An improved return-mapping scheme for nonsmooth yield surfaces: PART I - the Haigh-Westergaard coordinates

The paper is devoted to the numerical solution of elastoplastic constitutive initial value problems. An improved form of the implicit return-mapping scheme for nonsmooth yield surfaces is proposed that systematically builds on a subdifferential formulation of the flow rule. The main advantage of this approach is that the treatment of singular points, such as apices or edges at which the flow direction is multivalued involves only a uniquely defined set of non-linear equations, similarly to smooth yield surfaces. This paper (PART I) is focused on isotropic models containing: $a)$ yield surfaces with one or two apices (singular points) laying on the hydrostatic axis; $b)$ plastic pseudo-potentials that are independent of the Lode angle; $c)$ nonlinear isotropic hardening (optionally). It is shown that for some models the improved integration scheme also enables to a priori decide about a type of the return and investigate existence, uniqueness and semismoothness of discretized constitutive operators in implicit form. Further, the semismooth Newton method is introduced to solve incremental boundary-value problems. The paper also contains numerical examples related to slope stability with available Matlab implementation.Comment: 25 pages, 10 figure

Structural analysis of PLD3 reveals insights into the mechanism of lysosomal 5′ exonuclease-mediated nucleic acid degradation

The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3

Immunopeptidomics toolkit library (IPTK): a python-based modular toolbox for analyzing immunopeptidomics data

Background The human leukocyte antigen (HLA) proteins play a fundamental role in the adaptive immune system as they present peptides to T cells. Mass-spectrometry-based immunopeptidomics is a promising and powerful tool for characterizing the immunopeptidomic landscape of HLA proteins, that is the peptides presented on HLA proteins. Despite the growing interest in the technology, and the recent rise of immunopeptidomics-specific identification pipelines, there is still a gap in data-analysis and software tools that are specialized in analyzing and visualizing immunopeptidomics data. Results We present the IPTK library which is an open-source Python-based library for analyzing, visualizing, comparing, and integrating different omics layers with the identified peptides for an in-depth characterization of the immunopeptidome. Using different datasets, we illustrate the ability of the library to enrich the result of the identified peptidomes. Also, we demonstrate the utility of the library in developing other software and tools by developing an easy-to-use dashboard that can be used for the interactive analysis of the results. Conclusion IPTK provides a modular and extendable framework for analyzing and integrating immunopeptidomes with different omics layers. The library is deployed into PyPI at https://pypi.org/project/IPTKL/ and into Bioconda at https://anaconda.org/bioconda/iptkl , while the source code of the library and the dashboard, along with the online tutorials are available at https://github.com/ikmb/iptoolkit

MT1-MMP and ADAM10/17 exhibit a remarkable overlap of shedding properties

Membrane-type-I matrix metalloproteinase (MT1-MMP) is one of six human membrane-bound MMPs and is responsible for extracellular matrix remodelling by degrading several substrates like fibrillar collagens, including types I-III, or fibronectin. Moreover, MT1-MMP was described as a key player in cancer progression and it is involved in various inflammatory processes, as well as in the pathogenesis of Alzheimer's disease (AD). The membrane-tethered metalloprotease meprin β as well as a disintegrin and metalloproteinase 10 (ADAM10) and ADAM17 are also associated with these diseases. Interestingly, meprin β, ADAM10/17 and MT1-MMP also have a shared substrate pool including the interleukin-6 receptor and the amyloid precursor protein. We investigated the interaction of these proteases, focusing on a possible connection between MT1-MMP and meprin β, to elucidate the potential mutual regulations of both enzymes. Herein, we show that besides ADAM10/17, MT1-MMP is also able to shed meprin β from the plasma membrane, leading to the release of soluble meprin β. Mass spectrometry-based cleavage site analysis revealed that the cleavage of meprin β by all three proteases occurs between Pro602 and Ser603 , N-terminal of the EGF-like domain. Furthermore, only inactive human pro-meprin β is shed by MT1-MMP, which is again in accordance with the shedding capability observed for ADAM10/17. Vice versa, meprin β also appears to shed MT1-MMP, indicating a complex regulatory network. Further studies will elucidate this well-orchestrated proteolytic web under distinct conditions in health and disease and will possibly show whether the loss of one of the above-mentioned sheddases can be compensated by the other enzymes

Casein proteins: investigating their chaperone activity and amyloid fibril formation.

Molecular chaperones are a diverse group of proteins that interact and stabilise partially folded proteins, thereby preventing improper or incorrect interactions that would result in their misfolding and aggregation under conditions of cellular stress, e.g. elevated temperature. There are two alternative and distinct routes by which the aggregation of the protein may proceed, i.e. via the formation of disordered, amorphous aggregates or ordered amyloid fibrils. The latter is of considerable interest to researchers because of its intimate association (e.g. via the formation of proteinaceous deposits or amyloid plaques) with a wide range of debilitating diseases, including Alzheimer’s disease and type II diabetes. Amyloid-like plaques have also been identified in the mammary gland of various species within calcified stones known as corpora amylacea (CA). While the composition of the protein(s) involved in formation of these amyloid deposits has not been determined conclusively, immunoblotting and sequence analysis of peptides obtained from mammary CA indicate that fragments of several milk proteins, in particular caseins, are present. In vitro studies have shown that αs-, β- and κ-caseins, the major proteins in milk, are molecular chaperones, as they are able to stabilise heat-, light- and chemically-stressed target proteins by inhibiting their aggregation and precipitation. Casein chaperone-like activity is of biological importance since two of the four casein proteins, i.e. αs₂- and κ-casein, assemble into amyloid fibrils under physiological conditions, in vitro, which is inhibited by the chaperone action of the other milk caseins, αs₁- and β-casein. The chaperone-like activity of αS- and β-casein is of commercial interest due to their ability to stabilise other proteins during food processing, e.g. the heat treatment of milk during pasteurisation and the production of milk-related products. The work described in this thesis has two overall aims: (i) to further investigate caseins’ chaperone-like ability and (ii) to examine the propensity of the caseins to form amyloid fibrils. As such, αs- and β-casein, were dephosphorylated to determine the effect of phosphate groups on the ability of these caseins to act as molecular chaperones. Dephosphorylation of αs- and β-casein resulted in a decrease in the chaperone efficiency against both heat- and reduction-induced amorphously aggregating target proteins. Circular dichroism and fluorescence spectroscopic data indicated that the loss of negative charge associated with dephosphorylation led to an increase in ordered structure of αs- and β-casein (Chapter 2). The binding site of β-casein with reduced, partially folded α-lactalbumin, a milk whey protein, was explored using limited proteolysis and mass spectrometry to give insight into the mechanism of β-casein chaperone interaction with target proteins. It was concluded that the hydrophobic C-terminus of β-casein, from Ala⁹¹ to Trp¹ ⁴³, is involved in binding to reduced α-lactalbumin (Chapter 3). Amyloid fibrils were formed from reduced and carboxymethylated κ-casein and αs2-casein, and the amyloidogenic regions of both these proteins were identified using limited proteolysis and mass spectrometry. The residues from Tyr²⁵-Lys⁸⁶ and Ala⁸ ¹ -Lys1¹ ⁸ ¹ were determined to be incorporated into the core of κ-casein and αs2-casein fibrils respectively (Chapter 4). The oxidation of methionine residues is linked to the pathogenesis of several amyloid diseases. As such, the two methionine residues in κ-casein (Met-95 and Met-106) were oxidised and its effect on κ-casein structure and fibril-formation was investigated. Oxidation increased κ- casein’s fibril forming propensity and cellular toxicity. In addition, β-casein, which readily inhibits κ-casein fibril-formation in vitro, was less effective at suppressing fibril formation of oxidised κ-casein. As milk exists in an oxidative environment, this observation may have implications in vivo (Chapter 5).Thesis (Ph.D.) -- University of Adelaide, School of Chemistry and Physics, 201

Dephosphorylation of αs- and β -Caseins and Its Effect on Chaperone Activity: A Structural and Functional Investigation

Milk casein proteins can act as molecular chaperones: under conditions of stress, such as elevated temperature, molecular chaperones stabilize proteins from unfolding, aggregating, and precipitating. In this study, αS- and β-caseins were dephosphorylat

The use of rejuvenators as an effective way to restore aged binder properties

The amount of Reclaimed Asphalt recycling using rejuvenating agents has increased recently due to environmental and economic constraints. Rejuvenating agents are now regularly used to restore the aged binder properties similarly to those of a virgin binder. The current European specifications for asphalt paving bitumen use only empirical testing. Such procedure is appearing not sufficient to address the effect of rejuvenating agents. More advanced rheological analysis using DSR and BBR equipment appears promising. Parameters such as viscous to elastic transition (VET) from DSR or the difference between the creep stiffness and m-value critical temperature (ΔTCR) from BBR provide apparent indication of a binder’s flexibility and ability to relax stresses in cases of rejuvenation. This paper investigates the effect of three various rejuvenators on aged binder behaviour and shows that the rejuvenation process has a large impact on the material properties. Particularly rheological measurements demonstrated that as the aging process progresses the variance in rejuvenated binder behaviour increases especially in terms of performance at intermediate (VET) and low temperatures (ΔTCR)

C‑Terminal Charge-Reversal Derivatization and Parallel Use of Multiple Proteases Facilitates Identification of Protein C‑Termini by C‑Terminomics

The identification of protein C-termini in complex proteomes is challenging due to the poor ionization efficiency of the carboxyl group. Amidating the negatively charged C-termini with ethanolamine (EA) has been suggested to improve the detection of C-terminal peptides and allows for a directed depletion of internal peptides after proteolysis using carboxyl reactive polymers. In the present study, the derivatization with <i>N,N</i>-dimethylethylenediamine (DMEDA) and (4-aminobutyl)­guanidine (AG) leading to a positively charged C-terminus was investigated. C-terminal charge-reversed peptides showed improved coverage of b- and y-ion series in the MS/MS spectra compared to their noncharged counterparts. DMEDA-derivatized peptides resulted in many peptides with charge states of 3+, which benefited from ETD fragmentation. This makes the charge-reversal strategy particularly useful for the analysis of protein C-termini, which may also be post-translationally modified. The labeling strategy and the indirect enrichment of C-termini worked with similar efficiency for both DMEDA and EA, and their applicability was demonstrated on an E. coli proteome. Utilizing two proteases and different MS/MS activation mechanisms allowed for the identification of >400 C-termini, encompassing both canonical and truncated C-termini

Diazirine-functionalized mannosides for photoaffinity labeling : trouble with FimH

Photoaffinity labeling is frequently employed for the investigation of ligand-receptor interactions in solution. We have employed an interdisciplinary methodology to achieve facile photolabeling of the lectin FimH, which is a bacterial protein, crucial for adhesion, colonization and infection. Following our earlier work, we have here designed and synthesized diazirine-functionalized mannosides as high-affinity FimH ligands and performed an extensive study on photo-crosslinking of the best ligand (mannoside 3) with a series of model peptides and FimH. Notably, we have employed high-performance mass spectrometry to be able to detect radiation results with the highest possible accuracy. We are concluding from this study that photolabeling of FimH with sugar diazirines has only very limited success and cannot be regarded a facile approach for covalent modification of FimH