Molecular basis for the recognition of adenomatous polyposis coli by the Discs Large 1 protein

The human Discs Large 1 (DLG1) protein uses two of its three PDZ domains to interact with the C-terminal peptide of the Adenomatous Polyposis Coli (APC) tumor suppressor protein. The DLG1/APC complex inhibits the cell cycle progression from the G0/G1 to the S phase, regulates epithelial cell migration and morphogenesis, and is required for polarization of the microtubule cytoskeleton. However, the molecular details of how DLG1 recognizes APC is not clear. In this study, we performed biochemical and biophysical assays to investigate the interactions between PDZ domains of DLG1 and the C-terminal peptide of APC. In addition, we determined the crystal structures of the PDZ1 and PDZ2 domains of DLG1 each in complex with the C-terminal 11-residue peptide of APC. Our biochemical, biophysical, and structural results revealed structural elements and residues on PDZ1 and PDZ2 domains of DLG1 and on APC crucial for their mutual interaction. In particular, our results show that the β2/β3 loops of PDZ1 and PDZ2 play important roles in contributing to the binding affinities between PDZ domains and APC, through interacting with the residues upstream of the canonical PDZ-binding S/T-X-V motif. The results provide new insights into the binding mode of a defined C-terminal segment of APC by the PDZ domains of DLG1.     

Protein kinase Cα promotes cell migration through a PDZ-dependent interaction with its novel substrate discs large homolog 1 (DLG1)

Protein scaffolds maintain precision in kinase signaling by coordinating kinases with components of specific signaling pathways. Such spatial segregation is particularly important in allowing specificity of signaling mediated by the 10-member family of protein kinase C (PKC) isozymes. Here we identified a novel interaction between PKCα and the Discs large homolog (DLG) family of scaffolds that is mediated by a class I C-terminal PDZ (PSD-95, disheveled, and ZO1) ligand unique to this PKC isozyme. Specifically, use of a proteomic array containing 96 purified PDZ domains identified the third PDZ domains of DLG1/SAP97 and DLG4/PSD95 as interaction partners for the PDZ binding motif of PKCα. Co-immunoprecipitation experiments verified that PKCα and DLG1 interact in cells by a mechanism dependent on an intact PDZ ligand. Functional assays revealed that the interaction of PKCα with DLG1 promotes wound healing; scratch assays using cells depleted of PKCα and/or DLG1 have impaired cellular migration that is no longer sensitive to PKC inhibition, and the ability of exogenous PKCα to rescue cellular migration is dependent on the presence of its PDZ ligand. Furthermore, we identified Thr-656 as a novel phosphorylation site in the SH3-Hook region of DLG1 that acts as a marker for PKCα activity at this scaffold. Increased phosphorylation of Thr-656 is correlated with increased invasiveness in non-small cell lung cancer lines from the NCI-60, consistent with this phosphorylation site serving as a marker of PKCα-mediated invasion. Taken together, these data establish the requirement of scaffolding to DLG1 for PKCα to promote cellular migration.     

Ggamma13 interacts with PDZ domain-containing proteins

The G protein gamma13 subunit (Ggamma13) is expressed in taste and retinal and neuronal tissues and plays a key role in taste transduction. We identified PSD95, Veli-2, and other PDZ domain-containing proteins as binding partners for Ggamma13 by yeast two-hybrid and pull-down assays. In two-hybrid assays, Ggamma13 interacted specifically with the third PDZ domain of PSD95, the sole PDZ domain of Veli-2, and the third PDZ domain of SAP97, a PSD95-related protein. Ggamma13 did not interact with the other PDZ domains of PSD95. Coexpression of Ggamma13 with its Gbeta1 partner did not interfere with these two-hybrid interactions. The physical interaction of Ggamma13 with PSD95 in the cellular milieu was confirmed in pull-down assays following heterologous expression in HEK293 cells. The interaction of Ggamma13 with the PDZ domain of PSD95 was via the C-terminal CAAX tail of Ggamma13 (where AA indicates the aliphatic amino acid); alanine substitution of the CTAL sequence at the C terminus of Ggamma13 abolished its interactions with PSD95 in two-hybrid and pull-down assays. Veli-2 and SAP97 were identified in taste tissue and in Ggamma13-expressing taste cells. Coimmunoprecipitation of Ggamma13 and PSD95 from brain and of Ggamma13 and SAP97 from taste tissue indicates that Ggamma13 interacts with these proteins endogenously. This is the first demonstration that PDZ domain proteins interact with heterotrimeric G proteins via the CAAX tail of Ggamma subunits. The interaction of Ggamma13 with PDZ domain-containing proteins may provide a means to target particular Gbetagamma subunits to specific subcellular locations and/or macromolecular complexes involved in signaling pathways.     

Mutations in the DLG3 gene cause nonsyndromic X-linked mental retardation

We have identified truncating mutations in the human DLG3 (neuroendocrine dlg) gene in 4 of 329 families with moderate to severe X-linked mental retardation. DLG3 encodes synapse-associated protein 102 (SAP102), a member of the membrane-associated guanylate kinase protein family. Neuronal SAP102 is expressed during early brain development and is localized to the postsynaptic density of excitatory synapses. It is composed of three amino-terminal PDZ domains, an src homology domain, and a carboxyl-terminal guanylate kinase domain. The PDZ domains interact directly with the NR2 subunits of the NMDA glutamate receptor and with other proteins responsible for NMDA receptor localization, immobilization, and signaling. The mutations identified in this study all introduce premature stop codons within or before the third PDZ domain, and it is likely that this impairs the ability of SAP102 to interact with the NMDA receptor and/or other proteins involved in downstream NMDA receptor signaling pathways. NMDA receptors have been implicated in the induction of certain forms of synaptic plasticity, such as long-term potentiation and long-term depression, and these changes in synaptic efficacy have been proposed as neural mechanisms underlying memory and learning. The disruption of NMDA receptor targeting or signaling, as a result of the loss of SAP102, may lead to altered synaptic plasticity and may explain the intellectual impairment observed in individuals with DLG3 mutations.     

Direct isolation of specific RNA-interacting proteins using a novel affinity medium

Isolation of proteins that specifically interact with a given RNA or RNA regulation element is essential for studies on the molecular mechanisms of gene expression. Here, a novel method for direct isolation of such interacting proteins is described. It uses an affinity medium that consists of an interacting RNA with an artificially added ‘tail’, which is annealed to one end of a DNA ‘arm’, the other end of which is fixed covalently on the surface of aminosilanized glass powder. Thus the RNA itself is fully suspending, facilitating its interactions with proteins in its natural conformation. The proteins bound on the interacting RNA are eluted and subjected to SDS–PAGE, and the Coomassie-stained protein bands are cut and subjected to mass spectrometry (MS) analysis. Using this method, three proteins specifically interacting with the C/EBPβ 3′-untranslated region (3′-UTR) RNA were isolated and identified. This method is simple and convenient, and the DNA-glass powder medium can be used repeatedly.     

Divergent Evolution of a Protein–Protein Interaction Revealed through Ancestral Sequence Reconstruction and Resurrection

The postsynaptic density extends across the postsynaptic dendritic spine with discs large (DLG) as the most abundant scaffolding protein. DLG dynamically alters the structure of the postsynaptic density, thus controlling the function and distribution of specific receptors at the synapse. DLG contains three PDZ domains and one important interaction governing postsynaptic architecture is that between the PDZ3 domain from DLG and a protein called cysteine-rich interactor of PDZ3 (CRIPT). However, little is known regarding functional evolution of the PDZ3:CRIPT interaction. Here, we subjected PDZ3 and CRIPT to ancestral sequence reconstruction, resurrection, and biophysical experiments. We show that the PDZ3:CRIPT interaction is an ancient interaction, which was likely present in the last common ancestor of Eukaryotes, and that high affinity is maintained in most extant animal phyla. However, affinity is low in nematodes and insects, raising questions about the physiological function of the interaction in species from these animal groups. Our findings demonstrate how an apparently established protein–protein interaction involved in cellular scaffolding in bilaterians can suddenly be subject to dynamic evolution including possible loss of function.     

The viral nucleocapsid protein and the human RNA-binding protein Mex3A promote translation of the Andes orthohantavirus small mRNA

The capped Small segment mRNA (SmRNA) of the Andes orthohantavirus (ANDV) lacks a poly(A) tail. In this study, we characterize the mechanism driving ANDV-SmRNA translation. Results show that the ANDV-nucleocapsid protein (ANDV-N) promotes in vitro translation from capped mRNAs without replacing eukaryotic initiation factor (eIF) 4G. Using an RNA affinity chromatography approach followed by mass spectrometry, we identify the human RNA chaperone Mex3A (hMex3A) as a SmRNA-3’UTR binding protein. Results show that hMex3A enhances SmRNA translation in a 3’UTR dependent manner, either alone or when co-expressed with the ANDV-N. The ANDV-N and hMex3A proteins do not interact in cells, but both proteins interact with eIF4G. The hMex3A–eIF4G interaction showed to be independent of ANDV-infection or ANDV-N expression. Together, our observations suggest that translation of the ANDV SmRNA is enhanced by a 5’-3’ end interaction, mediated by both viral and cellular proteins.     

The PDZ protein mupp1 promotes Gi coupling and signaling of the Mt1 melatonin receptor

Intracellular signaling events are often organized around PDZ (PSD-95/Drosophila Disc large/ZO-1 homology) domain-containing scaffolding proteins. The ubiquitously expressed multi-PDZ protein MUPP1, which is composed of 13 PDZ domains, has been shown to interact with multiple viral and cellular proteins and to play important roles in receptor targeting and trafficking. In this study, we show that MUPP1 binds to the G protein-coupled MT(1) melatonin receptor and directly regulates its G(i)-dependent signal transduction. Structural determinants involved in this interaction are the PDZ10 domain of MUPP1 and the valine of the canonical class III PDZ domain binding motif DSV of the MT(1) carboxyl terminus. This high affinity interaction (K(d) approximately 4 nm), which is independent of MT(1) activation, occurs in the ovine pars tuberalis of the pituitary expressing both proteins endogenously. Although the disruption of the MT(1)/MUPP1 interaction has no effect on the subcellular localization, trafficking, or degradation of MT(1), it destabilizes the interaction between MT(1) and G(i) and abolishes G(i)-mediated signaling of MT(1). Our findings highlight a previously unappreciated role of PDZ proteins in promoting G protein coupling to receptors.     

Jointly They Edit: Examining the Impact of Community Identification on Political Interaction in Wikipedia

Background

In their 2005 study, Adamic and Glance coined the memorable phrase ‘divided they blog’, referring to a trend of cyberbalkanization in the political blogosphere, with liberal and conservative blogs tending to link to other blogs with a similar political slant, and not to one another. As political discussion and activity increasingly moves online, the power of framing political discourses is shifting from mass media to social media.

Methodology/Principal Findings

Continued examination of political interactions online is critical, and we extend this line of research by examining the activities of political users within the Wikipedia community. First, we examined how users in Wikipedia choose to display their political affiliation. Next, we analyzed the patterns of cross-party interaction and community participation among those users proclaiming a political affiliation. In contrast to previous analyses of other social media, we did not find strong trends indicating a preference to interact with members of the same political party within the Wikipedia community.

Conclusions/Significance

Our results indicate that users who proclaim their political affiliation within the community tend to proclaim their identity as a ‘Wikipedian’ even more loudly. It seems that the shared identity of ‘being Wikipedian’ may be strong enough to triumph over other potentially divisive facets of personal identity, such as political affiliation.     

Structural Insights into a Wildtype Domain of the Oncoprotein E6 and Its Interaction with a PDZ Domain

The high-risk human papilloma virus (HPV) oncoproteins E6 and E7 interact with key cellular regulators and are etiological agents for tumorigenesis and tumor maintenance in cervical cancer and other malignant conditions. E6 induces degradation of the tumor suppressor p53, activates telomerase and deregulates cell polarity. Analysis of E6 derived from a number of high risk HPV finally yielded the first structure of a wild-type HPV E6 domain (PDB 2M3L) representing the second zinc-binding domain of HPV 51 E6 (termed 51Z2) determined by NMR spectroscopy. The 51Z2 structure provides clues about HPV-type specific structural differences between E6 proteins. The observed temperature sensitivity of the well-folded wild-type E6 domain implies a significant malleability of the oncoprotein in vivo. Hence, the structural differences between individual E6 and their malleability appear, together with HPV type-specific surface exposed side-chains, to provide the structural basis for the different interaction networks reported for individual E6 proteins. Furthermore, the interaction of 51Z2 with a PDZ domain of hDlg was analyzed. Human Dlg constitutes a prototypic representative of the large family of PDZ proteins regulating cell polarity, which are common targets of high-risk HPV E6. Nine C-terminal residues of 51Z2 interact with the second PDZ domain of hDlg2. Surface plasmon resonance in conjunction with the NMR spectroscopy derived complex structure (PDB 2M3M) indicate that E6 residues N-terminal to the canonical PDZ-BM of E6 significantly contribute to this interaction and increase affinity. The structure of the complex reveals how residues outside of the classical PDZ-BM enhance the affinity of E6 towards PDZ domains. Such mechanism facilitates successful competition of E6 with cellular PDZ-binding proteins and may apply to PDZ-binding proteins of other viruses as well.     

Reticulophagy and nucleophagy: New findings and unsolved issues

Autophagy targets various intracellular components ranging from proteins and nucleic acids to organelles for their degradation in lysosomes or vacuoles. In selective types of autophagy, receptor proteins play central roles in target selection. These proteins bind or localize to specific targets, and also interact with Atg8 family proteins on forming autophagosomal membranes, leading to the efficient sequestration of the targets by the membranes. Our recent study revealed that yeast cells actively degrade the endoplasmic reticulum (ER) and even part of the nucleus via selective autophagy under nitrogen-deprived conditions. We identified novel receptors, Atg39 and Atg40, specific to these pathways. Here, we summarize our findings on ‘reticulophagy’ (or ‘ER-phagy’) and ‘nucleophagy’, and discuss key issues that remain to be solved in future studies.     

Different genome stability proteins underpin primed and na?ve adaptation in E. coli CRISPR-Cas immunity

CRISPR-Cas is a prokaryotic immune system built from capture and integration of invader DNA into CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, termed ‘Adaptation’, which is dependent on Cas1 and Cas2 proteins. In Escherichia coli, Cascade-Cas3 degrades invader DNA to effect immunity, termed ‘Interference’. Adaptation can interact with interference (‘primed’), or is independent of it (‘naïve’). We demonstrate that primed adaptation requires the RecG helicase and PriA protein to be present. Genetic analysis of mutant phenotypes suggests that RecG is needed to dissipate R-loops at blocked replication forks. Additionally, we identify that DNA polymerase I is important for both primed and naive adaptation, and that RecB is needed for naïve adaptation. Purified Cas1-Cas2 protein shows specificity for binding to and nicking forked DNA within single strand gaps, and collapsing forks into DNA duplexes. The data suggest that different genome stability systems interact with primed or naïve adaptation when responding to blocked or collapsed invader DNA replication. In this model, RecG and Cas3 proteins respond to invader DNA replication forks that are blocked by Cascade interference, enabling DNA capture. RecBCD targets DNA ends at collapsed forks, enabling DNA capture without interference. DNA polymerase I is proposed to fill DNA gaps during spacer integration.     

A dynamic model for replication protein A (RPA) function in DNA processing pathways

Processing of DNA in replication, repair and recombination pathways in cells of all organisms requires the participation of at least one major single-stranded DNA (ssDNA)-binding protein. This protein protects ssDNA from nucleolytic damage, prevents hairpin formation and blocks DNA reannealing until the processing pathway is successfully completed. Many ssDNA-binding proteins interact physically and functionally with a variety of other DNA processing proteins. These interactions are thought to temporally order and guide the parade of proteins that ‘trade places’ on the ssDNA, a model known as ‘hand-off’, as the processing pathway progresses. How this hand-off mechanism works remains poorly understood. Recent studies of the conserved eukaryotic ssDNA-binding protein replication protein A (RPA) suggest a novel mechanism by which proteins may trade places on ssDNA by binding to RPA and mediating conformation changes that alter the ssDNA-binding properties of RPA. This article reviews the structure and function of RPA, summarizes recent studies of RPA in DNA replication and other DNA processing pathways, and proposes a general model for the role of RPA in protein-mediated hand-off.     

Dynamic Hubs Show Competitive and Static Hubs Non-Competitive Regulation of Their Interaction Partners

Date hub proteins have 1 or 2 interaction interfaces but many interaction partners. This raises the question of whether all partner proteins compete for the interaction interface of the hub or if the cell carefully regulates aspects of this process? Here, we have used real-time rendering of protein interaction networks to analyse the interactions of all the 1 or 2 interface hubs of Saccharomyces cerevisiae during the cell cycle. By integrating previously determined structural and gene expression data, and visually hiding the nodes (proteins) and their edges (interactions) during their troughs of expression, we predict when interactions of hubs and their partners are likely to exist. This revealed that 20 out of all 36 one- or two- interface hubs in the yeast interactome fell within two main groups. The first was dynamic hubs with static partners, which can be considered as ‘competitive hubs’. Their interaction partners will compete for the interaction interface of the hub and the success of any interaction will be dictated by the kinetics of interaction (abundance and affinity) and subcellular localisation. The second was static hubs with dynamic partners, which we term ‘non-competitive hubs’. Regulatory mechanisms are finely tuned to lessen the presence and/or effects of competition between the interaction partners of the hub. It is possible that these regulatory processes may also be used by the cell for the regulation of other, non-cell cycle processes.     

In silico prediction of HIV-1-host molecular interactions and their directionality

Human immunodeficiency virus type 1 (HIV-1) continues to be a major cause of disease and premature death. As with all viruses, HIV-1 exploits a host cell to replicate. Improving our understanding of the molecular interactions between virus and human host proteins is crucial for a mechanistic understanding of virus biology, infection and host antiviral activities. This knowledge will potentially permit the identification of host molecules for targeting by drugs with antiviral properties. Here, we propose a data-driven approach for the analysis and prediction of the HIV-1 interacting proteins (VIPs) with a focus on the directionality of the interaction: host-dependency versus antiviral factors. Using support vector machine learning models and features encompassing genetic, proteomic and network properties, our results reveal some significant differences between the VIPs and non-HIV-1 interacting human proteins (non-VIPs). As assessed by comparison with the HIV-1 infection pathway data in the Reactome database (sensitivity > 90%, threshold = 0.5), we demonstrate these models have good generalization properties. We find that the ‘direction’ of the HIV-1-host molecular interactions is also predictable due to different characteristics of ‘forward’/pro-viral versus ‘backward’/pro-host proteins. Additionally, we infer the previously unknown direction of the interactions between HIV-1 and 1351 human host proteins. A web server for performing predictions is available at http://hivpre.cvr.gla.ac.uk/.     

Plastid Proteomic Analysis in Tomato Fruit Development

To better understand the mechanism of plastid differentiation from chloroplast to chromoplast, we examined proteome and plastid changes over four distinct developmental stages of ‘Micro-Tom’ fruit. Additionally, to discover more about the relationship between fruit color and plastid differentiation, we also analyzed and compared ‘Micro-Tom’ results with those from two other varieties, ‘Black’ and ‘White Beauty’. We confirmed that proteins related to photosynthesis remain through the orange maturity stage of ‘Micro-Tom’, and also learned that thylakoids no longer exist at this stage. These results suggest that at a minimum there are changes in plastid morphology occurring before all related proteins change. We also compared ‘Micro-Tom’ fruits with ‘Black’ and ‘White Beauty’ using two-dimensional gel electrophoresis. We found a decrease of CHRC (plastid-lipid-associated protein) and HrBP1 (harpin binding protein-1) in the ‘Black’ and ‘White Beauty’ varieties. CHRC is involved in carotenoid accumulation and stabilization. HrBP1 in Arabidopsis has a sequence similar to proteins in the PAP/fibrillin family. These proteins have characteristics and functions similar to lipocalin, an example of which is the transport of hydrophobic molecules. We detected spots of TIL (temperature-induced lipocalin) in 2D-PAGE results, however the number of spots and their isoelectric points differed between ‘Micro-Tom’ and ‘Black’/‘White Beauty’. Lipocalin has various functions including those related to environmental stress response, apoptosis induction, membrane formation and fixation, regulation of immune response, cell growth, and metabolism adjustment. Lipocalin related proteins such as TIL and HrBP1 could be related to the accumulation of carotenoids, fruit color and the differentiation of chromoplast.     

How do site-specific DNA-binding proteins find their targets?

Essentially all the biological functions of DNA depend on site-specific DNA-binding proteins finding their targets, and therefore ‘searching’ through megabases of non-target DNA. In this article, we review current understanding of how this sequence searching is done. We review how simple diffusion through solution may be unable to account for the rapid rates of association observed in experiments on some model systems, primarily the Lac repressor. We then present a simplified version of the ‘facilitated diffusion’ model of Berg, Winter and von Hippel, showing how non-specific DNA–protein interactions may account for accelerated targeting, by permitting the protein to sample many binding sites per DNA encounter. We discuss the 1-dimensional ‘sliding’ motion of protein along non-specific DNA, often proposed to be the mechanism of this multiple site sampling, and we discuss the role of short-range diffusive ‘hopping’ motions. We then derive the optimal range of sliding for a few physical situations, including simple models of chromosomes in vivo, showing that a sliding range of ~100 bp before dissociation optimizes targeting in vivo. Going beyond first-order binding kinetics, we discuss how processivity, the interaction of a protein with two or more targets on the same DNA, can reveal the extent of sliding and we review recent experiments studying processivity using the restriction enzyme EcoRV. Finally, we discuss how single molecule techniques might be used to study the dynamics of DNA site-specific targeting of proteins.     

Ribosomal protein L3 functions as a 'rocker switch' to aid in coordinating of large subunit-associated functions in eukaryotes and Archaea

Although ribosomal RNAs (rRNAs) comprise the bulk of the ribosome and carry out its main functions, ribosomal proteins also appear to play important structural and functional roles. Many ribosomal proteins contain long, nonglobular domains that extend deep into the rRNA cores. In eukaryotes and Archaea, ribosomal protein L3 contains two such extended domains tethered to a common globular hub, thus providing an excellent model to address basic questions relating to ribosomal protein structure/function relationships. Previous work in our laboratory identified the central ‘W-finger’ extension of yeast L3 in helping to coordinate ribosomal functions. New studies on the ‘N-terminal’ extension in yeast suggest that it works with the W-finger to coordinate opening and closing of the corridor through which the 3′ end of aa-tRNA moves during the process of accommodation. Additionally, the effect of one of the L3 N-terminal extension mutants on the interaction between C75 of the aa-tRNA and G2921 (Escherichia coli G2553) of 25S rRNA provides the first evidence of the effect of a ribosomal protein on aa-tRNA positioning and peptidyltransfer, possibly through the induced fit model. A model is presented describing how all three domains of L3 may function together as a ‘rocker switch’ to coordinate the stepwise processes of translation elongation.     

Can Natural Proteins Designed with ‘Inverted’ Peptide Sequences Adopt Native-Like Protein Folds?

We have carried out a systematic computational analysis on a representative dataset of proteins of known three-dimensional structure, in order to evaluate whether it would possible to ‘swap’ certain short peptide sequences in naturally occurring proteins with their corresponding ‘inverted’ peptides and generate ‘artificial’ proteins that are predicted to retain native-like protein fold. The analysis of 3,967 representative proteins from the Protein Data Bank revealed 102,677 unique identical inverted peptide sequence pairs that vary in sequence length between 5–12 and 18 amino acid residues. Our analysis illustrates with examples that such ‘artificial’ proteins may be generated by identifying peptides with ‘similar structural environment’ and by using comparative protein modeling and validation studies. Our analysis suggests that natural proteins may be tolerant to accommodating such peptides.