenchymal transition via mediating snail expression in pulmonary fibrosis

Epithelial–mesenchymal transition (EMT) is a complex biological program during which cells loss epithelial phenotype and acquire mesenchymal features. EMT is thought to be involved in the pathogenesis of various fibrotic diseases including pulmonary fibrosis (PF). Recent studies suggest that endoplasmic reticulum (ER) stress is associated with EMT in the progression of PF. However, the exact mechanism is unclear. Here, we developed a PF model with bleomycin (BLM) administration in rats and conducted several simulation experiments in alveolar epithelial cell (AECs) RLE-6TN to unravel the role of inositol-requiring protein 1 (IRE1) – X-box-binding protein 1 (XBP1) signal pathway in ER stress-induced EMT in PF. First, we observed that ER stress was occurred in type II AECs accompanied by EMT in BLM-induced PF. Then we explored the role of IRE1-XBP1-snail pathway in transforming growth factor (TGF)-β1/tunicamycin (TM)-induced EMT. When TGF-β1/TM was treated on AECs, IRE1 and XBP1 were overexpressed, meanwhile, snail expression was upregulated accompanied with EMT. However, when IRE1 or XBP1 was knockdown, TGF-β1/TM-induced EMT were blocked while the expression of snail was inhibited. Then we silenced snail and found that TGF-β1/TM-induced EMT were also suppressed, but it had no effect on the up-regulated expression of IRE1 and XBP1. Thus, we concluded that IRE1-XBP1 pathway promotes EMT via mediating snail expression in PF.     

Predicting protein–protein interactions from protein sequences using meta predictor

A novel method is proposed for predicting protein–protein interactions (PPIs) based on the meta approach, which predicts PPIs using support vector machine that combines results by six independent state-of-the-art predictors. Significant improvement in prediction performance is observed, when performed on Saccharomyces cerevisiae and Helicobacter pylori datasets. In addition, we used the final prediction model trained on the PPIs dataset of S. cerevisiae to predict interactions in other species. The results reveal that our meta model is also capable of performing cross-species predictions. The source code and the datasets are available at     

The crystal structure of human protein α1M reveals a chromophore-binding site and two putative protein–protein interfaces

Lipocalin α1-microglobulin (α1M) is a conserved glycoprotein present in plasma and in the interstitial fluids of all tissues. α1M is linked to a heterogeneous yellow–brown chromophore of unknown structure, and interacts with several target proteins, including α1-inhibitor-3, fibronectin, prothrombin and albumin. To date, there is little knowledge about the interaction sites between α1M and its partners. Here, we report the crystal structure of the human α1M. Due to the crystallization occurring in a low ionic strength solution, the unidentified chromophore with heavy electron density is observed at a hydrophobic inner tube of α1M. In addition, two conserved surface regions of α1M are proposed as putative protein–protein interface sites. Further study is needed to unravel the detailed information about the interaction between α1M and its partners.     

The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1α

P-Rex1 is a GEF (guanine-nucleotide-exchange factor) for the small G-protein Rac that is activated by PIP3 (phosphatidylinositol 3,4,5-trisphosphate) and Gβγ subunits and inhibited by PKA (protein kinase A). In the present study we show that PP1α (protein phosphatase 1α) binds P-Rex1 through an RVxF-type docking motif. PP1α activates P-Rex1 directly in vitro, both independently of and additively to PIP3 and Gβγ. PP1α also substantially activates P-Rex1 in vivo, both in basal and PDGF (platelet-derived growth factor)- or LPA (lysophosphatidic acid)-stimulated cells. The phosphatase activity of PP1α is required for P-Rex1 activation. PP1β, a close homologue of PP1α, is also able to activate P-Rex1, but less effectively. PP1α stimulates P-Rex1-mediated Rac-dependent changes in endothelial cell morphology. MS analysis of wild-type P-Rex1 and a PP1α-binding-deficient mutant revealed that endogenous PP1α dephosphorylates P-Rex1 on at least three residues, Ser834, Ser1001 and Ser1165. Site-directed mutagenesis of Ser1165 to alanine caused activation of P-Rex1 to a similar degree as did PP1α, confirming Ser1165 as a dephosphorylation site important in regulating P-Rex1 Rac-GEF activity. In summary, we have identified a novel mechanism for direct activation of P-Rex1 through PP1α-dependent dephosphorylation.     

Tomato SlSnRK1 protein interacts with and phosphorylates βC1, a pathogenesis protein encoded by a geminivirus β-satellite

The βC1 protein of tomato yellow leaf curl China β-satellite functions as a pathogenicity determinant. To better understand the molecular basis of βC1 in pathogenicity, a yeast two-hybrid screen of a tomato (Solanum lycopersicum) cDNA library was carried out using βC1 as bait. βC1 interacted with a tomato SUCROSE-NONFERMENTING1-related kinase designated as SlSnRK1. Their interaction was confirmed using a bimolecular fluorescence complementation assay in Nicotiana benthamiana cells. Plants overexpressing SnRK1 were delayed for symptom appearance and contained lower levels of viral and satellite DNA, while plants silenced for SnRK1 expression developed symptoms earlier and accumulated higher levels of viral DNA. In vitro kinase assays showed that βC1 is phosphorylated by SlSnRK1 mainly on serine at position 33 and threonine at position 78. Plants infected with βC1 mutants containing phosphorylation-mimic aspartate residues in place of serine-33 and/or threonine-78 displayed delayed and attenuated symptoms and accumulated lower levels of viral DNA, while plants infected with phosphorylation-negative alanine mutants contained higher levels of viral DNA. These results suggested that the SlSnRK1 protein attenuates geminivirus infection by interacting with and phosphorylating the βC1 protein.     

Phospho-tyrosine dependent protein–protein interaction network

Post-translational protein modifications, such as tyrosine phosphorylation, regulate protein–protein interactions (PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two-hybrid system employing human protein kinases for the analyses of phospho-tyrosine (pY)-dependent PPIs in a direct experimental, large-scale approach. We identified 292 mostly novel pY-dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co-immunoprecipitation experiments from mammalian cells. About one-sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY-PPIs are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY-mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY-dependent interactions of TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY-dependent PPIs dictate cellular cancer phenotypes.