Truncation Of Domain Research Articles

Quantitative Ubiquitylome Analysis Reveals the Specificity of RNF111/Arkadia E3 Ubiquitin Ligase for its Degradative Substrates SKI and SKIL/SnoN in TGF-β Signaling Pathway

RNF111/Arkadia is an E3 ubiquitin ligase that activates the transforming growth factor-β (TGF-β) pathway by degrading transcriptional repressors SKIL/SnoN and SKI. Truncations of the RING C-terminal domain of RNF111 that abolish its E3 function and subsequently activate TGF-β signaling are observed in some cancers. In the present study, we sought to perform a comprehensive analysis of RNF111 endogenous substrates upon TGF-β signaling activation using an integrative proteomic approach. In that aim, we carried out label-free quantitative proteomics after the enrichment of ubiquitylated proteins (ubiquitylome) in parental U2OS cell line compared with U2OS CRISPR engineered clones expressing a truncated form of RNF111 devoid of its C-terminal RING domain. We compared two methods of enrichment for ubiquitylated proteins before proteomics analysis by mass spectrometry, the diGlycine (diGly) remnant peptide immunoprecipitation with a K-ε-GG antibody, and a novel approach using protein immunoprecipitation with a ubiquitin pan nanobody that recognizes all ubiquitin chains and monoubiquitylation. Although we detected SKIL ubiquitylation among 108 potential RNF111 substrates with the diGly method, we found that the ubiquitin pan nanobody method also constitutes a powerful approach because it enabled the detection of 52 potential RNF111 substrates including SKI, SKIL, and RNF111. Integrative comparison of the RNF111-dependent proteome and ubiquitylomes enabled the identification of SKI and SKIL as the only targets ubiquitylated and degraded by RNF111 E3 ligase function in the presence of TGF-β. Our results indicate that lysine 343 localized in the SAND domain of SKIL constitutes a target for RNF111 ubiquitylation and demonstrate that RNF111 E3 ubiquitin ligase function specifically targets SKI and SKIL ubiquitylation and degradation upon TGF-β pathway activation.

Recombinant expression and purification of pentameric ligand-gated ion channels for Cryo-EM structural studies.

Pentameric ligand-gated ion channels (pLGICs) are central players in synaptic neurotransmission and are targets to a range of drugs used to treat neurological disorders and pain. pLGICs are intrinsically dynamic membrane proteins that upon stimulation by neurotransmitters, undergo global conformational changes across multiple domains spanning a distance of over 165Å. The inter-domain flexibility, a feature crucial for their function as signal transducers in chemical synapses, has been problematic in the efforts toward determining high-resolution structures. Earlier structural studies tackled this issue with a variety of strategies that included partial truncation of flexible domains and the use of antibodies and small-molecule inhibitors to restrict domain movement. With the recent advances in cryo-electron microscopy and single-particle analysis, many of these limitations have been overcome. Here, we describe the methods used in the recombinant expression and purification of full-length constructs of two members of the pentameric ligand-gated ion channel family and the approaches used for capturing multiple conformations in cryo-EM imaging.

Accurate Spectral Collocation Computation of High Order Eigenvalues for Singular Schrödinger Equations

We are concerned with the study of some classical spectral collocation methods, mainly Chebyshev and sinc as well as with the new software system Chebfun in computing high order eigenpairs of singular and regular Schrödinger eigenproblems. We want to highlight both the qualities as well as the shortcomings of these methods and evaluate them in conjunction with the usual ones. In order to resolve a boundary singularity, we use Chebfun with domain truncation. Although it is applicable with spectral collocation, a special technique to introduce boundary conditions as well as a coordinate transform, which maps an unbounded domain to a finite one, are the special ingredients. A challenging set of “hard”benchmark problems, for which usual numerical methods (f. d., f. e. m., shooting, etc.) fail, were analyzed. In order to separate “good”and “bad”eigenvalues, we have estimated the drift of the set of eigenvalues of interest with respect to the order of approximation and/or scaling of domain parameter. It automatically provides us with a measure of the error within which the eigenvalues are computed and a hint on numerical stability. We pay a particular attention to problems with almost multiple eigenvalues as well as to problems with a mixed spectrum.

Tuning of adsorption of enzymes to polymer.

In the past years, several serine hydrolases such as cutinases, esterases and lipases have shown the ability to degrade not only natural polymers but also synthetic polyesters, even aromatic representatives like polyethylene terephthalate (PET). Hence, cutinases and related ester hydrolases have become very important to be applied in the biocatalytic plastic recycling as green alternative to chemical recycling as well as to the functionalization of polyester surfaces in order to change superficial properties like hydrophobicity or hydrophilicity. Sorption characteristics of the enzymes to the polymers have turned out to be a crucial process for efficient polymer hydrolysis. Hence, special attention was paid on tuning the sorption of the enzymes to the hydrophobic polymers. Engineering of the enzyme surface, fusion of hydrophobic substrate-binding domains or truncation of domains hindering the access of the polymer to the enzyme has led to significant improvement of sorption processes and consequently increased activity on the bulky substrate. Finally, the combination of engineering approaches has proved that they can bring additional advantages in improving the enzyme activity when used in a synergistic manner.

Clamping, bending, and twisting inter-domain motions in the misfold-recognizing portion of UDP-glucose: Glycoprotein glucosyltransferase

SummaryUDP-glucose:glycoprotein glucosyltransferase (UGGT) flags misfolded glycoproteins for ER retention. We report crystal structures of full-length Chaetomium thermophilum UGGT (CtUGGT), two CtUGGT double-cysteine mutants, and its TRXL2 domain truncation (CtUGGT-ΔTRXL2). CtUGGT molecular dynamics (MD) simulations capture extended conformations and reveal clamping, bending, and twisting inter-domain movements. We name “Parodi limit” the maximum distance on the same glycoprotein between a site of misfolding and an N-linked glycan that can be reglucosylated by monomeric UGGT in vitro, in response to recognition of misfold at that site. Based on the MD simulations, we estimate the Parodi limit as around 70–80 Å. Frequency distributions of distances between glycoprotein residues and their closest N-linked glycosylation sites in glycoprotein crystal structures suggests relevance of the Parodi limit to UGGT activity in vivo. Our data support a “one-size-fits-all adjustable spanner” UGGT substrate recognition model, with an essential role for the UGGT TRXL2 domain.

Yeast Nucleolin Nsr1 Impedes Replication and Elevates Genome Instability at an Actively Transcribed Guanine-Rich G4 DNA-Forming Sequence.

A significant increase in genome instability is associated with the conformational shift of a guanine-run-containing DNA strand into the four-stranded G-quadruplex (G4) DNA. The mechanism underlying the recombination and genome rearrangements following the formation of G4 DNA in vivo has been difficult to elucidate but has become better clarified by the identification and functional characterization of several key G4 DNA-binding proteins. Mammalian nucleolin (NCL) is a highly specific G4 DNA-binding protein with a well-defined role in the transcriptional regulation of genes with associated G4 DNA-forming sequence motifs at their promoters. The consequence of the in vivo interaction between G4 DNA and nucleolin in respect to the genome instability has not been previously investigated. We show here that the yeast nucleolin Nsr1 is enriched at a G4 DNA-forming sequence in vivo and is a major factor in inducing the genome instability associated with the cotranscriptionally formed G4 DNA in the yeast genome. We also show that Nsr1 results in impeding replication past such a G4 DNA-forming sequence. The G4-associated genome instability and the G4 DNA-binding in vivo require the arginine-glycine-glycine (RGG) repeats located at the C-terminus of the Nsr1 protein. Nsr1 with the deletion of RGG domain supports normal cell growth and is sufficient for its pre-rRNA processing function. However, the truncation of the RGG domain of Nsr1 significantly weakens its interaction with G4 DNA in vivo and restores unhindered replication, overall resulting in a sharp reduction in the genome instability associated with a guanine-rich G4 DNA-forming sequence. Our data suggest that the interaction between Nsr1 with the intact RGG repeats and G4 DNA impairs genome stability by precluding the access of G4-resolving proteins and impeding replication.

Epidermal growth factor receptor association with β1-adrenergic receptor is mediated via its juxtamembrane domain

β1-adrenergic receptor (β1AR)-mediated transactivation of epidermal growth factor receptor (EGFR) engages downstream signaling events that impact numerous cellular processes including growth and survival. While association of these receptors has been shown to occur basally and be important for relaying transactivation-specific intracellular events, the mechanism by which they do so is unclear and elucidation of which would aid in understanding the consequence of disrupting their interaction. Using fluorescence resonance energy transfer (FRET) and immunoprecipitation (IP) analyses, we evaluated the impact of C-terminal truncations of EGFR on its ability to associate with β1AR. While loss of the last 230 amino acid C-terminal phosphotyrosine-rich domain did not disrupt the ability of EGFR to associate with β1AR, truncation of the entire intracellular domain of EGFR resulted in almost complete loss of its interaction with β1AR, suggesting that either the kinase domain or juxtamembrane domain (JMD) may be required for this association. Treatment with the EGFR antagonist gefitinib did not prevent β1AR-EGFR association, however, treatment with a palmitoylated peptide encoding the first 20 amino acids of the JMD domain (JMD-A) disrupted β1AR-EGFR association over time and prevented β1AR-mediated ERK1/2 phosphorylation, both in general and specifically in association with EGFR. Conversely, neither a mutated JMD-A peptide nor a palmitoylated peptide fragment consisting of the subsequent 18 amino acids of the JMD domain (JMD-B) were capable of doing so. Altogether, the proximal region of the JMD of EGFR is responsible for its association with β1AR, and its disruption prevents β1AR-mediated transactivation, thus providing a new tool to study the functional consequences of disrupting β1AR-EGFR downstream signaling.

CSIG-10. GENOTYPE – KINOME GUIDED DEVELOPMENT OF PRECISION EGFR-TARGETED THERAPEUTICS FOR GLIOBLASTOMA

Abstract Glioblastoma (GBM) is an aggressive primary brain tumor with poor survival and limited treatment options. However, it is an attractive candidate for precision therapeutic approaches due to the frequency of amplification and/or activating mutations in the epidermal growth factor receptor (EGFR) gene and the availability of several brain penetrant second- and third-generation EGFR tyrosine kinase inhibitors (TKI). We used comprehensive molecular profiling of a panel of genetically engineered mouse astrocyte models to examine whether mutational profiles, particularly EGFR and PTEN status, could be used to identify kinases upregulated in specific mutational backgrounds. Using RNA-seq and multiplex inhibitor bead/mass spectrometry (MIB-MS) to analyze the kinase transcriptomes and proteomes, respectively, we have identified several potential targets for combination therapy. Overexpression of wild type EGFR in immortalized, Cdkn2a-/- astrocytes resulted in mild rewiring of the GBM kinome. Only 5 kinases aside from EGFR itself were overexpressed on either the transcript or protein levels. One overexpressed kinase, Hck, has been shown to be involved in cell survival, proliferation, adhesion, and migration. In contrast, overexpression of EGFRvIII, a constitutively active, extracellular domain truncation mutant of EGFR, resulted in significant alteration of the GBM kinome – 81 kinases showed differential expression, with 27 upregulated. One potentially attractive target among these was Cdk6, a drug-targetable, prognostically significant cyclin-dependent kinase implicated in proliferation, migration, and invasion. Finally, overexpression of EGFRvIII in cells lacking Pten dysregulated 46 kinases, including 15 upregulated. One particularly interesting target in these cells was Ddr2, a tyrosine kinase involved in migration, invasion, and extracellular matrix remodeling. We conclude that Hck, Cdk6, and Ddr2 represent attractive targets for therapeutic intervention in their relevant genetic contexts. These findings also suggest that molecular diagnostics for EGFR and PTEN status may be useful in guiding development of rational, EGFR TKI-centric drug combinations.

Zrsr2 Deficient Zebrafish Display Hematopoietic Defects and U12-Type Intron Retention in mRNA Processing Genes

ZRSR2 is a small nuclear riboprotein necessary for assembly of the minor spliceosome and subsequent splicing of U12-type introns. It is involved in the recognition of 3' splice sites during the assembly of the spliceosome. Somatic mutations in ZRSR2 gene are present in hematopoietic malignancies, most notably myelodysplastic syndrome (MDS). These mutations are spread throughout the gene, indicating that its function is critical for hematopoiesis. In spite of these clinical associations, the role of ZRSR2 in hematopoiesis has not been well studied. Zebrafish make an excellent animal model for investigating this gene as all of the functional domains in the human protein are conserved with a high level of protein similarity in zebrafish. Additionally, hematopoietic development is well understood in this species. We used CRISPR/Cas9 to generate a zebrafish zrsr2 knockout model with an 11 base pair deletion (zrsr2Δ11) that results in frameshift with premature truncation and loss of all functional domains (p.W167fs*175). The zrsr2Δ11/Δ11 mutants began to appear morphologically different from their siblings by 4 days post fertilization (dpf), showing aberrant development in the mandible and pharyngeal arches. The zrsr2Δ11/Δ11 mutants developed mild to severe edema by 6 dpf and died by 8 dpf. To understand its role in hematopoiesis, we performed o-dianisidine staining and whole mount in situ hybridization (WISH) with lineage-specific markers during embryonic development. Our data showed that zrsr2Δ11/Δ11 embryos exhibit mild anemia by 2 dpf, suggesting that primitive hematopoiesis is defective. WISH showed that zrsr2Δ11/Δ11 mutants have normal early hematopoietic stem cell (HSC) emergence (c-myb) at 36 hours post fertilization. However, these cells are absent through 3 and 5 dpf. At 5 dpf zrsr2Δ11/Δ11 mutants had severely reduced expression of hematopoietic markers for erythroid (hbae1), lymphoid (rag1), and myeloid (mpo) lineages. This verifies that knockout of zrsr2 in zebrafish disrupted normal hematopoiesis and leads to cytopenias - emulating the symptoms of MDS. We then investigated the transcriptional and splicing changes underlying these phenotypes using RNA sequencing on zrsr2Δ11/Δ11 mutant embryos at 3dpf. We found a total of 285 intron retention events in zrsr2Δ11/Δ11 mutants as compared to the control embryos, with about 76% of those events occurring within U12-type introns. Genes with retained introns were associated with mRNA decay and destabilization. We also found that genes related to cell cycle arrest were upregulated in zrsr2Δ11/Δ11 mutants . These results indicate that aberrant mRNA splicing and cell cycle deregulation contribute to arrested hematopoiesis when zrsr2 is knocked out in zebrafish. Disclosures No relevant conflicts of interest to declare.

Semi-implicit Hermite–Galerkin Spectral Method for Distributed-Order Fractional-in-Space Nonlinear Reaction–Diffusion Equations in Multidimensional Unbounded Domains

In this paper, we construct an efficient Hermite–Galerkin spectral method for the nonlinear reaction–diffusion equations with distributed-order fractional Laplacian in multidimensional unbounded domains. By applying Gauss–Legendre quadrature rule for the distributed integral term, we first approximate the original distributed-order fractional problem by the multi-term fractional-in-space differential equation. Applying Hermite–Galerkin spectral method in space and backward difference method in time, we establish semi-implicit fully discrete scheme. For two- and three-dimensional cases of the original fractional problem, the linear systems are solved by the preconditioned conjugate gradients method. The main advantage of our method is that the original fractional problem is directly solved in the unbounded domains, thus avoiding the errors introduced by the domain truncations. The stability analysis is rigourously established, which shows that our scheme is unconditionally stable under suitable assumption on the nonlinear term. Several numerical examples are presented to validate both stability and accuracy of the numerical method. The numerical results of the fractional Allen–Cahn, Gray–Scott, and Belousov–Zhabotinskii models show that our semi-implicit methods produce good numerical solutions.

A nonlocal boundary condition for domain truncation in frequency-domain Helmholtz problems

Numerical resolution of exterior Helmholtz problems requires some approach to domain truncation. As an alternative to approximate nonreflecting boundary conditions and invocation of the Dirichlet-to-Neumann map, we introduce a new, nonlocal boundary condition. This condition is exact and requires the evaluation of layer potentials involving the free space Green's function. However, it seems towork in general unstructured geometry, and Galerkin finite element discretization leads to convergence under some mesh constraintsimposed by Garding-type inequalities. We will sketch this method, show how we have integrated Firedrake with pytential (a fast-multipole code), and discuss what issues this raises for extending Firedrake to work with more general nonlocal operators.

Complete radiation boundary conditions for acoustic waves

The radiation of energy to the far field is a central feature of acoustics. As such, efficient, convergent domain truncation algorithms are a necessary component of any software for simulating acoustic waves in the time domain. Complete radiation boundary conditions are, in our view, an ideal solution to this problem. In particular, they are provably spectrally convergent, depending on parameters which can be chosen automatically to guarantee any required accuracy; as they directly approximate both propagating and evanescent modes, the computational boundary can be placed arbitrarily close to scatterers or other inhomogeneities. In this talk we will outline the theory behind the method for scattering and waveguide problems with a homogeneous far field, discuss the simple implementation of the method via the solution of a coupled system wave equations in a thin double absorbing boundary layer, and consider its application in more complex settings including advective acoustics, stratified media, and random media. Numerical experiments in three space dimensions using high-order methods will be shown.

A comparison between off and on-body control surfaces for the FW-H equation: Application to a non-compact landing gear wheel

This work is a contribution to the ongoing debate about the role of quadrupoles in low Mach number flows, studied through the use of solid and permeable surface Ffowcs-Williams & Hawkings (FW-H) integrals for landing gear numerical noise predictions. It rests upon the key idea that the dominance of surface sources over volume sources can only be guaranteed when the compact-source condition is met. We propose here to express this property as a condition on the smallness of the product between the Mach and Strouhal numbers, or formally as MSt < 1. We consider a canonical isolated wheel, that basically consists in a shallow circular cavity inscribed in a coin-like cylinder, thus presenting few different length scales, as compared to a full landing gear assembly. Zonal Detached Eddy Simulations are performed on several gradually refined grids to assess the grid convergence of the numerical result. In particular, important computational effort is put into the accurate resolution of acoustic waves up to Strouhal numbers such that MSt > 1. Overall, determining the real significance of quadrupoles was challenging as numerical errors or misleading effects such as near-field terms, surface discretization, or source domain truncation biased the initial permeable surface results. Even when these sources of bias are removed, non-negligible differences are found between the solid and permeable noise spectra at non-compact St values, while both formulations are equivalent at lower Strouhal numbers. The analysis of these differences is extended to the determination of their impact on frequency-domain noise maps obtained with the DAMAS algorithm fed with far-field signals computed with both FW-H approaches. A tentative interpretation of the wheel noise sources is finally proposed, highlighting the dominant role of the scattering of aerodynamic sources by the cavity downstream edge at low Strouhal numbers, while wake sources dominate at higher Strouhal numbers.

The rice NLR pair Pikp-1/Pikp-2 initiates cell death through receptor cooperation rather than negative regulation.

Plant NLR immune receptors are multidomain proteins that can function as specialized sensor/helper pairs. Paired NLR immune receptors are generally thought to function via negative regulation, where one NLR represses the activity of the second and detection of pathogen effectors relieves this repression to initiate immunity. However, whether this mechanism is common to all NLR pairs is not known. Here, we show that the rice NLR pair Pikp-1/Pikp-2, which confers resistance to strains of the blast pathogen Magnaporthe oryzae (syn. Pyricularia oryzae) expressing the AVR-PikD effector, functions via receptor cooperation, with effector-triggered activation requiring both NLRs to trigger the immune response. To investigate the mechanism of Pikp-1/Pikp-2 activation, we expressed truncated variants of these proteins, and made mutations in previously identified NLR sequence motifs. We found that any domain truncation, in either Pikp-1 or Pikp-2, prevented cell death in the presence of AVR-PikD, revealing that all domains are required for activity. Further, expression of individual Pikp-1 or Pikp-2 domains did not result in cell death. Mutations in the conserved P-loop and MHD sequence motifs in both Pikp-1 and Pikp-2 prevented cell death activation, demonstrating that these motifs are required for the function of the two partner NLRs. Finally, we showed that Pikp-1 and Pikp-2 associate to form homo- and hetero-complexes in planta in the absence of AVR-PikD; on co-expression the effector binds to Pikp-1 generating a tri-partite complex. Taken together, we provide evidence that Pikp-1 and Pikp-2 form a fine-tuned system that is activated by AVR-PikD via receptor cooperation rather than negative regulation.

Efficient calculation of transient eddy current response from multi-layer cylindrical conductive media.

The transient response from a transmitter-receiver coil system inside a multi-layer cylindrical conductive configuration is obtained. The particular set-up applies to well logging as well as to eddy current tube testing. In this work, a number of improvements are presented to existing models for an efficient calculation of the induced voltage. These include: domain truncation, novel treatment of arbitrary number of layers in order to avoid computational overflows and efficient time response calculation. The latter is based on a combination of Laplace inversion techniques for short- and long-time transient responses. This article is part of the theme issue 'Advanced electromagnetic non-destructive evaluation and smart monitoring'.

Research Note: A workaround for the corner problem in numerically exact non‐reflecting boundary conditions

ABSTRACTSimulations of wave propagation in the Earth usually require truncation of a larger domain to the region of interest to keep computational cost acceptable. This introduces artificial boundaries that should not generate reflected waves. Most existing boundary conditions are not able to completely suppress all the reflected energy, but suffice in practice except when modelling subtle events such as interbed multiples. Exact boundary conditions promise better performance but are usually formulated in terms of the governing wave equation and, after discretization, still may produce unwanted artefacts. Numerically exact non‐reflecting boundary conditions are instead formulated in terms of the discretized wave equation. They have the property that the numerical solution computed on a given domain is the same as one on a domain enlarged to the extent that waves reflected from the boundary do not have the time to reach the original truncated domain. With a second‐ or higher‐order finite‐difference scheme for the one‐dimensional wave equation, these boundary conditions follow from a recurrence relation. In its generalization to two or three dimensions, a recurrence relation was only found for a single non‐reflecting boundary on one side of the domain or two of them at opposing ends. The other boundaries should then be zero Dirichlet or Neumann. If two non‐reflecting boundaries meet at a corner, translation invariance is lost and a simple recurrence relation could not be found.Here, a workaround is presented that restores translation invariance by imposing classic, approximately non‐reflecting boundary conditions on the other sides and numerically exact ones on the two opposing sides that otherwise would create the strongest reflected waves with the classic condition. The exact ones can also be applied independently. As a proof of principle, the method is applied to the two‐dimensional acoustic wave equation, discretized on a rectangular domain with a second‐order finite‐difference scheme and first‐order Enquist–Majda boundary conditions as approximate ones. The method is computationally costly but has the advantage that it can be reused on a sequence of problems as long as the time step and the sound speed values next to the boundary are kept fixed.

YejM Controls LpxC Levels by Regulating Protease Activity of the FtsH/YciM Complex of Escherichia coli.

LpxC is a deacetylase that catalyzes the first committed step of lipid A biosynthesis in Escherichia coli LpxC competes for a common precursor, R-3-hydroxymyristoyl-UDP-GlcNAc, with FabZ, whose dehydratase activity catalyzes the first committed step of phospholipid biosynthesis. To maintain the optimum flow of the common precursor to these two competing pathways, the LpxC level is controlled by FtsH/YciM-mediated proteolysis. It is not known whether this complex or another protein senses the status of lipid A synthesis to control LpxC proteolysis. The work carried out in this study began with a novel mutation, yejM1163, which causes hypersensitivity to large antibiotics such as vancomycin and erythromycin. Isolates resistant to these antibiotics carried suppressor mutations in the ftsH and yciM genes. Western blot analysis showed a dramatically reduced LpxC level in the yejM1163 background, while the presence of ftsH or yciM suppressor mutations restored LpxC levels to different degrees. Based on these observations, it is proposed that YejM is a sensor of lipid A synthesis and controls LpxC levels by modulating the activity of the FtsH/YciM complex. The truncation of the periplasmic domain in the YejM1163 protein causes unregulated proteolysis of LpxC, thus diverting a greater pool of R-3-hydroxymyristoyl-UDP-GlcNAc toward phospholipid synthesis. This imbalance in lipid synthesis perturbs the outer membrane permeability barrier, causing hypersensitivity toward vancomycin and erythromycin. yejM1163 suppressor mutations in ftsH and yciM lower the proteolytic activity toward LpxC, thus restoring lipid homeostasis and the outer membrane permeability barrier.IMPORTANCE Lipid homeostasis is critical for proper envelope functions. The level of LpxC, which catalyzes the first committed step of lipopolysaccharide (LPS) synthesis, is controlled by an essential protease complex comprised of FtsH and YciM. Work carried out here suggests YejM, an essential envelope protein, plays a central role in sensing the state of LPS synthesis and controls LpxC levels by regulating the activity of FtsH/YciM. All four essential proteins are attractive targets of therapeutic development.

Hsp22 with an N-Terminal Domain Truncation Mediates a Reduction in Tau Protein Levels.

Misfolding, aggregation and accumulation of proteins are toxic elements in the progression of a broad range of neurodegenerative diseases. Molecular chaperones enable a cellular defense by reducing or compartmentalizing these insults. Small heat shock proteins (sHsps) engage proteins early in the process of misfolding and can facilitate their proper folding or refolding, sequestration, or clearance. Here, we evaluate the effects of the sHsp Hsp22, as well as a pseudophosphorylated mutant and an N-terminal domain deletion (NTDΔ) variant on tau aggregation in vitro and tau accumulation and aggregation in cultured cells. Hsp22 wild-type (WT) protein had a significant inhibitory effect on heparin-induced aggregation in vitro and the pseudophosphorylated mutant Hsp22 demonstrated a similar effect. When co-expressed in a cell culture model with tau, these Hsp22 constructs significantly reduced soluble tau protein levels when transfected at a high ratio relative to tau. However, the Hsp22 NTDΔ protein drastically reduced the soluble protein expression levels of both tau WT and tau P301L/S320F even at lower transfection ratios, which resulted in a correlative reduction of the triton-insoluble tau P301L/S320F aggregates.

Wide-angle mode parabolic equations for the modelling of horizontal refraction in underwater acoustics and their numerical solution on unbounded domains

The modelling of sound propagation in the ocean by the solution of mode parabolic equations is discussed. Mode parabolic equations can be obtained as the one-way approximation to horizontal refraction equations for modal amplitudes. Their wide-angle capabilities depend on the order of the Padé approximation of the involved pseudo-differential operators. Various aspects of numerical solution methods for wide-angle mode parabolic equations are considered in detail, including artificial domain truncation and Cauchy initial data for the point source field approximation. The capabilities of the discussed numerical approaches are demonstrated in several important test cases, including the problems of sound propagation in a penetrable wedge and in a sea with an underwater canyon.

Elements of the Endomucin Extracellular Domain Essential for VEGF-Induced VEGFR2 Activity.

Endomucin (EMCN) is the type I transmembrane glycoprotein, mucin-like component of the endothelial cell glycocalyx. We have previously shown that EMCN is necessary for vascular endothelial growth factor (VEGF)-induced VEGF receptor 2 (VEGFR2) internalization and downstream signaling. To explore the structural components of EMCN that are necessary for its function and the molecular mechanism of EMCN in VEGF-induced endothelial functions, we generated a series of mouse EMCN truncation mutants and examined their ability to rescue VEGF-induced endothelial functions in human primary endothelial cells (EC) in which endogenous EMCN had been knocked down using siRNA. Expression of the mouse full-length EMCN (FL EMCN) and the extracellular domain truncation mutants ∆21-81 EMCN and ∆21-121 EMCN, but not the shortest mutant ∆21-161 EMCN, successfully rescued the VEGF-induced EC migration, tube formation, and proliferation. ∆21-161 EMCN failed to interact with VEGFR2 and did not facilitate VEGFR2 internalization. Deletion of COSMC (C1GalT1C1) revealed that the abundant mucin-type O-glycans were not required for its VEGFR2-related functions. Mutation of the two N-glycosylation sites on ∆21-121 EMCN abolished its interaction with VEGFR2 and its function in VEGFR2 internalization. These results reveal ∆21-121 EMCN as the minimal extracellular domain sufficient for VEGFR2-mediated endothelial function and demonstrate an important role for N-glycosylation in VEGFR2 interaction, internalization, and angiogenic activity.