Well-structured Domains Research Articles

KDM6A mutations promote acute cytoplasmic DNA release, DNA damage response and mitosis defects

BackgroundKDM6A, encoding a histone demethylase, is one of the top ten mutated epigenetic cancer genes. The effect of mutations on its structure and function are however poorly characterized.MethodsDatabase search identified nonsense and missense mutations in the N-terminal TPR motifs and the C-terminal, catalytic JmjC domain, but also in the intrinsically disordered region connecting both these two well-structured domains. KDM6A variants with cancer-derived mutations were generated using site directed mutagenesis and fused to eGFP serving as an all-in-one affinity and fluorescence tag to study demethylase activity by an ELISA-based assay in vitro, apoptosis by FACS, complex assembly by Co-immunoprecipitation and localization by microscopy in urothelial cells and apoptosis by FACS.ResultsIndependent of the mutation and demethylase activity, all KDM6A variants were detectable in the nucleus. Truncated KDM6A variants displayed changes in complex assemblies affecting (1) known interactions with the COMPASS complex component RBBP5 and (2) KDM6A-DNA associated assemblies with the nuclear protein Nucleophosmin. Some KDM6A variants induced a severe cellular phenotype characterized by multiple acute effects on nuclear integrity, namely, release of nuclear DNA into the cytoplasm, increased level of DNA damage indicators RAD51 and p-γH2A.X, and mitosis defects. These damaging effects were correlated with increased cell death.ConclusionThese observations reveal novel effects of pathogenic variants pointing at new specific functions of KDM6A variants. The underlying mechanisms and affected pathways have to be investigated in future research to understand how tumor cells cope with and benefit from KDM6A truncations.

Quantitative live-cell imaging and 3D modeling reveal critical functional features in the cytosolic complex of phagocyte NADPH oxidase

Phagocyte NADPH oxidase produces superoxide anions, a precursor of reactive oxygen species (ROS) critical for host responses to microbial infections. However, uncontrolled ROS production contributes to inflammation, making NADPH oxidase a major drug target. It consists of two membranous (Nox2 and p22phox) and three cytosolic subunits (p40phox, p47phox, and p67phox) that undergo structural changes during enzyme activation. Unraveling the interactions between these subunits and the resulting conformation of the complex could shed light on NADPH oxidase regulation and help identify inhibition sites. However, the structures and the interactions of flexible proteins comprising several well-structured domains connected by intrinsically disordered protein segments are difficult to investigate by conventional techniques such as X-ray crystallography, NMR, or cryo-EM. Here, we developed an analytical strategy based on FRET-fluorescence lifetime imaging (FLIM) and fluorescence cross-correlation spectroscopy (FCCS) to structurally and quantitatively characterize NADPH oxidase in live cells. We characterized the inter- and intramolecular interactions of its cytosolic subunits by elucidating their conformation, stoichiometry, interacting fraction, and affinities in live cells. Our results revealed that the three subunits have a 1:1:1 stoichiometry and that nearly 100% of them are present in complexes in living cells. Furthermore, combining FRET data with small-angle X-ray scattering (SAXS) models and published crystal structures of isolated domains and subunits, we built a 3D model of the entire cytosolic complex. The model disclosed an elongated complex containing a flexible hinge separating two domains ideally positioned at one end of the complex and critical for oxidase activation and interactions with membrane components.

Cognitive Load Theory and the Use of Worked Examples as an Instructional Strategy in Physics for Distance Learners: A Preliminary Study

This article revisits the cognitive load theory to explore the use of worked examples to teach a selected topic in a higher level undergraduate physics course for distance learners at the School of Distance Education, Universiti Sains Malaysia. With a break of several years from receiving formal education and having only minimum science background, distance learners need an appropriate instructional strategy for courses that require complex conceptualization and mathematical manipulations. As the working memory is limited, distance learners need to acquire domain specific knowledge in stages to lessen cognitive load. This article charts a learning task with a lower cognitive load to teach Fermi-Dirac distribution and demonstrates the use of sequential worked examples. Content taught in stages using worked examples can be presented as a form of didactic conversation to reduce transactional distance. This instructional strategy can be applied to similar challenging topics in other well-structured domains in a distance learning environment.

The Hsp70 interdomain linker is a dynamic switch that enables allosteric communication between two structured domains

Hsp70 molecular chaperones play key roles in cellular protein homeostasis by binding to exposed hydrophobic regions of incompletely folded or aggregated proteins. This crucial Hsp70 function relies on allosteric communication between two well-structured domains: an N-terminal nucleotide-binding domain (NBD) and a C-terminal substrate-binding domain (SBD), which are tethered by an interdomain linker. ATP or ADP binding to the NBD alters the substrate-binding affinity of the SBD, triggering functionally essential cycles of substrate binding and release. The interdomain linker is a well-structured participant in the interdomain interface in ATP-bound Hsp70s. By contrast, in the ADP-bound state, exemplified by the Escherichia coli Hsp70 DnaK, the interdomain linker is flexible. Hsp70 interdomain linker sequences are highly conserved; moreover, mutations in this region compromise interdomain allostery. To better understand the role of this region in Hsp70 allostery, we used molecular dynamics simulations to explore the conformational landscape of the interdomain linker in ADP-bound DnaK and supported our simulations by strategic experimental data. We found that while the interdomain linker samples many conformations, it behaves as three relatively ordered segments connected by hinges. As a consequence, the distances and orientations between the NBD and SBD are limited. Additionally, the C-terminal region of the linker forms previously unreported, transient interactions with the SBD, and the predominant linker-docking site is available in only one allosteric state, that with high affinity for substrate. This preferential binding implicates the interdomain linker as a dynamic allosteric switch. The linker-binding site on the SBD is a potential target for small molecule modulators of the Hsp70 allosteric cycle.

NMR Spectroscopic and Bioinformatic Analyses of the LTBP1 C-Terminus Reveal a Highly Dynamic Domain Organisation

Proteins from the LTBP/fibrillin family perform key structural and functional roles in connective tissues. LTBP1 forms the large latent complex with TGFβ and its propeptide LAP, and sequesters the latent growth factor to the extracellular matrix. Bioinformatics studies suggest the main structural features of the LTBP1 C-terminus are conserved through evolution. NMR studies were carried out on three overlapping C-terminal fragments of LTBP1, comprising four domains with characterised homologues, cbEGF14, TB3, EGF3 and cbEGF15, and three regions with no homology to known structures. The NMR data reveal that the four domains adopt canonical folds, but largely lack the interdomain interactions observed with homologous fibrillin domains; the exception is the EGF3-cbEGF15 domain pair which has a well-defined interdomain interface. 15N relaxation studies further demonstrate that the three interdomain regions act as flexible linkers, allowing a wide range of motion between the well-structured domains. This work is consistent with the LTBP1 C-terminus adopting a flexible “knotted rope” structure, which may facilitate cell matrix interactions, and the accessibility to proteases or other factors that could contribute to TGFβ activation.

Providing sufficient opportunity to learn: a response to Grehaigne, Caty and Godbout

Background: Over the last 30 years, traditional skill-based game teaching models have gradually been supplemented by instruction under an inclusive banner of ‘Teaching Games for Understanding’ (TGfU). This approach focuses on developing tactical understanding through modified games and a philosophy that places the learner rather than the game at the centre of instruction. A recent paper by Grehaigne, Caty, and Godbout, ‘Modelling Ball Circulation in Invasion Team Sports: A Way to Promote Learning Games Through Understanding’, had a threefold focus.• to report the results of a qualitative study on various offensive configurations of football play observed in Physical Education (PE) lessons with young novice players• to propose a model of game play based on the analysis of such configurations of play• to promote a radical constructivist teaching approach based on ‘learning games through understanding’ that challenges the long-established TGfU methodologyPurpose: This paper critically examines the contention of Grehaigne, Caty, and Godbout that the presentation of tactical data collected through the observation of novices playing sport in ill-structured domains, e.g. team games such as football, represent a useful pedagogical model that promotes ‘learning games through understanding’. In rewording the familiar TGfU approach, and calling it ‘learning games through understanding’, Grehaigne, Caty, and Godbout challenge the evolving TGfU approach as too solutions based and not sufficiently student centred.Discussion: This paper challenges the use of radical constructivism as a construct for the development of a philosophy for instructing novices in team games, in this instance, football. It defines ill-structured and well-structured learning domains and suggests that effective pedagogy, the art of teaching, requires flexible attitudes towards the choice of pedagogy in games. By inference, it also challenges Grehaigne, Caty, and Godbout's assumption that the TGfU models previously published, e.g. TGfU, Game Sense, Play Practice, and interpreted in various texts, e.g. Transforming Play, Teaching Tactics and Game Sense, are not student-centred and teacher dominated.Conclusions: Grehaigne, Caty, and Godbout's work is important in the evolution of models of game instruction for use in PE contexts. The concept of ‘learning games through understanding’ is a timely reminder of the importance of pedagogies, for example, guided or discovery learning that induce effortful thinking. However, if we consider TGfU in its true philosophical light, that is, as a holistic and experiential approach to teaching, then it already encompasses ‘learning games through understanding’. Because no two students learn or conceive knowledge in exactly the same way, teaching contexts require a flexible approach to instruction, based on a methodological continuum of empirical to radical constructivism. In short, providing novice learners with sufficient opportunities to learn, requires flexibility and a holistic experiential approach to teaching that is appropriate for the learner, activity and context.

A Single Disulfide Bond Disruption in the β3 Integrin Subunit Promotes Thiol/Disulfide Exchange, a Molecular Dynamics Study

The integrins are a family of membrane receptors that attach a cell to its surrounding and play a crucial function in cell signaling. The combination of internal and external stimuli alters a folded non-active state of these proteins to an extended active configuration. The β3 subunit of the platelet αIIbβ3 integrin is made of well-structured domains rich in disulfide bonds. During the activation process some of the disulfides are re-shuffled by a mechanism requiring partial reduction of some of these bonds; any disruption in this mechanism can lead to inherent blood clotting diseases. In the present study we employed Molecular Dynamics simulations for tracing the sequence of structural fluctuations initiated by a single cysteine mutation in the β3 subunit of the receptor. These simulations showed that in-silico protein mutants exhibit major conformational deformations leading to possible disulfide exchange reactions. We suggest that any mutation that prevents Cys560 from reacting with one of the Cys567–Cys581 bonded pair, thus disrupting its ability to participate in a disulfide exchange reaction, will damage the activation mechanism of the integrin. This suggestion is in full agreement with previously published experiments. Furthermore, we suggest that rearrangement of disulfide bonds could be a part of a natural cascade of thiol/disulfide exchange reactions in the αIIbβ3 integrin, which are essential for the native activation process.

Ensemble Structure of the Modular and Flexible Full-Length Vesicular Stomatitis Virus Phosphoprotein

The phosphoprotein (P) is an essential component of the viral replication machinery of non-segmented negative-strand RNA viruses, connecting the viral polymerase to its nucleoprotein-RNA template and acting as a chaperone of the nucleoprotein by preventing nonspecific encapsidation of cellular RNAs. The phosphoprotein of vesicular stomatitis virus (VSV) forms homodimers and possesses a modular organization comprising two stable, well-structured domains concatenated with two intrinsically disordered regions. Here, we used a combination of nuclear magnetic resonance spectroscopy and small-angle X-ray scattering to depict VSV P as an ensemble of continuously exchanging conformers that captures the dynamic character of this protein. We discuss the implications of the dynamics and the large conformational space sampled by VSV P in the assembly and functioning of the viral transcription/replication machinery.

Molecular dynamics studies of the nucleoprotein of influenza A virus: role of the protein flexibility in RNA binding.

The influenza viruses contain a segmented, negative stranded RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP). X-ray structures have shown that NP contains well-structured domains juxtaposed with regions of missing electron densities corresponding to loops. In this study, we tested if these flexible loops gated or promoted RNA binding and RNA-induced oligomerization of NP. We first performed molecular dynamics simulations of wt NP monomer and trimer in comparison with the R361A protein mutated in the RNA binding groove, using the H1N1 NP as the initial structure. Calculation of the root-mean-square fluctuations highlighted the presence of two flexible loops in NP trimer: loop 1 (73–90), loop 2 (200–214). In NP, loops 1 and 2 formed a 10–15 Å-wide pinch giving access to the RNA binding groove. Loop 1 was stabilized by interactions with K113 of the adjacent β-sheet 1 (91–112) that interacted with the RNA grove (linker 360–373) via multiple hydrophobic contacts. In R361A, a salt bridge formed between E80 of loop 1 and R208 of loop 2 driven by hydrophobic contacts between L79 and W207, due to a decreased flexibility of loop 2 and loop 1 unfolding. Thus, RNA could not access its binding groove in R361A; accordingly, R361A had a much lower affinity for RNA than NP. Disruption of the E80-R208 interaction in the triple mutant R361A-E80A-E81A increased its RNA binding affinity and restored its oligomerization back to wt levels in contrast with impaired levels of R361A. Our data suggest that the flexibility of loops 1 and 2 is required for RNA sampling and binding which likely involve conformational change(s) of the nucleoprotein.

The orientation of the C-terminal domain of the Saccharomyces cerevisiae Rap1 protein is determined by its binding to DNA.

Rap1 is an essential DNA-binding factor from the yeast Saccharomyces cerevisiae involved in transcription and telomere maintenance. Its binding to DNA targets Rap1 at particular loci, and may optimize its ability to form functional macromolecular assemblies. It is a modular protein, rich in large potentially unfolded regions, and comprising BRCT, Myb and RCT well-structured domains. Here, we present the architectures of Rap1 and a Rap1/DNA complex, built through a step-by-step integration of small angle X-ray scattering, X-ray crystallography and nuclear magnetic resonance data. Our results reveal Rap1 structural adjustment upon DNA binding that involves a specific orientation of the C-terminal (RCT) domain with regard to the DNA binding domain (DBD). Crystal structure of DBD in complex with a long DNA identifies an essential wrapping loop, which constrains the orientation of the RCT and affects Rap1 affinity to DNA. Based on our structural information, we propose a model for Rap1 assembly at telomere.

Hybrid model for learner modelling and feedback prioritisation in exploratory learning

Individual and/or hybrid AI techniques are often used in learning environments for well-structured domains to perform learner diagnosis, create and update a learner model and provide support at individual or group level. This paper presents a concept

Learning how to use a computer-based concept-mapping tool: Self-explaining examples helps

In initial skill acquisition in well-structured domains, example-based learning typically leads to better learning outcomes than learning by doing. Cognitive Load Theory explains this result by the worked-example effect: Example-based learning prevents learners from using load-intensive strategies and focuses their attention on the principles to-be-learned. In two experiments, we investigated the use of examples for acquiring a new learning strategy, namely computer-based concept mapping. Experiment 1 compared learners who studied two examples on how to construct a concept map with learners who practiced concept mapping by constructing two concept maps on their own. We did not find significant differences in learning outcomes. Therefore, in Experiment 2, we introduced a third group of learners who studied examples with the additional support of self-explanation prompts. Self-explaining examples led to better learning outcomes than learning with examples without prompts or practicing. With respect to cognitive load, we found that examples without prompts released learners’ working memory compared to practicing, whereas self-explaining examples led to a higher cognitive load compared to examples without self-explanation.

Finding and fixing errors in worked examples: Can this foster learning outcomes?

Learning from worked examples is an effective learning method in well-structured domains. Can its effectiveness be further enhanced when errors are included? This was tested by determining whether a combination of correct and incorrect solutions in worked examples enhances learning outcomes in comparison to correct solutions only, and whether a mixture of correct and incorrect solutions is more effective when the errors are highlighted. In addition, the effectiveness of fostering self-explanations was assessed. In Experiment 1, the participants learned to solve probability problems under six conditions that constituted a 2 × 3-factorial design (Factor 1: correct and incorrect solutions with highlighting the errors vs. correct and incorrect solutions without highlighting the errors vs. correct solutions only; Factor 2: prompting written self-explanations vs. no prompts). An aptitude-treatment interaction was found: providing correct and incorrect solutions fostered far transfer performance if learners had favourable prior knowledge; if learners had poor prior knowledge correct solutions only were more favourable. Experiment 2 replicated this interaction effect. Thus, a mixture of correct and incorrect solutions in worked examples enhanced learning outcomes only for “good” learners. In addition, Experiment 2 showed that confronting learners with incorrect solutions changed the quality of their self-explanations: on the one hand, new types of effective self-explanations could be observed, but on the other hand the amount of the very important principle-based self-explanations was substantially reduced. A possible measure to prevent this negative side effect of incorrect solutions is discussed.

Learning argumentation skills through the use of prompts for self-explaining examples.

Learning with self-explaining examples is an effective method in well-structured domains. The authors analyzed this method in teaching the complex skill of argumentation, experimentally comparing 4 conditions (N = 71 student teachers) that differed with respect to whether and how the processing of the examples was supported by self-explanation prompts. They found that examples of argumentation could be successfully used to teach declarative knowledge about argumentation. However, when the skill of argumentation is to be fostered, prompts that direct the learners' attention to the principles of argumentation should be used. The authors demonstrate that learning with self-explaining examples is also a promising method of enhancing skills in ill-structured domains such as argumentation.

Transitioning From Studying Examples to Solving Problems: Effects of Self-Explanation Prompts and Fading Worked-Out Steps.

Although research has demonstrated that successively fading or successively removing more and more worked-out solution steps as learners transition from relying on examples to independent problem solving reliably fosters performance on near-transfer tasks—relative to example–problem pairs—this effect is not reliable on far-transfer tasks. To address this, the authors combined fading with the introduction of prompts designed to encourage learners to identify the underlying principle illustrated in each worked-out solution step. Across 2 experiments, this combination produced medium to large effects on near and far transfer without requiring additional time on task. Thus, the instructional procedure is highly recommendable because it (a) is relatively straightforward to implement, (b) does not prolong learning time, and (c) fosters both near- and far-transfer performance. Worked-out examples typically consist of a problem formulation, solution steps, and the final answer itself. Research indicates that exposure to worked-out examples is critical when learners are in the initial stages of learning a new cognitive skill in wellstructured domains such as mathematics, physics, and computer programming (Anderson, Fincham, & Douglass, 1997). Moreover, studies performed by Sweller and his colleagues (e.g., Sweller &

Learning mathematics from worked-out examples: Analyzing and fostering self-explanations

Recent research has shown that learning from worked-out examples is of major importance for initial skill acquisition in well-structured domains such as mathematics. However, only those learners who actively process the presented examples profit noticeably from this learning mode. Specifically, the learning outcomes depend on how well the learners explain the solution steps presented in the examples to themselves (‘self-explanation effect”). In a series of studies on learning mathematics from examples, learners’ spontaneous self-explanations and instructional means used to encourage self-explanations were investigated. In this research, the following main findings were obtained. Most learners were rather passive with respect to their spontaneous self-explanations. Among the active and successful learners, two subgroups employing different self-explanation styles could be identified. With regard to the instructional means used to induce effective example processing, it turned out that to employ “learning by teaching” in order to stimulate explanation activities was of very limited use. Attempts to directly train for or elicit certain types of self-explanations were more successful. However, even in the latter case, self-explanations had inherent deficits (e.g., proneness to errors). Thus, we sought to design learning arrangements that try to integrate self-explanations with well-timed and well-adapted instructional explanations (e.g., from tutors) in order to enhance students’ problem-solving skills.

Extending the Rule Space Methodology to a Semantically-Rich Domain: Diagnostic Assessment in Architecture

This paper presents a technique for applying the Rule Space methodology of cognitive diagnosis to assessment in a semantically-rich domain. Previous applications of Rule Space—all in simple, well-structured domains—based diagnosis on examinees’ ability to perform individual problem-solving steps. In a complex domain, however, test items might be so different from one another that the problem-solving steps used for one item are unrelated to the steps used to solve another item. The technique presented herein extends Rule Space’s applicability by basing diagnosis on item characteristics that are more abstract than individual problem-solving steps. A cognitive model of problem-solving motivates selection of characteristics in order to maintain the connection between an examinee’s problem-solving skill and his/her diagnosis. To test the extended Rule Space procedure, data were collected from 122 architects of three ability levels (students, architecture interns, and professional architects) on a 22-item test of architectural knowledge. Rule Space provided diagnostic reporting for between 40 and 90% of examinees. The findings support the effectiveness of Rule Space in a complex domain.

An internal ribosomal entry mechanism promotes translation of murine leukemia virus gag polyprotein precursors.

The genomic retroviral RNA is the messenger for the translation of the gag and pol genes encoding the precursors to the major structural proteins and enzymes, respectively, of the virion core. The long 5' untranslated region, the leader, is formed of independent well-structured domains involved in key steps of the viral life cycle such as the initiation of proviral DNA synthesis, genomic RNA dimerization and packaging, and the initiation of gag translation. These functional features and the presence of stable secondary structures between the cap and the gag initiation codon suggested that translation initiation of gag might proceed through a mechanism different from the canonical ribosome scanning process. Interestingly enough, murine leukemia viruses code also for a glycosylated gag precursor, named glyco-gag, initiated at a CUG codon upstream and in the same open reading frame as the AUGgag. We have investigated the translation initiation of gag and glyco-gag precursors of Friend murine leukemia virus (F-MLV) in the rabbit reticulocyte lysate system and in murine cells. Through site-directed mutagenesis of gag and glyco-gag initiation codons, we show that initiation of gag and glyco-gag synthesis does not utilize the classical ribosome scanning. When poliovirus protease 2A is coexpressed in murine cells, expression of MLV-lacZ RNA is not modified, indicating that translation initiation of MLV gag precursors is a cap-independent mechanism. In addition, the F-MLV leader was inserted between two genes in a dicistronic neo-MLV-lacZ mRNA, and its ability to promote expression was examined in vitro and in vivo. Results obtained demonstrate that an internal ribosome entry mechanism promotes translation of F-MLV gag precursors. This finding led us to construct a new dicistronic retroviral vector in which the F-MLV leader can promote both packaging of recombinant genomic RNA and expression of the 3' gene.