Protein Complementation Assay Research Articles

Abstract A25: Insights into TIMP-1/CD63 interaction and their functional relationship with MT1-MMP in cancer progression

Abstract Purpose: To unveil multi-faceted roles of Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) in cancer progression, particularly, those apart from Matrix Metalloproteinase (MMP) inhibition. Introduction: Clinical studies show increased TIMP-1 to associate with poor prognoses in many types of cancer, contrary to its well-known anti-invasive activity via MMP inhibition. Consistent with clinical data, we have made novel findings that TIMP-1 induces epithelial cell survival signaling and epithelial-mesenchymal transition (EMT) phenotype independent of MMP-inhibitory activity. Importantly, TIMP-1 signaling is dependent on interaction with the tetraspanin CD63, on the cell surface. Interestingly, CD63 was previously shown to bind and induce endocytosis of Membrane-type 1 MMP (MT1-MMP) to the lysosome. We have recently shown that over-expression of TIMP-1 can lead to increased MT1-MMP expression/activity which contribute to the resultant EMT phenotype. However, it has not yet been clarified if TIMP-1 and MT1-MMP have cooperative or competitive interactions with CD63. Methods/Results: To map the interacting domains of TIMP-1, or MT1-MMP with CD63, we have utilized yeast-2-hybrid (Y2H) and protein complementation assay (PCA). For Y2H, the proteins were fused to complementary fragments of the GAL-4 transcription factor. For PCA, partner proteins were fused to complementary fragments of a luciferase enzyme. Our results indicate that the Large Extracellular Loop (LEL) of CD63 may be responsible for TIMP-1 interaction; while the N-terminus or else transmembrane domains interact with MT1-MMP. For TIMP-1, the C-terminal end of the protein shows importance for CD63 interaction; which our preliminary results show can be specifically interrupted by use of a blocking antibody. Interestingly, despite differences in direct binding sites, TIMP-1 and MT1-MMP display competitive and not mutual interaction with CD63 according to our preliminary results. Conclusions: Taken together, we present a novel finding that TIMP-1/CD63 interaction and MT1-MMP/CD63 interaction are not compatible in their occurrence with each other. And that TIMP-1 over-expression in cancer therefore may disrupt the MT1-MMP/CD63 complex which offers MT1-MMP protection from lysosomal degradation. The C-terminus of TIMP-1 is critical to CD63 interaction and may provide a non-MMP-inhibitory site for therapeutically targeting TIMP-1 in cancer. Collectively, our findings suggest a model in which TIMP-1 functions as a signaling molecule via CD63 interaction and as an indirect regulator of MT1-MMP, in addition to its well-known function as an inhibitor of MMPs. This concept represents a paradigm shift in the current view of TIMP-1/MT1-MMP interactions and functions during cancer development/progression. [Support from: Ruth L. Kirschtein National Research Service Award T-32 CA009531, Thomas C. Rumble University Graduate Fellowship, and NIH/NCI RO1 089113] Citation Format: Richard B. Warner, Young-Suk Jung, Hyeong-Reh C. Kim. Insights into TIMP-1/CD63 interaction and their functional relationship with MT1-MMP in cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A25.

In-Frame cDNA Library Combined with Protein Complementation Assay Identifies ARL11-Binding Partners

The cDNA expression libraries that produce correct proteins are essential in facilitating the identification of protein-protein interactions. The 5′-untranslated regions (UTRs) that are present in the majority of mammalian and non-mammalian genes are predicted to alter the expression of correct proteins from cDNA libraries. We developed a novel cDNA expression library from which 5′-UTRs were removed using a mixture of polymerase chain reaction primers that complement the Kozak sequences we refer to as an “in-frame cDNA library.” We used this library with the protein complementation assay to identify two novel binding partners for ras-related ADP-ribosylation factor-like 11 (ARL11), cellular retinoic acid binding protein 2 (CRABP2), and phosphoglycerate mutase 1 (PGAM1). Thus, the in-frame cDNA library without 5′-UTRs we describe here increases the chance of correctly identifying protein interactions and will have wide applications in both mammalian and non-mammalian detection systems.

Wheat truncated hemoglobin interacts with photosystem I PSK-I subunit and photosystem II subunit PsbS1

Recently, the truncated hemoglobin gene (trHb) was discovered in plant species, however, its role has not yet been determined. In this study, the gene expression of wheat trHb (TatrHb) was analyzed under various biotic and abiotic stresses. TatrHb transcript levels increased in NaCl-treated leaves and gibberellic acid (GA3)-treated roots. In addition, sodium nitroprusside (SNP), a nitric oxide donor, induced an increase in TatrHb transcript levels in roots and leaves. A yeast two-hybrid assay (YIIH) was used to screen a hypoxia-treated wheat seedling library with the goal of determining the putative function of TatrHb. In this YIIH assay, photosynthesis-related genes that showed high homology to the Hordeum vulgare chloroplast photosystem I PSK-I subunit and Zea mays photosystem II subunit PsbS1 were detected and their interactions with TatrHb were confirmed. Subcellular localization of a TatrHb-green fluorescent protein (GFP) fusion protein and bimolecular fluorescence complementation (BiFC) assay suggested that TatrHb is involved in photosynthesis. The TatrHb-GFP fusion protein was localized in the plastids and the yellow fluorescent protein signal indicated that the TatrHb protein interacted with PSK-I and PsbS1 in the chloroplast.

Comparative analysis of virus–host interactomes with a mammalian high-throughput protein complementation assay based on Gaussia princeps luciferase

Comparative interactomics is a strategy for inferring potential interactions among orthologous proteins or “interologs”. Herein we focus, in contrast to standard homology-based inference, on the divergence of protein interaction profiles among closely related organisms, showing that the approach can correlate specific traits to phenotypic differences. As a model, this new comparative interactomic approach was applied at a large scale to human papillomaviruses (HPVs) proteins. The oncogenic potential of HPVs is mainly determined by the E6 and E7 early proteins. We have mapped and overlapped the virus–host protein interaction networks of E6 and E7 proteins from 11 distinct HPV genotypes, selected for their different tropisms and pathologies. We generated robust and comprehensive datasets by combining two orthogonal protein interaction assays: yeast two-hybrid (Y2H), and our recently described “high-throughput Gaussia princeps protein complementation assay” (HT-GPCA). HT-GPCA detects protein interaction by measuring the interaction-mediated reconstitution of activity of a split G. princeps luciferase. Hierarchical clustering of interaction profiles recapitulated HPV phylogeny and was used to correlate specific virus–host interaction profiles with pathological traits, reflecting the distinct carcinogenic potentials of different HPVs. This comparative interactomics constitutes a reliable and powerful strategy to decipher molecular relationships in virtually any combination of microorganism–host interactions.

IRF8 Interacts with the BCL6 Co-repressor, BCOR (109.5)

Abstract BCOR has been discovered as a BCL-6 interacting co-repressor, but little is known about its biological activities in normal B cell development and function. Previously, we found that the interferon regulatory factor 8 (IRF8), also known as interferon consensus sequence-binding protein, ICSBP, directly targets a large number of genes in germinal center (GC) B cells including BCL6. In this study, we screened potential binding partners of IRF8 using a retrovirus-based protein complementation assay in a mouse pre-B cell line. We found that IRF8 interacts directly with BCOR and the alpha helical region of IRF8 is required for this interaction. Using a siRNA-mediated IRF8 knockdown mouse B cell lymphoma cell line, we showed that IRF8 represses BCOR and enhances BCL6 transcription. Taken together, these data suggest that a complex comprised of BCOR-IRF8 modulates BCL6 associated transcriptional regulation of B cell function. Additional studies to evaluate the possible role of BCOR-IRF8 complex are in progress.

Disrupting the PIKE-A/Akt interaction inhibits glioblastoma cell survival, migration, invasion and colony formation.

The cyclin-dependent kinase 4 (CDK4) amplicon is frequently amplified in numerous human cancers including gliomas. PIKE-A, a proto-oncogene that is one of the important components of the CDK4 amplicon, binds to and enhances the kinase activity of Akt, thereby promoting cancer progression. To define the roles of the PIKE-A/Akt interaction in glioblastoma multiform (GBM) progression, we used biochemical protein/protein interaction (PPI) assays and live cell fluorescence-based protein complementation assays to search for small peptide antagonist from these proteins that were able to block their interaction. Here, we show that disruption of the interaction between PIKE-A and Akt by the small peptides significantly reduces glioblastoma cell proliferation, colony formation, migration and invasion. Disruption of PIKE-A/Akt association potently suppressed GBM cell proliferation and sensitized the cells to two clinical drugs that are currently used to treat GBM. Interestingly, GBM cells containing the CDK4 amplicon were more responsive to the inhibition of the PIKE-A/Akt interaction than GBM cells lacking this amplicon. Taken together, our findings provide proof-of-principle that blocking a PPI that is essential for cancer progression provides a valuable strategy for therapeutic discovery.

A modification of the split-tobacco etch virus method for monitoring interactions between membrane proteins in mammalian cells

Despite progress in the development of methods to monitor protein interactions, studies of interactions between membrane proteins in mammalian cells remain challenging. Protein complementation assays (PCAs) are commonly used to study interactions between proteins due to their simplicity. They are based on interaction-mediated reconstitution of a reporter protein, which can be easily monitored. Recently, a protein complementation method named split-TEV (tobacco etch virus) has been developed and is based on the functional reconstitution of TEV protease and subsequent proteolytic-mediated activation of reporters. In this work, we have developed a modification of the split-TEV method to study the interactions between membrane proteins with increased specificity. This assay was validated by addressing the interactions between different membrane proteins, including G protein-coupled receptors (GPCRs) and ion channels. By comparing it with another PCA, we found that this new method showed a higher sensitivity.

Mapping of Chikungunya Virus Interactions with Host Proteins Identified nsP2 as a Highly Connected Viral Component

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that has been responsible for an epidemic outbreak of unprecedented magnitude in recent years. Since then, significant efforts have been made to better understand the biology of this virus, but we still have poor knowledge of CHIKV interactions with host cell components at the molecular level. Here we describe the extensive use of high-throughput yeast two-hybrid (HT-Y2H) assays to characterize interactions between CHIKV and human proteins. A total of 22 high-confidence interactions, which essentially involved the viral nonstructural protein nsP2, were identified and further validated in protein complementation assay (PCA). These results were integrated to a larger network obtained by extensive mining of the literature for reports on alphavirus-host interactions. To investigate the role of cellular proteins interacting with nsP2, gene silencing experiments were performed in cells infected by a recombinant CHIKV expressing Renilla luciferase as a reporter. Collected data showed that heterogeneous nuclear ribonucleoprotein K (hnRNP-K) and ubiquilin 4 (UBQLN4) participate in CHIKV replication in vitro. In addition, we showed that CHIKV nsP2 induces a cellular shutoff, as previously reported for other Old World alphaviruses, and determined that among binding partners identified by yeast two-hybrid methods, the tetratricopeptide repeat protein 7B (TTC7B) plays a significant role in this activity. Altogether, this report provides the first interaction map between CHIKV and human proteins and describes new host cell proteins involved in the replication cycle of this virus.

Identification of new interacting partners of the shuttling protein ubinuclein (Ubn-1)

We have previously characterized ubinuclein (Ubn-1) as a NACos (Nuclear and Adherent junction Complex components) protein which interacts with viral or cellular transcription factors and the tight junction (TJ) protein ZO-1. The purpose of the present study was to get more insights on the binding partners of Ubn-1, notably those present in the epithelial junctions. Using an in vivo assay of fluorescent protein-complementation assay (PCA), we demonstrated that the N-terminal domains of the Ubn-1 and ZO-1 proteins triggered a functional interaction inside the cell. Indeed, expression of both complementary fragments of venus fused to the N-terminal parts of Ubn-1 and ZO-1 was able to reconstitute a fluorescent venus protein. Furthermore, nuclear expression of the chimeric Ubn-1 triggered nuclear localization of the chimeric ZO-1. We could localize this interaction to the PDZ2 domain of ZO-1 using an in vitro pull-down assay. More precisely, a 184-amino acid region (from amino acids 39 to 223) at the N-terminal region of Ubn-1 was responsible for the interaction with the PDZ2 domain of ZO-1. Co-imunoprecipitation and confocal microscopy experiments also revealed the tight junction protein cingulin as a new interacting partner of Ubn-1. A proteomic approach based on mass spectrometry analysis (MS) was then undertaken to identify further binding partners of GST-Ubn-1 fusion protein in different subcellular fractions of human epithelial HT29 cells. LYRIC (Lysine-rich CEACAM1-associated protein) and RACK-1 (receptor for activated C-kinase) proteins were validated as bona fide interacting partners of Ubn-1. Altogether, these results suggest that Ubn-1 is a scaffold protein influencing protein subcellular localization and is involved in several processes such as cell–cell contact signalling or modulation of gene activity.

Development of a split SNAP-tag protein complementation assay for visualization of protein–protein interactions in living cells

A split SNAP-tag protein complementation assay was developed for visualization of protein-protein interactions in living cells. Split SNAP-tagμs, fragments of divided SNAP-tag between amino acid residues 91 and 92, were fused to proteins that can interact with each other. After incubation with a fluorescent SNAP-tag substrate, cells that expressed split SNAP-tag fusion proteins generated fluorescent signals when these proteins interacted. Moreover, by combination with the split CLIP-tag (SNAP-tag mutant), simultaneous labeling was achieved. This split SNAP-tag labeling method should be a useful tool for visualization of protein-protein interaction processes.

Visualizing protein partnerships in living cells and organisms

In recent years, scientists have expanded their focus from cataloging genes to characterizing the multiple states of their translated products. One anticipated result is a dynamic map of the protein association networks and activities that occur within the cellular environment. While in vitro-derived network maps can illustrate which of a multitude of possible protein-protein associations could exist, they supply a falsely static picture lacking the subtleties of subcellular location (where) or cellular state (when). Generating protein association network maps that are informed by both subcellular location and cell state requires novel approaches that accurately characterize the state of protein associations in living cells and provide precise spatiotemporal resolution. In this review, we highlight recent advances in visualizing protein associations and networks under increasingly native conditions. These advances include second generation protein complementation assays (PCAs), chemical and photo-crosslinking techniques, and proximity-induced ligation approaches. The advances described focus on background reduction, signal optimization, rapid and reversible reporter assembly, decreased cytotoxicity, and minimal functional perturbation. Key breakthroughs have addressed many challenges and should expand the repertoire of tools useful for generating maps of protein interactions resolved in both time and space.

Gaussia princeps Luciferase as a Reporter for Transcriptional Activity, Protein Secretion, and Protein-Protein Interactions in Salmonella enterica Serovar Typhimurium

Gaussia princeps luciferase (Gluc) is widely used as a reporter in eukaryotes, but data about its applicability in bacteria are very limited. Here we show that a codon-optimized Gluc gene can be efficiently expressed in Salmonella enterica serovar Typhimurium. To test different Gluc variants as transcriptional reporters, we used the siiA promoter of Salmonella pathogenicity island 4 (SPI-4) driving expression of either an episomal or a chromosomally integrated Gluc gene. Most reliable results were obtained from lysates of single-copy Gluc reporter strains. Given the small size, high activity, and cofactor independence of Gluc, it might be especially suited to monitor secretion of bacterial proteins. We demonstrate its usefulness by luminescence detection of fusion proteins of Gluc and C-terminal portions of the SPI-4-encoded, type I-secreted adhesin SiiE in supernatants. The SiiE C-terminal moiety including immunoglobulin (Ig) domain 53 is essential and sufficient for mediating type I-dependent secretion of Gluc. In eukaryotes, protein-protein interaction studies based on split-Gluc protein complementation assays (PCA) could be established. We adapted these methods for use in Salmonella, demonstrating the interaction between the SPI-1-encoded effector SipA and its cognate secretion chaperone InvB. In conclusion, the versatile Gluc can be used to address a variety of biological questions, thus representing a valuable addition to the toolbox of modern molecular biology and microbiology.

HIV-1 integrase SUMOylation and viral replication

Background HIV-1 integrase catalyzes the integration of the reverse transcribed viral cDNA into the cellular genome, a key event of retroviral replication that is targeted by novel anti-HIV therapeutic agents. Numerous studies have contributed to understand of the molecular basis of integrase catalytic functions. Besides integration, HIV-1 integrase participates in other steps of viral replication such as reverse transcription and/or uncoating, PIC nuclear import and virion morphology. However, the underlying mechanisms are still not fully elucidated. Cellular and viral factors assist integrase in performing its multiple activities. Moreover, post-translational modifications (i.e. acetylation, ubiquitination and phosphorylation), which represent a common, rapid and generally reversible mechanism for fine tuning of protein activities, have also been shown to regulate integrase functions. Materials and methods and results By in vitro SUMOylation assay and purification on NiNTA beads in denaturing conditions, we show that HIV1 integrase is covalently modified by the three SUMO paralogues. By mutating SUMO-acceptor lysine residues within phylogenetically conserved SUMOylation consensus motifs identified in silico, we demonstrate that they represent major sites of SUMO conjugation. We introduced the same changes in the integrase sequence of a plasmid encoding the viral genome, which was used to generate mutant viral particles, and we compare their infectivity to that of WT HIV-1. We find that viruses harboring SUMOylation-defective integrase mutants are less infectious that the WT counterpart. Real-time PCR analysis of viral cDNA synthesis reveals that cells infected with mutant viruses display similar content of traverse transcripts, but a lower number of integrated proviruses, than WT HIV-1-infected cells. This integration defect at the integration step is not due to impairment of integrase binding to LEDGF/p7S, a key chromatin-tethering factor of HIV-1 pre-integration complex. Indeed, both WT and SUMOylation-defective integrase proteins coprecipitated with similar efficiency with LEDGF/p75. Finally, we establish that SUMOylation-defective integrase mutants retained WT catalytic activity as determined by Vprfusion protein complementation assay. Conclusions Here, we show that HIV-1 IN is modified by SUMO proteins and that phylogenetically conserved SUMOylation consensus motifs represent major SUMO-acceptor sites. Viruses harboring SUMOylation-site IN mutants displayed a replication defect that was mapped during the early stages of infection, before integration but after reverse transcription. Since SUMOylation-defective IN mutants retained WT catalytic activity as well as LEDGF-binding, we hypothesize that SUMOylation might regulate the affinity of IN for co-factors, contributing to efficient HIV-1 replication.

Abstract A41: The ASK1/IKK signaling axis mediates crosstalk between inflammation and apoptosis

Abstract Inflammation plays a well-established role in the promotion of tumorigenesis. The inflammatory response is mediated in large part by the inhibitor of kappa b (IKK) and nuclear factor κB (NfκB) signaling pathway. This signaling pathway can promote inflammation induced-tumorigenesis and inhibit apoptosis through protection against reactive oxygen species (ROS). The action of ROS is in part controlled by the apoptosis signal regulating kinase 1 (ASK1). ASK1 activation can initiate pro-apoptotic signaling cascades through mitogen activated protein kinases (MAPKs) Jun N-terminal Kinase (JNK) and p38. Improper regulation of ASK1 has been implicated in a wide array of diseases, including cancer. NfκB promotes tumor growth through activation of pro-growth and survival genes and decreases apoptosis through impairing JNK activation. This work has identified an unexpected, direct, non-genetic method of cell death inhibition by an upstream regulator of NfκB, IKK. We have found that IKK can phosphorylate ASK1 Ser967, which leads to the recruitment of 14–3–3 proteins, decreased ASK1 kinase activity and ASK1-mediated apoptosis. To further elucidate the relationship between ASK1 and IKK, the interaction interface was identified and the consequences on ASK1-mediated signaling were studied. IKK and ASK1 were shown to interact in cells by immunoprecipitation of both overexpressed and endogenous proteins. Furthermore, the direct interaction between these kinases was confirmed through time resolved – fluorescence resonance energy transfer (TR-FRET). The ASK1/IKK interaction interface was dissected through iterative deletional mutagenesis and protein complementation assays. ASK1 activity was assessed by measuring H2O2 induced apoptosis, neurite outgrowth of ASK1-inducible cells, and phosphorylation of ASK1 Ser967 and downstream targets under several IKK states. IKK prevented stress-induced dephosphorylation of ASK1 and inhibited ASK1 mediated apoptosis. These data suggest the existence of an IKK/ASK1 signaling node that negatively regulates apoptosis in response to pro-survival conditions. Characterization of this interaction could lead to development of targeted agents for ASK1 and IKK related diseases. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A41.

Nerve growth factor receptor TrkA exists as a preformed, yet inactive, dimer in living cells

The tropomyosin-related kinase A (TrkA) receptor and its ligand, nerve growth factor (NGF), play crucial roles in the development and function of the nervous system. NGF is believed to activate TrkA by bridging two TrkA monomers, leading to TrkA transphosphorylation. However, here we show that the majority of TrkA receptors exist as preformed, yet inactive, homodimers prior to NGF binding by using three different approaches such as chemical crosslinking and enzyme fragment complementation assay. Furthermore, TrkA homodimers are formed in endoplasmic reticulum before newly synthesized receptors reach the cell surface. These findings shed light on molecular mechanisms underlying transmembrane signaling by TrkA. Structured summaryTrkAphysically interacts with TrkA by protein complementation assay (View interaction)TrkAphysically interacts with TrkA by bimolecular fluorescence complementation (View interaction)TrkAphysically interacts with TrkA by cross-linking study (View interaction)

Therapeutic Targeting of Myc

Protein-protein interactions between members of the Myc transcription factor network are potential targets of small molecule inhibitors and stabilizers. Diverse screening strategies, including fluorescence resonance energy transfer, fluorescence polarization, two hybrid and protein complementation assays have identified several lead compounds that inhibit Myc-Max dimerization and one compound that stabilizes the Max homodimer. Representative compounds interfere with Myc-induced transcriptional activation, Myc-mediated oncogenic transformation, Myc-driven cellular replication and DNA binding of Myc. For the best characterized compounds, specific binding sites have been determined, and molecular mechanisms of action have been documented. This knowledge of small molecule - protein interaction is currently applied to highly targeted approaches that seek to identify novel compounds with improved potency.

Multimerization of Staufen1 in live cells

Transport of mRNA is an efficient mechanism to target proteins to specific regions of a cell. Although it is well documented that mRNAs are transported in ribonucleoprotein (RNP) complexes, several of the mechanisms involved in complex formation and localization are poorly understood. Staufen (Stau) 1, a double-stranded RNA-binding protein, is a well accepted marker of mRNA transport complexes. In this manuscript, we provide evidence that Stau1 self-associates in live cells using immunoprecipitation and bioluminescence resonance energy transfer (BRET) assays. The double-stranded RNA-binding domains dsRBD3 and dsRBD4 contributed about half of the signal, suggesting that Stau1 RNA-binding activity is involved in Stau1 self-association. Protein-protein interaction also occurred, via dsRBD5 and dsRBD2, as shown by in vitro pull-down, yeast two-hybrid, and BRET assays in live cells. Interestingly, Stau1 self-association contributes to the formation of oligomeric complexes as evidenced by the coexpression of split Renilla luciferase halves covalently linked to Stau1 in a protein complementation assay (PCA) combined with a BRET assay with Stau1-YFP. Moreover, we showed that these higher-order Stau1-containing complexes carry RNAs when the RNA stain SYTO 14 was used as the energy acceptor in the PCA/BRET assay. The oligomeric composition of Stau1-containing complexes and the presence of specific mRNAs have been confirmed by biochemical approaches involving two successive immunoprecipitations of Stau1-tagged molecules followed by qRT-PCR amplification. Altogether, these results indicate that Stau1 self-associates in mRNPs via its multiple functional domains that can select mRNAs to be transported and establish protein-protein interaction.

Brain-Permeable Small-Molecule Inhibitors of Hsp90 Prevent α-Synuclein Oligomer Formation and Rescue α-Synuclein-Induced Toxicity

Aggregation of alpha-synuclein (alphasyn) is a hallmark of sporadic and familial Parkinson's disease (PD) and dementia with Lewy bodies. Lewy bodies contain alphasyn and several heat shock proteins (Hsp), a family of molecular chaperones up-regulated by the cell under stress. We have previously shown that direct expression of Hsp70 and pharmacological up-regulation of Hsp70 by geldanamycin, an Hsp90 inhibitor, are protective against alphasyn-induced toxicity and prevent aggregation in culture. Here, we use a novel protein complementation assay to screen a series of small-molecule Hsp90 inhibitors for their ability to prevent alphasyn oligomerization and rescue toxicity. By use of this assay, we found that several compounds prevented alphasyn oligomerization as measured by decreased luciferase activity, led to a reduction in high-molecular-mass oligomeric alphasyn, and protected against alphasyn cytotoxicity. A lead compound, SNX-0723 (2-fluoro-6-[(3S)-tetrahydrofuran-3-ylamino]-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indol-1-yl)benzamide) was determined to have an EC(50) for inhibition of alphasyn oligomerization of approximately 48 nM and was able to rescue alphasyn-induced toxicity. In vivo assessment of SNX-0723 showed significant brain concentrations along with induction of brain Hsp70. With a low EC(50), brain permeability, and oral availability, these novel inhibitors represent an exciting new therapeutic strategy for PD.

Cross Talk among the Glycoproteins Involved in Herpes Simplex Virus Entry and Fusion: the Interaction between gB and gH/gL Does Not Necessarily Require gD

The gD, gB, and gH/gL glycoprotein quartet constitutes the basic apparatus for herpes simplex virus (HSV) entry into the cell and fusion. gD serves as a receptor binding glycoprotein and trigger of fusion. The conserved gB and gH/gL execute fusion. Central to understanding HSV entry/fusion has become the dissection of how the four glycoproteins engage in cross talk. While the independent interactions of gD with gB and gD with gH/gL have been documented, less is known of the interaction of gB with gH/gL. So far, this interaction has been detected only in the presence of gD by means of a split green fluorescent protein complementation assay. Here, we show that gB interacts with gH/gL in the absence of gD. The gB-gH/gL complex was best detected with a form of gB in which the endocytosis and phosphorylation motif have been deleted; this form of gB persists in the membranes of the exocytic pathway and is not endocytosed. The gB-gH/gL interaction was detected both in whole transfected cells by means of a split yellow fluorescent protein complementation assay and, biochemically, by a pull-down assay. Results with a panel of chimeric forms of gB, in which portions of the glycoprotein bracketed by consecutive cysteines were replaced with the corresponding portions from human herpesvirus 8 gB, favor the view that gB carries multiple sites for interaction with gH/gL, and one of these sites is located in the pleckstrin-like domain 1 carrying the bipartite fusion loop.

A Computationally Guided Protein-Interaction Screen Uncovers Coiled-Coil Interactions Involved in Vesicular Trafficking

Mapping protein–protein interactions at a domain or motif level can provide structural annotation of the interactome. The α-helical coiled coil is among the most common protein-interaction motifs, and proteins predicted to contain coiled coils participate in diverse biological processes. Here, we introduce a combined computational/experimental screening strategy that we used to uncover coiled-coil interactions among proteins involved in vesicular trafficking in Saccharomyces cerevisiae. A number of coiled-coil complexes have already been identified and reported to play important roles in this important biological process. We identify additional examples of coiled coils that can form physical associations. The computational strategy used to prioritize coiled-coil candidates for testing dramatically improved the efficiency of discovery in a large experimental screen. As assessed by comprehensive yeast two-hybrid assays, computational prefiltering retained 90% of positive interacting pairs and eliminated > 60% of negatives from a set of interaction candidates. The coiled-coil-mediated interaction network elucidated using the combined computational/experimental approach comprises 80 coiled-coil associations between 58 protein pairs, among which 21 protein interactions have not been previously reported in interaction databases and 26 interactions were previously known at the protein level but have now been localized to the coiled-coil motif. The coiled-coil-mediated interactions were specific rather than promiscuous, and many interactions could be recapitulated in a green fluorescent protein complementation assay. Our method provides an efficient route to discovering new coiled-coil interactions and uncovers a number of associations that may have functional significance for vesicular trafficking.