The BAR domain proteins: Molding membranes in fission, fusion, and phagy

The Bin1/amphiphysin/Rvs167 (BAR) domain proteins are a ubiquitous protein family. Genes encoding members of this family have not yet been found in the genomes of prokaryotes, but within eukaryotes, BAR domain proteins are found universally from unicellular eukaryotes such as yeast through to plants, insects, and vertebrates. BAR domain proteins share an N-terminal BAR domain with a high propensity to adopt alpha-helical structure and engage in coiled-coil interactions with other proteins. BAR domain proteins are implicated in processes as fundamental and diverse as fission of synaptic vesicles, cell polarity, endocytosis, regulation of the actin cytoskeleton, transcriptional repression, cell-cell fusion, signal transduction, apoptosis, secretory vesicle fusion, excitation-contraction coupling, learning and memory, tissue differentiation, ion flux across membranes, and tumor suppression. What has been lacking is a molecular understanding of the role of the BAR domain protein in each process. The three-dimensional structure of the BAR domain has now been determined and valuable insight has been gained in understanding the interactions of BAR domains with membranes. The cellular roles of BAR domain proteins, characterized over the past decade in cells as distinct as yeasts, neurons, and myocytes, can now be understood in terms of a fundamental molecular function of all BAR domain proteins: to sense membrane curvature, to bind GTPases, and to mold a diversity of cellular membranes.

The β domain is required for Vps4p oligomerization into a functionally active ATPase

Endocytic and biosynthetic trafficking pathways to the lysosome/vacuole converge at the prevacuolar endosomal compartment. During transport through this compartment, integral membrane proteins that are destined for delivery to the lysosome/vacuole lumen undergo multivesicular body (MVB) sorting into internal vesicles formed by invagination of the endosomal limiting membrane. Vps4 is an AAA family ATPase which plays a key role in MVB sorting and facilitates transport through endosomes. It possesses an N-terminal microtubule interacting and trafficking domain required for recruitment to endosomes and an AAA domain with an ATPase catalytic site. The recently solved 3D structure revealed a P domain, which protrudes from the AAA domain, and a final C-terminal alpha-helix. However, the in vivo roles of these domains are not known. In this study, we have identified motifs in these domains that are highly conserved between yeast and human Vps4. We have mutated these motifs and studied the effect on yeast Vps4p function in vivo and in vitro. We show that the P domain of the budding yeast Vps4p is not required for recruitment to endosomes, but is essential for all Vps4p endocytic functions in vivo. We also show that the P domain is required for Vps4p homotypic interaction and for full ATPase activity. In addition, it is required for interaction with Vta1p, which works in concert with Vps4p in vivo. Our studies suggest that assembly of a Vps4p oligomeric complex with full ATPase activity that interacts with Vta1p is essential for normal endosome function.

Structure and dynamics of ASC2, a pyrin domain-only protein that regulates inflammatory signaling

Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain ( DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-KB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone N-15 NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding alpha 2-alpha 3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the alpha 2-alpha 3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the alpha 2-alpha 3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions.

Effect of site-specific mutations in different phosphotransfer domains of the chemosensory protein ChpA on Pseudomonas aeruginosa motility

The virulence of Pseudomonas aeruginosa and other surface pathogens involves the coordinate expression of a wide range of virulence determinants, including type IV pili. These surface filaments are important for the colonization of host epithelial tissues and mediate bacterial attachment to, and translocation across, surfaces by a process known as twitching motility. This process is controlled in part by a complex signal transduction system whose central component, ChpA, possesses nine potential sites of phosphorylation, including six histidine-containing phosphotransfer (HPt) domains, one serine-containing phosphotransfer domain, one threonine-containing phosphotransfer domain, and one CheY-like receiver domain. Here, using site-directed mutagenesis, we show that normal twitching motility is entirely dependent on the CheY-like receiver domain and partially dependent on two of the HPt domains. Moreover, under different assay conditions, point mutations in several of the phosphotransfer domains of ChpA give rise to unusual "swarming" phenotypes, possibly reflecting more subtle perturbations in the control of P. aeruginosa motility that are not evident from the conventional twitching stab assay. Together, these results suggest that ChpA plays a central role in the complex regulation of type IV pilus-mediated motility in P. aeruginosa

How does your model grow? An empirical investigation of the effects of ontological clarity and application domain size on query performance

Semantic data models provide a map of the components of an information system. The characteristics of these models affect their usefulness for various tasks (e.g., information retrieval). The quality of information retrieval has obvious important consequences, both economic and otherwise. Traditionally, data base designers have produced parsimonious logical data models. In spite of their increased size, ontologically clearer conceptual models have been shown to facilitate better performance for both problem solving and information retrieval tasks in experimental settings. The experiments producing evidence of enhanced performance for ontologically clearer models have, however, used application domains of modest size. Data models in organizational settings are likely to be substantially larger than those used in these experiments. This research used an experiment to investigate whether the benefits of improved information retrieval performance associated with ontologically clearer models are robust as the size of the application domains increase. The experiment used an application domain of approximately twice the size as tested in prior experiments. The results indicate that, relative to the users of the parsimonious implementation, end users of the ontologically clearer implementation made significantly more semantic errors, took significantly more time to compose their queries, and were significantly less confident in the accuracy of their queries.

The DHR1 domain of DOCK180 binds to SNX5 and regulates cation-independent mannose 6-phosphate receptor transport

DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.

Identifying a novel functional domain within the p115 tethering factor required for Golgi ribbon assembly and membrane trafficking

The tethering factor p115 has been shown to facilitate Golgi biogenesis and membrane traffic in cells in culture. However, the role of p115 within an intact animal is largely unknown. Here, we document that RNAi-mediated depletion of p115 in C. elegans causes accumulation of the yolk protein (YP170) in body cavity and the retention of the yolk receptor RME-2 in the ER and the Golgi within oocytes.Structure-function analyses of p115 have identified two homology (H1-2) regions within the N-terminal globular head and the coiled-coil 1 (CC1) domain as essential for p115 function. We identify a novel C-terminal domain of p115 as necessary for Golgi ribbon formation and cargo trafficking. We show that p115 mutants lacking the fourth CC domain (CC4) act in a dominant negative manner to disrupt Golgi and prevent cargo trafficking in cells containing endogenous p115. Furthermore, using RNAi-mediated "replacement" strategy we show that CC4 is necessary for Golgi ribbon formation and membrane trafficking in cells depleted of endogenous p115.p115 has been shown to bind a subset of ER-Golgi SNAREs through CC1 and CC4 domains (Shorter et al., 2002). Our findings show that CC4 is required for p115 function and suggest that both the CC1 and the CC4 SNARE-binding motifs may participate in p115-mediated membrane tethering.

Determining the temperature from Cauchy data in corner domains

We consider the problem of reconstruction of the temperature from knowledge of the temperature and heat flux on a part of the boundary of a bounded planar domain containing corner points. An iterative method is proposed involving the solution of mixed boundary value problems for the heat equation (with time-dependent conductivity). These mixed problems are shown to be well-posed in a weighted Sobolev space.

On an indirect integral equation approach for stationary heat transfer in semi-infinite layered domains in R3 with cavities

Regions containing internal boundaries such as composite materials arise in many applications.We consider a situation of a layered domain in IR3 containing a nite number of bounded cavities. The model is stationary heat transfer given by the Laplace equation with piecewise constant conductivity. The heat ux (a Neumann condition) is imposed on the bottom of the layered region and various boundary conditions are imposed on the cavities. The usual transmission (interface) conditions are satised at the interface layer, that is continuity of the solution and its normal derivative. To eciently calculate the stationary temperature eld in the semi-innite region, we employ a Green's matrix technique and reduce the problem to boundary integral equations (weakly singular) over the bounded surfaces of the cavities. For the numerical solution of these integral equations, we use Wienert's approach [20]. Assuming that each cavity is homeomorphic with the unit sphere, a fully discrete projection method with super-algebraic convergence order is proposed. A proof of an error estimate for the approximation is given as well. Numerical examples are presented that further highlights the eciency and accuracy of the proposed method.

The multiple pheromone ant clustering algorithm and its application to real world domains

The Multiple Pheromone Ant Clustering Algorithm (MPACA) models the collective behaviour of ants to find clusters in data and to assign objects to the most appropriate class. It is an ant colony optimisation approach that uses pheromones to mark paths linking objects that are similar and potentially members of the same cluster or class. Its novelty is in the way it uses separate pheromones for each descriptive attribute of the object rather than a single pheromone representing the whole object. Ants that encounter other ants frequently enough can combine the attribute values they are detecting, which enables the MPACA to learn influential variable interactions. This paper applies the model to real-world data from two domains. One is logistics, focusing on resource allocation rather than the more traditional vehicle-routing problem. The other is mental-health risk assessment. The task for the MPACA in each domain was to predict class membership where the classes for the logistics domain were the levels of demand on haulage company resources and the mental-health classes were levels of suicide risk. Results on these noisy real-world data were promising, demonstrating the ability of the MPACA to find patterns in the data with accuracy comparable to more traditional linear regression models. © 2013 Polish Information Processing Society.

A procedure for the temperature reconstruction in corner domains from Cauchy data

An iterative procedure is proposed for the reconstruction of a stationary temperature field from Cauchy data given on a part of the boundary of a bounded plane domain where the boundary is smooth except for a finite number of corner points. In each step, a series of mixed well-posed boundary value problems are solved for the heat operator and its adjoint. Convergence is proved in a weighted L2-space. Numerical results are included which show that the procedure gives accurate and stable approximations in relatively few iterations.

Large-scale molecular dynamics simulations of HLA-A*0201 complexed with a tumor-specific antigenic peptide:can the α3 and β2m domains be neglected?

Large-scale massively parallel molecular dynamics (MD) simulations of the human class I major histo-compatibility complex (MHC) protein HLA-A*0201 bound to a decameric tumor-specific antigenic peptide GVY-DGREHTV were performed using a scalable MD code on high-performance computing platforms. Such computational capabilities put us in reach of simulations of various scales and complexities. The supercomputing resources available Large-scale massively parallel molecular dynamics (MD) simulations of the human class I major histocompatibility complex (MHC) protein HLA-A*0201 bound to a decameric tumor-specific antigenic peptide GVYDGREHTV were performed using a scalable MD code on high-performance computing platforms. Such computational capabilities put us in reach of simulations of various scales and complexities. The supercomputing resources available for this study allow us to compare directly differences in the behavior of very large molecular models; in this case, the entire extracellular portion of the peptide–MHC complex vs. the isolated peptide binding domain. Comparison of the results from the partial and the whole system simulations indicates that the peptide is less tightly bound in the partial system than in the whole system. From a detailed study of conformations, solvent-accessible surface area, the nature of the water network structure, and the binding energies, we conclude that, when considering the conformation of the α1–α2 domain, the α3 and β2m domains cannot be neglected. © 2004 Wiley Periodicals, Inc. J Comput Chem 25: 1803–1813, 2004

A Domain-Independent Otology-based Approach to Representation of Courseware Knowledge

This paper proposes an ontology-based approach to representation of courseware knowledge in different domains. The focus is on a three-level semantic graph, modeling respectively the course as a whole, its structure, and domain contents itself. The authors plan to use this representation for flexibie e- learning and generation of different study plans for the learners.

Domain Modeling to Support Anti-cyber Crime Education

This paper describes an approach to a computer-based learning of educational material. We define a model for the class of subjects of our interest - teaching of investigation and prevention of computer crimes, (those including both theoretical and practical issues). From this model, specific content outlines can be derived as subclasses and then instanced into actual domains. The last step consists in generating interactive documents, which use the instanced domain. Students can explore these documents through a web browser. Thus, an interactive learning scenario is created. This approach allows reusing and adapting the contents to a variety of situations, students and teaching purposes.

When does a Bounded Domain Cover a Projective Manifold? (Survey)

* The research has been partially supported by Bulgarian Funding Organizations, sponsoring the Algebra Section of the Mathematics Institute, Bulgarian Academy of Sciences, a Contract between the Humboldt Univestit¨at and the University of Sofia, and Grant MM 412 / 94 from the Bulgarian Board of Education and Technology