Shared Binding Site Research Articles

Competition between α-actinin and Ca2+-Calmodulin Controls Surface Retention of the L-type Ca2+ Channel CaV1.2

Regulation of neuronal excitability and cardiac excitation-contraction coupling requires the proper localization of L-type Ca²⁺ channels. We show that the actin-binding protein α-actinin binds to the C-terminal surface targeting motif of α11.2, the central pore-forming Ca(V)1.2 subunit, in order to foster its surface expression. Disruption of α-actinin function by dominant-negative or small hairpin RNA constructs reduces Ca(V)1.2 surface localization in human embryonic kidney 293 and neuronal cultures and dendritic spine localization in neurons. We demonstrate that calmodulin displaces α-actinin from their shared binding site on α11.2 upon Ca²⁺ influx through L-type channels, but not through NMDAR, thereby triggering loss of Ca(V)1.2 from spines. Coexpression of a Ca²⁺-binding-deficient calmodulin mutant does not affect basal Ca(V)1.2 surface expression but inhibits its internalization upon Ca²⁺ influx. We conclude that α-actinin stabilizes Ca(V)1.2 at the plasma membrane and that its displacement by Ca²⁺-calmodulin triggers Ca²⁺-induced endocytosis of Ca(V)1.2, thus providing an important negative feedback mechanism for Ca²⁺ influx.

Abstract 2313: Differences in estrogen receptor signaling in normal mammary epithelial cells and ER-positive primary breast tumors and metastases.

Abstract Background: The estrogen receptor (ER) is expressed in ≈70% of sporadic breast cancer and activates genes driving cell proliferation and tumorigenesis. We have previously performed genome-wide analysis of ER binding sites in MCF-7 breast cancer cells and identified distinct mechanisms of tumorigenic ER signaling. Using EpCAM and CD49f as markers to enrich for ER-positive (ER+) cells obtained from primary non-malignant breast tissue, we seek to elucidate differences in ER signaling between normal and primary and metastatic ER+ breast cancer cells. Methods: Viable breast epithelial cells were obtained from patients undergoing reduction mammoplasties, and ER+ breast cancer cells from primary tumors and metastatic effusions. Following dissociation into single cells, EpCAM+ cell subpopulations were isolated and stimulated with estradiol. Gene expression microarray analysis, chromatin immunoprecipitation and DNA sequencing (ChIP-seq) on transcription factors and histone modifications as well as DNAse I hypersensitivity assays (DHS) were performed, and compared to MCF-7 breast cancer cells. Results: Triplicates of normal, ER+ breast cancers, and metastatic ER+ cancer were analyzed. Gene expression profiles revealed differences in estradiol regulated genes between primary normal, breast tumor, and metastatic ER+ breast cancer cells. Genes that promote cell cycling and cell proliferation were downregulated in non-malignant tissue but were upregulated in breast cancer cells. Our ChIP-seq results showed differential binding of ER between normal and ER+ breast cancer with little common overlap, and motif analysis of these binding sites demonstrated the enrichment of ERE motifs in common sites, TCF12 motifs in unique normal sites, and FOXA1 motifs in unique breast cancer sites. Analyses of the distribution of histone modifications and DHS regions demonstrated distinct patterns at shared, normal, and breast cancer ER binding sites, suggesting functionality and further validating differential ER binding. Conclusions: There are contrasting differences in ER signaling between normal mammary and ER+ breast cancer cells, with estrogen appearing to have anti-proliferative effects in normal luminal cells compared to pro-proliferative effects in BC. ER ChIP-seq has identified unique motifs, distribution of histone modifications, and DHS regions specific to unique normal, cancer and shared ER binding sites. Our studies point to TCF12 as a potential ER pioneer cofactor in non-malignant breast tissue and provide more data in support of FOXA1 as an important ER coregulator in ER+ breast cancer. Our data provides evidence for key alterations in ER-signaling during tumorigenesis and could lead to the identification of novel strategies to target breast cancer specific ER signaling. Citation Format: David Chi, Housheng He, Tony Yeung, Rinath Jeselsohn, Stuart Schnitt, Judy Garber, Andrea Richardson, Elgene Lim, Myles Brown. Differences in estrogen receptor signaling in normal mammary epithelial cells and ER-positive primary breast tumors and metastases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2313. doi:10.1158/1538-7445.AM2013-2313

Hepatocyte Nuclear Factor 4 Alpha and Farnesoid X Receptor Co-regulates Gene Transcription in Mouse Livers on a Genome-Wide Scale

Farnesoid X receptor (Fxr) is a ligand-activated nuclear receptor critical for liver function. Reports indicate that the functions of Fxr in the liver may overlap with those of hepatocyte nuclear factor 4α (Hnf4α), but studies of their precise genome-wide interaction to regulate gene transcription in the liver are lacking. Thus, we compared the genome-wide binding of Fxr and Hnf4α in the liver of mice and characterized their cooperative activity on binding to and activating target gene transcription. Genome-wide ChIP-Seq data of Fxr and Hnf4α in mouse liver were analyzed by MACS, BEDTools, and DAVID. Co-immunoprecipitation, ChIP-qPCR, and luciferase assays were done to test for protein-protein interaction and cooperative binding. ChIP-seq analysis showed nearly 50% binding sites of Fxr and Hnf4α in mouse liver overlap and Hnf4α bound to shared target sites upstream and in close proximity to Fxr. Moreover, genes co-bound by Fxr and Hnf4α are enriched in complement and coagulation cascades and drug metabolism. A direct Fxr-Hnf4α protein interaction dependent on Fxr activity was detected and transcriptional assays suggest that Hnf4α can increase Fxr transcriptional activity. Conversely, binding assays showed Hnf4α can be either Fxr-dependent or -independent at different shared binding sites. Our results showed that Fxr cooperates with Hnf4α in the liver to modulate gene transcription. This study provides the first evidence on a genome-wide scale of both cooperative and independent interactions between Fxr and Hnf4α in regulating gene transcription in the liver.

A new method to customize protein expression vectors for fast, efficient and background free parallel cloning.

BackgroundExpression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. Parallel vector series that cover all variations are available, but not without compromise. We have established a fast, efficient and absolutely background free cloning approach that can be applied to any selected vector.ResultsHere we describe a method to tailor selected expression vectors for parallel Sequence and Ligation Independent Cloning. SLIC cloning enables precise and sequence independent engineering and is based on joining vector and insert with 15–25 bp homologies on both DNA ends by homologous recombination. We modified expression vectors based on pET, pFastBac and pTT backbones for parallel PCR-based cloning and screening in E.coli, insect cells and HEK293E cells, respectively. We introduced the toxic ccdB gene under control of a strong constitutive promoter for counterselection of insert less vector. In contrast to DpnI treatment commonly used to reduce vector background, ccdB used in our vector series is 100% efficient in killing parental vector carrying cells and reduces vector background to zero. In addition, the 3’ end of ccdB functions as a primer binding site common to all vectors. The second shared primer binding site is provided by a HRV 3C protease cleavage site located downstream of purification and solubility enhancing tags for tag removal. We have so far generated more than 30 different parallel expression vectors, and successfully cloned and expressed more than 250 genes with this vector series. There is no size restriction for gene insertion, clone efficiency is > 95% with clone numbers up to 200. The procedure is simple, fast, efficient and cost-effective. All expression vectors showed efficient expression of eGFP and different target proteins requested to be produced and purified at our Core Facility services.ConclusionThis new expression vector series allows efficient and cost-effective parallel cloning and thus screening of different protein constructs, tags and expression hosts.

Structures of a Na + -coupled, substrate-bound MATE multidrug transporter

Multidrug transporters belonging to the multidrug and toxic compound extrusion (MATE) family expel dissimilar lipophilic and cationic drugs across cell membranes by dissipating a preexisting Na(+) or H(+) gradient. Despite its clinical relevance, the transport mechanism of MATE proteins remains poorly understood, largely owing to a lack of structural information on the substrate-bound transporter. Here we report crystal structures of a Na(+)-coupled MATE transporter NorM from Neisseria gonorrheae in complexes with three distinct translocation substrates (ethidium, rhodamine 6G, and tetraphenylphosphonium), as well as Cs(+) (a Na(+) congener), all captured in extracellular-facing and drug-bound states. The structures revealed a multidrug-binding cavity festooned with four negatively charged amino acids and surprisingly limited hydrophobic moieties, in stark contrast to the general belief that aromatic amino acids play a prominent role in multidrug recognition. Furthermore, we discovered an uncommon cation-π interaction in the Na(+)-binding site located outside the drug-binding cavity and validated the biological relevance of both the substrate- and cation-binding sites by conducting drug resistance and transport assays. Additionally, we uncovered potential rearrangement of at least two transmembrane helices upon Na(+)-induced drug export. Based on our structural and functional analyses, we suggest that Na(+) triggers multidrug extrusion by inducing protein conformational changes rather than by directly competing for the substrate-binding amino acids. This scenario is distinct from the canonical antiport mechanism, in which both substrate and counterion compete for a shared binding site in the transporter. Collectively, our findings provide an important step toward a detailed and mechanistic understanding of multidrug transport.

Bacillus thuringiensis Cry34Ab1/Cry35Ab1 Interactions with Western Corn Rootworm Midgut Membrane Binding Sites

Background Bacillus thuringiensis (Bt) Cry34Ab1/Cry35Ab1 are binary insecticidal proteins that are co-expressed in transgenic corn hybrids for control of western corn rootworm, Diabrotica virgifera virgifera LeConte. Bt crystal (Cry) proteins with limited potential for field-relevant cross-resistance are used in combination, along with non-transgenic corn refuges, as a strategy to delay development of resistant rootworm populations. Differences in insect midgut membrane binding site interactions are one line of evidence that Bt protein mechanisms of action differ and that the probability of receptor-mediated cross-resistance is low.Methodology/Principal FindingsBinding site interactions were investigated between Cry34Ab1/Cry35Ab1 and coleopteran active insecticidal proteins Cry3Aa, Cry6Aa, and Cry8Ba on western corn rootworm midgut brush border membrane vesicles (BBMV). Competitive binding of radio-labeled proteins to western corn rootworm BBMV was used as a measure of shared binding sites. Our work shows that 125I-Cry35Ab1 binds to rootworm BBMV, Cry34Ab1 enhances 125I-Cry35Ab1 specific binding, and that 125I-Cry35Ab1 with or without unlabeled Cry34Ab1 does not share binding sites with Cry3Aa, Cry6Aa, or Cry8Ba. Two primary lines of evidence presented here support the lack of shared binding sites between Cry34Ab1/Cry35Ab1 and the aforementioned proteins: 1) No competitive binding to rootworm BBMV was observed for competitor proteins when used in excess with 125I-Cry35Ab1 alone or combined with unlabeled Cry34Ab1, and 2) No competitive binding to rootworm BBMV was observed for unlabeled Cry34Ab1 and Cry35Ab1, or a combination of the two, when used in excess with 125I-Cry3Aa, or 125I-Cry8Ba.Conclusions/SignificanceCombining two or more insecticidal proteins active against the same target pest is one tactic to delay the onset of resistance to either protein. We conclude that Cry34Ab1/Cry35Ab1 are compatible with Cry3Aa, Cry6Aa, or Cry8Ba for deployment as insect resistance management pyramids for in-plant control of western corn rootworm.

Comprehensive meta-analysis of Signal Transducers and Activators of Transcription (STAT) genomic binding patterns discerns cell-specific cis-regulatory modules.

BackgroundCytokine-activated transcription factors from the STAT (Signal Transducers and Activators of Transcription) family control common and context-specific genetic programs. It is not clear to what extent cell-specific features determine the binding capacity of seven STAT members and to what degree they share genetic targets. Molecular insight into the biology of STATs was gained from a meta-analysis of 29 available ChIP-seq data sets covering genome-wide occupancy of STATs 1, 3, 4, 5A, 5B and 6 in several cell types.ResultsWe determined that the genomic binding capacity of STATs is primarily defined by the cell type and to a lesser extent by individual family members. For example, the overlap of shared binding sites between STATs 3 and 5 in T cells is greater than that between STAT5 in T cells and non-T cells. Even for the top 1,000 highly enriched STAT binding sites, ~15% of STAT5 binding sites in mouse female liver are shared by other STATs in different cell types while in T cells ~90% of STAT5 binding sites are co-occupied by STAT3, STAT4 and STAT6. In addition, we identified 116 cis-regulatory modules (CRM), which are recognized by all STAT members across cell types defining a common JAK-STAT signature. Lastly, in liver STAT5 binding significantly coincides with binding of the cell-specific transcription factors HNF4A, FOXA1 and FOXA2 and is associated with cell-type specific gene transcription.ConclusionsOur results suggest that genomic binding of STATs is primarily determined by the cell type and further specificity is achieved in part by juxtaposed binding of cell-specific transcription factors.

Interplay between signal sequence recognition and N‐terminal protein modification at the ribosome exit site

Proteins destined for secretion are targeted to the endoplasmic reticulum by N‐terminal signal sequences. In contrast, the majority of cytosolic proteins are modified at their extreme N‐termini by methionine‐aminopeptidase (MetAP) and/or N‐acetylation. Both of these processes typically occur co‐translationally soon after the nascent chain emerges from the ribosome exit site.Proteins targeted by the Signal Recognition Particle‐independent targeting pathway have a strong bias against N‐terminal processing and mutations that induce such processing inhibit translocation. Detailed examination of SRP‐dependent substrates reveals that mutations that induce N‐terminal processing have no effect on ER targeting. In the context of signal anchor proteins with a long N‐terminal cytoplasmic region, methionine processing and acetylation can occur, but has no effect on translocation. In contrast, signal anchor sequences with short N‐terminal regions are inefficiently processed at their N‐termini. This is rationalized by the finding that SRP and MetAP compete for a shared binding site on the ribosome. Furthermore, we find that additional ribosome‐associated factors regulate the differential recruitment of these two factors to secretory and cytosolic ribosome nascent chains. Hence a tightly regulated interplay exists to ensure the fidelity of the early events of nascent chain processing.This work is funded by the Biotechnology and Biological Sciences Research Council.

Drug repositioning through incomplete bi-cliques in an integrated drug–target–disease network

Recently, there has been much interest in gene-disease networks and polypharmacology as a basis for drug repositioning. Here, we integrate data from structural and chemical databases to create a drug-target-disease network for 147 promiscuous drugs, their 553 protein targets, and 44 disease indications. Visualizing and analyzing such complex networks is still an open problem. We approach it by mining the network for network motifs of bi-cliques. In our case, a bi-clique is a subnetwork in which every drug is linked to every target and disease. Since the data are incomplete, we identify incomplete bi-cliques, whose completion introduces novel, predicted links from drugs to targets and diseases. We demonstrate the power of this approach by repositioning cardiovascular drugs to parasitic diseases, by predicting the cancer-related kinase PIK3CG as a novel target of resveratrol, and by identifying for five drugs a shared binding site in four serine proteases and novel links to cancer, cardiovascular, and parasitic diseases.

Expanding the conformational selection paradigm in protein-ligand docking.

Conformational selection emerges as a theme in macromolecular interactions. Data validate it as a prevailing mechanism in protein-protein, protein-DNA, protein-RNA, and protein-small molecule drug recognition. This raises the question of whether this fundamental biomolecular binding mechanism can be used to improve drug docking and discovery. Actually, in practice this has already been taking place for some years in increasing numbers. Essentially, it argues for using not a single conformer, but an ensemble. The paradigm of conformational selection holds that because the ensemble is heterogeneous, within it there will be states whose conformation matches that of the ligand. Even if the population of this state is low, since it is favorable for binding the ligand, it will bind to it with a subsequent population shift toward this conformer. Here we suggest expanding it by first modeling all protein interactions in the cell by using Prism, an efficient motif-based protein-protein interaction modeling strategy, followed by ensemble generation. Such a strategy could be particularly useful for signaling proteins, which are major targets in drug discovery and bind multiple partners through a shared binding site, each with some-minor or major-conformational change.

Modulation of gene transcription noise by competing transcription factors

Sequence specific transcription factors (TFs) are critical to ensuring that genes are transcribed in the right cell at the right time. Often, the gene promoter is flanked by multiple binding sites, some of which can be bound by different types of TFs in the cell. To investigate how the transcription noise is modulated by the competition of these TFs at their shared binding sites, we model gene transcription as a renewal process where the time spent in each transcription cycle is assumed to be independently and identically distributed. With the help of the elementary renewal theorem and the central limit theorem, we prove that the stationary noise strength Φ of transcription frequency equals the noise η (2) of the time spent in a single transcription cycle. Subsequent analysis shows that competitive TF binding could produce an unbounded spectrum of Φ, in sharp contrast to the estimate 1/3 ≤ Φ < for single binding pattern activated transcription. We predict several mechanisms by which genes could stay away from abnormally noisy transcription while living with multiple binding patterns. The most efficient one is to maintain a relatively long engaged time by transcription pausing, interrupting, or other means. Alternatively, high noise strength is prevented if all binding patterns activate transcription strongly. When some binding patterns activate transcription weakly, low noise strength is ensured if the binding pattern with the weakest activation strength is utilized frequently.

Pulsed Electron Spin Resonance Resolves the Coordination Site of Cu2+ Ions in α1-Glycine Receptor

In this talk, we will discuss the coordination environment of copper ions (Cu2+) in human α1 glycine receptor (GlyR). GlyRs are members of the pentameric ligand-gated ion channel (pLGIC) superfamily that mediate fast signaling at synapses. Metal ions like Zn2+ and Cu2+ significantly modulate the activity of pLGICs, and in vivo results suggest that metal ion coordination might be essential for proper physiological postsynaptic inhibition by GlyR. In order to better understand the molecular basis of this effect we have used pulsed electron spin resonance (ESR) methods like electron spin echo envelope modulation (ESEEM) and double electron electron resonance (DEER) spectroscopy to directly examine Cu2+ coordination with GlyR. We show, that Cu2+ has one binding site per α1 subunit, and that five Cu2+ can be coordinated per GlyR. Cu2+ binds to GlyR with a 40 μM apparent dissociation constant, and the metal ion are coordinated to E192 and H215 in each subunit. This is consistent with earlier functional measurements. However, the coordination site does not include residues around the agaonist/antagonist binding site, such as T112, R131, R65, E157 that were previously suggested to have roles in Cu2+ coordination by functional measurements on site-directed mutants. Intriguingly, the E192 and H215 are also known to bind Zn2+. The binding of Zn2+ at this site potentiates channel activity, whereas Cu2+ has an inhibitory effect. The opposing modulatory actions of these similar metal divalent cations at a shared binding site highlight the sensitive allosteric nature of GlyR. This work is supported by NIH (5R01NS053788). S.R. is supported by a Long-term Postdoctoral Fellowship awarded by the EMBO.

Contactins: Structural aspects in relation to developmental functions in brain disease

The contactins are members of a protein subfamily of neural immunoglobulin (Ig) domain-containing cell adhesion molecules. Their architecture is based on six N-terminal Ig domains, four fibronectin type III domains, and a C-terminal glycophosphatidylinositol (GPI)-anchor to the extracellular part of the cell membrane. Genetics of neuropsychiatric disorders, particularly autism spectrum disorders, have pinpointed contactin-4, -5, and -6 (CNTN4, -5, and -6) as potential disease genes in neurodevelopmental disorders and suggested that they participate in pathways important for appropriate brain development. These contactins have distinct but overlapping patterns of brain expression, and null-mutation causes subtle morphological and functional defects in the brain. The molecular basis of their neurodevelopmental functions is likely conferred by heterophilic protein interactions. Cntn4, -5, and -6 interact with protein tyrosine phosphatase receptor gamma (Ptptg) using a shared binding site that spans their second and third Ig repeats. Interactions with amyloid precursor protein (APP), Notch, and other IgCAMs have also been indicated. The present data indicate that Cntn4, -5, and -6 proteins may be part of heteromeric receptor complexes as well as serve as ligands themselves.

Pulsed Electron Spin Resonance Resolves the Coordination Site of Cu 2+ Ions in α1-Glycine Receptor

Herein, we identify the coordination environment of Cu 2+ in the human α1-glycine receptor (GlyR). GlyRs are members of the pentameric ligand-gated ion channel superfamily (pLGIC) that mediate fast signaling at synapses. Metal ions like Zn 2+ and Cu 2+ significantly modulate the activity of pLGICs, and metal ion coordination is essential for proper physiological postsynaptic inhibition by GlyR in vivo. Zn 2+ can either potentiate or inhibit GlyR activity depending on its concentration, while Cu 2+ is inhibitory. To better understand the molecular basis of the inhibitory effect we have used electron spin resonance to directly examine Cu 2+ coordination and stoichiometry. We show that Cu 2+ has one binding site per α1 subunit, and that five Cu 2+ can be coordinated per GlyR. Cu 2+ binds to E192 and H215 in each subunit of GlyR with a 40 μM apparent dissociation constant, consistent with earlier functional measurements. However, the coordination site does not include several residues of the agonist/antagonist binding site that were previously suggested to have roles in Cu 2+ coordination by functional measurements. Intriguingly, the E192/H215 site has been proposed as the potentiating Zn 2+ site. The opposing modulatory actions of these cations at a shared binding site highlight the sensitive allosteric nature of GlyR.

Binding site alteration is responsible for field-isolated resistance to Bacillus thuringiensis Cry2A insecticidal proteins in two Helicoverpa species.

BackgroundEvolution of resistance by target pests is the main threat to the long-term efficacy of crops expressing Bacillus thuringiensis (Bt) insecticidal proteins. Cry2 proteins play a pivotal role in current Bt spray formulations and transgenic crops and they complement Cry1A proteins because of their different mode of action. Their presence is critical in the control of those lepidopteran species, such as Helicoverpa spp., which are not highly susceptible to Cry1A proteins. In Australia, a transgenic variety of cotton expressing Cry1Ac and Cry2Ab (Bollgard II) comprises at least 80% of the total cotton area. Prior to the widespread adoption of Bollgard II, the frequency of alleles conferring resistance to Cry2Ab in field populations of Helicoverpa armigera and Helicoverpa punctigera was significantly higher than anticipated. Colonies established from survivors of F2 screens against Cry2Ab are highly resistant to this toxin, but susceptible to Cry1Ac.Methodology/Principal FindingsBioassays performed with surface-treated artificial diet on neonates of H. armigera and H. punctigera showed that Cry2Ab resistant insects were cross-resistant to Cry2Ae while susceptible to Cry1Ab. Binding analyses with 125I-labeled Cry2Ab were performed with brush border membrane vesicles from midguts of Cry2Ab susceptible and resistant insects. The results of the binding analyses correlated with bioassay data and demonstrated that resistant insects exhibited greatly reduced binding of Cry2Ab toxin to midgut receptors, whereas no change in 125I-labeled-Cry1Ac binding was detected. As previously demonstrated for H. armigera, Cry2Ab binding sites in H. punctigera were shown to be shared by Cry2Ae, which explains why an alteration of the shared binding site would lead to cross-resistance between the two Cry2A toxins.Conclusion/SignificanceThis is the first time that a mechanism of resistance to the Cry2 class of insecticidal proteins has been reported. Because we found the same mechanism of resistance in multiple strains representing several field populations, we conclude that target site alteration is the most likely means that field populations evolve resistance to Cry2 proteins in Helicoverpa spp. Our work also confirms the presence in the insect midgut of specific binding sites for this class of proteins. Characterizing the Cry2 receptors and their mutations that enable resistance could lead to the development of molecular tools to monitor resistance in the field.

MutLα and Proliferating Cell Nuclear Antigen Share Binding Sites on MutSβ

MutSβ (MSH2-MSH3) mediates repair of insertion-deletion heterologies but also triggers triplet repeat expansions that cause neurological diseases. Like other DNA metabolic activities, MutSβ interacts with proliferating cell nuclear antigen (PCNA) via a conserved motif (QXX(L/I)XXFF). We demonstrate that MutSβ-PCNA complex formation occurs with an affinity of ∼0.1 μm and a preferred stoichiometry of 1:1. However, up to 20% of complexes are multivalent under conditions where MutSβ is in molar excess over PCNA. Conformational studies indicate that the two proteins associate in an end-to-end fashion in solution. Surprisingly, mutation of the PCNA-binding motif of MutSβ not only abolishes PCNA binding, but unlike MutSα, also dramatically attenuates MutSβ-MutLα interaction, MutLα endonuclease activation, and bidirectional mismatch repair. As predicted by these findings, PCNA competes with MutLα for binding to MutSβ, an effect that is blocked by the cell cycle regulator p21CIP1. We propose that MutSβ-MutLα interaction is mediated in part by residues ((L/I)SRFF) embedded within the MSH3 PCNA-binding motif. To our knowledge this is the first case where residues important for PCNA binding also mediate interaction with a second protein. These findings also indicate that MutSβ- and MutSα-initiated repair events differ in fundamental ways.

Identification and Characterization of Unique Proline-rich Peptides Binding to the Mitochondrial Fission Protein hFis1

Mammalian mitochondrial fission requires at least two proteins, hFis1 and the dynamin-like GTPase DLP1/Drp1. The mitochondrial protein hFis1 is anchored at the outer membrane by a C-terminal transmembrane domain. The cytosolic domain of hFis1 contains six alpha helices [alpha1-alpha6] out of which [alpha2-alpha5] form tetratricopeptide repeat (TPR)-like motifs. DLP1 and possibly other proteins are thought to interact with the hFis1 TPR region during the fission process. It has also been suggested that the alpha1-helix regulates protein-protein interactions at the TPR. We performed random peptide phage display screening using the hFis1[alpha2-alpha6] as the target and identified ten different peptide sequences. Phage ELISA using mutant hFis1 indicates that the peptide binding requires the alpha2 and alpha3 helices and the intact TPR structure. Competition experiments and surface plasmon resonance analyses confirmed that a subset of free peptides enriched with proline residues directly bind to the target. Two of these peptides bind to the alpha1-containing intact cytosolic domain of hFis1 with decreased affinity. Peptide microinjection into cells abolished the mitochondrial swelling induced by overexpression of alpha1-deleted hFis1, and significantly decreased cytochrome c release from mitochondria upon apoptotic induction. Our data demonstrate that hFis1 can bind to multiple amino acid sequences selectively, and that the TPR constitutes the main binding region of hFis1, providing a first insight into the hFis1 TPR as a potential therapeutic target.

Structural interpretation of protein-protein interaction network

BackgroundCurrently a huge amount of protein-protein interaction data is available from high throughput experimental methods. In a large network of protein-protein interactions, groups of proteins can be identified as functional clusters having related functions where a single protein can occur in multiple clusters. However experimental methods are error-prone and thus the interactions in a functional cluster may include false positives or there may be unreported interactions. Therefore correctly identifying a functional cluster of proteins requires the knowledge of whether any two proteins in a cluster interact, whether an interaction can exclude other interactions, or how strong the affinity between two interacting proteins is.MethodsIn the present work the yeast protein-protein interaction network is clustered using a spectral clustering method proposed by us in 2006 and the individual clusters are investigated for functional relationships among the member proteins. 3D structural models of the proteins in one cluster have been built – the protein structures are retrieved from the Protein Data Bank or predicted using a comparative modeling approach. A rigid body protein docking method (Cluspro) is used to predict the protein-protein interaction complexes. Binding sites of the docked complexes are characterized by their buried surface areas in the docked complexes, as a measure of the strength of an interaction.ResultsThe clustering method yields functionally coherent clusters. Some of the interactions in a cluster exclude other interactions because of shared binding sites. New interactions among the interacting proteins are uncovered, and thus higher order protein complexes in the cluster are proposed. Also the relative stability of each of the protein complexes in the cluster is reported.ConclusionsAlthough the methods used are computationally expensive and require human intervention and judgment, they can identify the interactions that could occur together or ones that are mutually exclusive. In addition indirect interactions through another intermediate protein can be identified. These theoretical predictions might be useful for crystallographers to select targets for the X-ray crystallographic determination of protein complexes.

Relationship between gene co-expression and sharing of transcription factor binding sites in Drosophila melanogaster

In functional genomics, it is frequently useful to correlate expression levels of genes to identify transcription factor binding sites (TFBS) via the presence of common sequence motifs. The underlying assumption is that co-expressed genes are more likely to contain shared TFBS and, thus, TFBS can be identified computationally. Indeed, gene pairs with a very high expression correlation show a significant excess of shared binding sites in yeast. We have tested this assumption in a more complex organism, Drosophila melanogaster, by using experimentally determined TFBS and microarray expression data. We have also examined the reverse relationship between the expression correlation and the extent of TFBS sharing. Pairs of genes with shared TFBS show, on average, a higher degree of co-expression than those with no common TFBS in Drosophila. However, the reverse does not hold true: gene pairs with high expression correlations do not share significantly larger numbers of TFBS. Exception to this observation exists when comparing expression of genes from the earliest stages of embryonic development. Interestingly, semantic similarity between gene annotations (Biological Process) is much better associated with TFBS sharing, as compared to the expression correlation. We discuss these results in light of reverse engineering approaches to computationally predict regulatory sequences by using comparative genomics.

In-vitro-Diagnostik und molekulare Grundlagen von IgE-vermittelten Nahrungsmittelallergien

Detection of allergen-specific IgE represents the most important tool for in vitro diagnostic testing of food allergy. Applied methods vary in design (single allergen sources or mixtures, panel tests, single allergen components) and diagnostic efficacy. High specific IgE to hens egg, cows milk, peanut and fish is associated with an increased risk for clinical reactions, but rarely supersedes oral challenge tests. Cellular tests with basophil leukocytes, demonstrating IgE-mediated sensitizations indirectly, are useful only in selected cases. Particular protein families (i.e. Bet v 1 homologs, lipid transfer proteins and profilins) consist of allergenic molecules with similar sequence and structure. Their shared IgE binding sites form the basis of cross-reactivity. Cross-reactive carbohydrate determinants (CCD), frequently from plant origin, can also bind IgE being rarely clinically relevant. Allergy to plants (i.e. tree nuts, fruits, legumes) and animals (cows milk, hens egg, fish) is diagnosed with extracts, if their quality is sufficient. Detecting IgE to single allergenic components allows molecule-specific diagnoses, their value varies for each allergen source and single allergen component. Allergen-specific IgE detections are indicated in case of suspected food-allergic reactions despite uncertain history and skin tests, sensitizations to foods not applicable for skin testing, severe reactions to foods, conditions hampering skin testing or its interpretation, and in children. Interpreting should consider incorrect results due to inferior reagents or laboratory errors and clinically irrelevant results due to highly elevated total IgE, low assay thresholds or cross-reactive allergens (errors of interpretation). Positive test results indicate allergen-specific sensitizations, being clinically relevant only in case of corresponding symptoms. The following methods are not useful for diagnosing food allergy: bioresonance, electroacupuncture, kinesiology, cytotoxic food allergy test (methods without validity and/or evidence), lymphocyte stimulation test, food-specific IgG and IgG4 (methods with invalid interpretation).