MPB2C,a microtubule-associated plant protein binds to and interferes with cell-to-cell transport of tobacco mosaic virus movement protein

The movement protein of tobacco mosaic virus, MP30, mediates viral cell-to-cell transport via plasmodesmata. The complex MP30 intra- and intercellular distribution pattern includes localization to the endoplasmic reticulum, cytoplasmic bodies, microtubules, and plasmodesmata and likely requires interaction with plant endogenous factors. We have identified and analyzed an MP30-interacting protein, MPB2C, from the host plant Nicotiana tabacum. MPB2C constitutes a previously uncharacterized microtubule-associated protein that binds to and colocalizes with MP30 at microtubules. In vivo studies indicate that MPB2C mediates accumulation of MP30 at microtubules and interferes with MP30 cell-to-cell movement. In contrast, intercellular transport of a functionally enhanced MP30 mutant, which does not accumulate and colocalize with MP30 at microtubules, is not impaired by MPB2C. Together, these data support the concept that MPB2C is not required for MP30 cell-to-cell movement but may act as a negative effector of MP30 cell-to-cell transport activity.     

Rabies virus P protein binds to TBK1 and interferes with the formation of innate immunity-related liquid condensates

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A blocking monoclonal antibody to endothelial-leukocyte adhesion molecule-1 (ELAM1).

ELAM1 is a leukocyte adhesion molecule induced on human umbilical vein endothelial cells (HUVECs) by inflammatory cytokines. Balb/C mice were immunized with COS cells transiently expressing cell-surface ELAM1 after transfection with ELAM1 cDNA. After fusion, ELAM1-specific monoclonal antibodies (Mabs) were identified by selective adhesion to ELAM1-expressing, but not control, CHO cells, and to cytokine-treated but not untreated HUVECs. One Mab, designated BB11, binds to and immunoprecipitates ELAM1 expressed on HUVECs, COS and CHO cells. BB11 blocks the interaction of ELAM1 with human PMN, the human myelomonocytic cell line HL60, and the human colon carcinoma line HT29.     

A peptide derived from LFA-1 protein that modulates T-cell adhesion binds to soluble ICAM-1 protein

Leukocyte function associated antigen 1 (LFA-1) and intercellular adhesion molecule 1 (ICAM-1) have been shown to be critical for adhesion process and immune response. Modulation or inhibition of the interaction between LFA-1/ICAM-1 interactions can result in therapeutic effects. Our group and others have shown that peptides derived from ICAM-1 or LFA-1 inhibit adhesion in a homotypic T-cell adhesion assay. It is likely that the peptides derived from ICAM-1 bind to LFA-1 and peptides derived from LFA-1 bind to ICAM-1 and inhibit the adhesion interaction. However, there are no concrete experimental evidence to show that peptides bind to either LFA-1 or ICAM-1 and inhibit the adhesion. Using NMR, CD and docking studies we have shown that an LFA-1 derived peptide binds to soluble ICAM-1. Docking studies using "autodock" resulted in LFA-1 peptide interacting with the ICAM-1 protein near Glu34. The proposed model based on our experimental data indicated that the LFA-1 peptide interacts with the protein via three intermolecular hydrogen bonds. Hydrophobic interactions also play a role in stabilizing the complex.     

Staurosporine, a protein kinase C inhibitor interferes with proliferation of arterial smooth muscle cells

Staurosporine (10 ng/ml and more), a protein kinase C inhibitor, inhibited the serum-stimulated growth of smooth muscle cells. This inhibitory effect proved to be linked to the inhibition of transition from the G0 to the S phase of the cell cycle, as measured by 5-bromodeoxyuridine incorporation into the nuclei. As this inhibitory effect of staurosporine was restricted to the first 10 hours of the stimulation, reactions which are essential for the signal transduction may be associated with actions of protein kinase C. The possibility that staurosporine may inhibit cell growth via inhibition of this step warrants further attention.     

The protein kinase C inhibitor bisindolyl maleimide 2 binds with reversed orientations to different conformations of protein kinase A

As the key mediators of eukaryotic signal transduction, the protein kinases often cause disease, and in particular cancer, when disregulated. Appropriately selective protein kinase inhibitors are sought after as research tools and as therapeutic drugs; several have already proven valuable in clinical use. The AGC subfamily protein kinase C (PKC) was identified early as a cause of cancer, leading to the discovery of a variety of PKC inhibitors. Despite its importance and early discovery, no crystal structure for PKC has yet been reported. Therefore, we have co-crystallized PKC inhibitor bisindolyl maleimide 2 (BIM2) with PKA variants to study its binding interactions. BIM2 co-crystallized as an asymmetric pair of kinase-inhibitor complexes. In this asymmetric unit, the two kinase domains have different lobe configurations, and two different inhibitor conformers bind in different orientations. One kinase molecule (A) is partially open with respect to the catalytic conformation, the other (B) represents the most open conformation of PKA reported so far. In monomer A, the BIM2 inhibitor binds tightly via an induced fit in the ATP pocket. The indole moieties are rotated out of the plane with respect to the chemically related but planar inhibitor staurosporine. In molecule B a different conformer of BIM2 binds in a reversed orientation relative to the equivalent maleimide atoms in molecule A. Also, a critical active site salt bridge is disrupted, usually indicating the induction of an inactive conformation. Molecular modeling of the clinical phase III PKC inhibitor LY333531 into the electron density of BIM2 reveals the probable binding mechanism and explains selectivity properties of the inhibitor.     

Complement factor H binds to denatured rather than to native pentameric C-reactive protein

Binding of the complement regulatory protein, factor H, to C-reactive protein has been reported and implicated as the biological basis for association of the H402 polymorphic variant of factor H with macular degeneration. Published studies utilize solid-phase or fluid-phase binding assays to show that the factor H Y402 variant binds C-reactive protein more strongly than H402. Diminished binding of H402 variant to C-reactive protein in retinal drusen is posited to permit increased complement activation, driving inflammation and pathology. We used well validated native human C-reactive protein and pure factor H Y402H variants to test interactions. When factor H variants were incubated with C-reactive protein in the fluid phase at physiological concentrations, no association occurred. When C-reactive protein was immobilized on plastic, either non-specifically by adsorption in the presence of Ca(2+) to maintain its native fold and pentameric subunit assembly or by specific Ca(2+)-dependent binding to immobilized natural ligands, no specific binding of either factor H variant from the fluid phase was observed. In contrast, both factor H variants reproducibly bound to C-reactive protein immobilized in the absence of Ca(2+), conditions that destabilize the native fold and pentameric assembly. Both factor H variants strongly bound C-reactive protein that was denatured by heat treatment before immobilization, confirming interaction with denatured but not native C-reactive protein. We conclude that the reported binding of factor H to C-reactive protein results from denaturation of the C-reactive protein during immobilization. Differential binding to C-reactive protein, thus, does not explain association of the Y402H polymorphism with macular degeneration.     

Bovine plasma protein C inhibitor with structural and functional homologous properties to human plasma protein C inhibitor

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)     

CRMP-2 directly binds to cytoplasmic dynein and interferes with its activity

The active transport of proteins and organelles is critical for cellular organization and function in eukaryotic cells. A substantial portion of long-distance transport depends on the opposite polarity of the kinesin and dynein family molecular motors to move cargo along microtubules. It is increasingly clear that many cargo molecules are moved bi-directionally by both sets of motors; however, the regulatory mechanism that determines the directionality of transport remains unclear. We previously reported that collapsin response mediator protein-2 (CRMP-2) played key roles in axon elongation and neuronal polarization. CRMP-2 was also found to associate with the anterograde motor protein Kinesin-1 and was transported with other cargoes toward the axon terminal. In this study, we investigated the association of CRMP-2 with a retrograde motor protein, cytoplasmic dynein. Immunoprecipitation assays showed that CRMP-2 interacted with cytoplasmic dynein heavy chain. Dynein heavy chain directly bound to the N-terminus of CRMP-2, which is the distinct side of CRMP-2's kinesin light chain-binding region. Furthermore, over-expression of the dynein-binding fragments of CRMP-2 prevented dynein-driven microtubule transport in COS-7 cells. Given that CRMP-2 is a key regulator of axon elongation, this interference with cytoplasmic dynein function by CRMP-2 might have an important role in axon formation, and neuronal development.     

The translocation inhibitor CAM741 interferes with vascular cell adhesion molecule 1 signal peptide insertion at the translocon

The cyclopeptolide CAM741 selectively inhibits cotranslational translocation of vascular cell adhesion molecule 1 (VCAM1), a process that is dependent on its signal peptide. In this study we identified the C-terminal (C-) region upstream of the cleavage site of the VCAM1 signal peptide as most critical for inhibition of translocation by CAM741, but full sensitivity to the compound also requires residues of the hydrophobic (h-) region and the first amino acid of the VCAM1 mature domain. The murine VCAM1 signal peptide, which is less susceptible to translocation inhibition by CAM741, can be converted into a fully sensitive signal peptide by two amino acid substitutions identified as critical for compound sensitivity of the human VCAM1 signal peptide. Using cysteine substitutions of non-critical residues in the human VCAM1 signal peptide and chemical cross-linking of targeted short nascent chains we show that, in the presence of CAM741, the N- and C-terminal segments of the VCAM1 signal peptide could be cross-linked to the cytoplasmic tail of Sec61beta, indicating altered positioning of the VCAM1 signal peptide relative to this translocon component. Moreover, translocation of a tag fused N-terminal to the VCAM1 signal peptide is selectively inhibited by CAM741. Our data indicate that the compound inhibits translocation of VCAM1 by interfering with correct insertion of its signal peptide into the translocon.     

Hepatitis B virus regulatory HBx protein binds to adaptor protein IPS-1 and inhibits the activation of beta interferon

Hepatitis B virus (HBV) encodes the regulatory HBx protein, which is required for virus replication, although its specific role(s) in the replication cycle remains under investigation. An immunoprecipitation/mass spectrometry approach was used to identify four novel HBx binding proteins from the cytoplasmic fraction of HBx transgenic mouse livers. One of these HBx binding partners is beta interferon promoter stimulator 1 (IPS-1), an adaptor protein that plays a critical role in mediating retinoic acid-inducible gene I (RIG-I) signaling, which leads to the activation of beta interferon (IFN-β). The HBx-IPS-1 protein interaction was confirmed in plasmid-transfected HepG2 cells by reciprocal coimmunoprecipitation and Western blotting. We hypothesized that HBx might alter IPS-1 function since proteins of hepatitis C virus and hepatitis A virus similarly bind IPS-1 and target it for inactivation. The effect of HBx on IPS-1-mediated IFN-β signaling was tested in transfected 293T and HepG2 cells, and we show that HBx inhibits double-stranded DNA (dsDNA)-mediated IFN-β activation in a dose-dependent manner when expressed either alone or within the context of HBV replication. However, HBx does not inhibit poly(I:C)-activated IFN-β signaling. These results demonstrate that HBx interferes with the RIG-I pathway of innate immunity. Hepatitis B virus now joins hepatitis C virus and hepatitis A virus in targeting the same innate immune response pathway, presumably as a shared strategy to benefit replication of these viruses in the liver.     

A minimal regulatory domain in the C terminus of STIM1 binds to and activates ORAI1 CRAC channels

Store-operated Ca²⁺ entry (SOCE) is a universal mechanism to increase intracellular Ca²⁺ concentrations in non-excitable cells. It is initiated by the depletion of ER Ca²⁺ stores, activation of stromal interaction molecule (STIM) 1 and gating of the Ca²⁺ release activated Ca²⁺ (CRAC) channel ORAI1 in the plasma membrane. We identified a minimal activation domain in the cytoplasmic region of STIM1 (CCb9) which activated Ca²⁺ influx and CRAC currents (I_CRAC) in the absence of store depletion similar to but more potently than the entire C terminus of STIM1. A STIM1 fragment (CCb7) that is longer by 31 amino acids than CCb9 at its C terminal end showed reduced ability to constitutively activate I_CRAC consistent with our observation that CCb9 but not CCb7 efficiently colocalized with and bound to ORAI1. Intracellular application of a 31 amino acid peptide contained in CCb7 but not CCb9 inhibited constitutive and store-dependent CRAC channel activation. In summary, these findings suggest that CCb9 represents a minimal ORAI1 activation domain within STIM1 that is masked by an adjacent 31 amino acid peptide preventing efficient CRAC channel activation in cells with replete Ca²⁺ stores.     

Tissue factor pathway inhibitor binds to platelet thrombospondin-1

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine proteinase inhibitor that down-regulates tissue factor-initiated blood coagulation. The most biologically active pool of TFPI is associated with the vascular endothelium, however, the biochemical mechanisms responsible for its cellular binding are not entirely defined. Proposed cellular binding sites for TFPI include nonspecific association with cell surface glycosaminoglycans and binding to glycosyl phosphatidylinositol-anchored proteins. Here, we report that TFPI binds specifically and saturably to thrombospondin-1 (TSP-1) purified from platelet alpha-granules with an apparent K(D) of approximately 7.5 nm. Binding is inhibited by polyclonal antibodies against TFPI and partially inhibited by the B-7 monoclonal anti-TSP-1 antibody. TFPI bound to immobilized TSP-1 remains an active proteinase inhibitor. Additionally, in solution phase assays measuring TFPI inhibition of factor VIIa/tissue factor catalytic activity, the rate of factor Xa generation was decreased 55% in the presence of TSP-1 compared with TFPI alone. Binding experiments done in the presence of heparin and with altered forms of TFPI suggest that the basic C-terminal region of TFPI is required for TSP-1 binding. The data provide a mechanism for the recruitment and localization of TFPI to extravascular surfaces within a bleeding wound, where it can efficiently down-regulate the procoagulant activity of tissue factor and allow subsequent aspects of platelet-mediated healing to proceed.     

An anti-endotoxin peptide that generates from the amino-terminal domain of complement regulatory protein C1 inhibitor

C1 inhibitor (C1INH), a complement regulatory protein, prevents endotoxin shock via a direct interaction of the amino-terminal domain with gram-negative bacterial lipopolysaccharide (LPS). Importantly, the cleaved, inactive C1INH still is an anti-endotoxin effector indicating the anti-endotoxin peptide that generates from the amino-terminal domain of C1INH. In this study, we first identified that a cleaved fragment within the major part of the amino-terminal domain in in vitro proteolytic analysis of C1INH had an ability to bind to LPS. We synthesized several peptides overlapping the C1INH cleaved fragment. Among these synthetic peptides, a 13-mer derivative peptide at position from 18 to 30, named N2((18-30)), exhibited the most powerful anti-endotoxin activity in vitro, enlightening that it was most strong at binding to LPS, inhibiting the interaction of LPS with LPS-binding protein (LBP), blocking LPS binding to CD14(+) cells, and suppressing production of tumor necrosis factor (TNF)-alpha by murine macrophages, RAW 264.7. In the murine endotoxin shock model, the peptide N2((18-30)) protected mice from LPS-induced lethal septic shock by inhibiting macrophage activation. These data indicate that the peptide N2((18-30)) derived from the amino-terminal region of C1INH is anti-endotoxin.     

Glutamate receptor-interacting protein 1 protein binds to the microtubule-associated protein

To identify the interaction proteins for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit glutamate receptor-interacting protein 1 (GRIP1), GRIP1 interactions with microtubule-associated protein (MAP)-1B light chain (LC) were investigated. GRIP1 interacts with MAP-1A and MAP-1B in the yeast two-hybrid assay, as is indicated also by glutathione S-transferase (GST) pull-down and coimmunoprecipitation with MAP-1B LC antibody in brain fractions. These results suggest a novel mechanism for localizing AMPA receptors to synaptic sites.