Characterization of the REC114-MEI4-IHO1 complex regulating meiotic DNA double-strand break formation.

Meiotic recombination is initiated by the formation of DNA double-strand breaks (DSBs), essential for fertility and genetic diversity. In the mouse, DSBs are formed by the catalytic TOPOVIL complex consisting of SPO11 and TOPOVIBL. To preserve genome integrity, the activity of the TOPOVIL complex is finely controlled by several meiotic factors including REC114, MEI4, and IHO1, but the underlying mechanism is poorly understood. Here, we report that mouse REC114 forms homodimers, that it associates with MEI4 as a 2:1 heterotrimer that further dimerizes, and that IHO1 forms coiled-coil-based tetramers. Using AlphaFold2 modeling combined with biochemical characterization, we uncovered the molecular details of these assemblies. Finally, we show that IHO1 directly interacts with the PH domain of REC114 by recognizing the same surface as TOPOVIBL and another meiotic factor ANKRD31. These results provide strong evidence for the existence of a ternary IHO1-REC114-MEI4 complex and suggest that REC114 could act as a potential regulatory platform mediating mutually exclusive interactions with several partners.

Selenite reduced uptake/translocation of cadmium via regulation of assembles and interactions of pectins, hemicelluloses, lignins, callose and Casparian strips in rice roots.

Selenium (Se) can reduce cadmium (Cd) uptake/translocation via regulating pectins, hemicelluloses and lignins of plant root cell walls, but the detailed molecular mechanisms are not clear. In this study, six hydroponic experiments were set up to explore the relationships of uptake/translocation inhibition of Cd by selenite (Se(IV)) with cell wall component (CWC) synthesis and/or interactions. Cd and Se was supplied (alone or combinedly) at 1.0 mg L-1 and 0.5 mg L-1, respectively, with the treatment without Cd and Se as the control. When compared to the Cd1 treatment, the Se0.5Cd1 treatment 1) significantly increased total sugar concentrations in pectins, hemicelluloses and callose, suggesting an enhanced capacity of binding Cd or blocking Cd translocation; 2) stimulated the deposition of Casparian strips (CS) in root endodermis and exodermis to block Cd translocation; 3) stimulated the release of C-O-C (-OH- or -O-) and CO (carboxyl, carbonyl, or amide) to combine Cd; 4) regulated differential expression genes (DEGs) and metabolites (DMs) correlated with synthesis and/or interactions of CWSs to affect cell wall net structure to affect root cell division, subsequent root morphology and finally elemental uptake; and 5) stimulated de-methylesterification of pectins via reducing expression abundances of many DMs and DEGs in the Yang Cycle to reduce supply of methyls to homogalacturonan, and regulated gene expressions of pectin methylesterase to release carboxyls to combine Cd; and 6) down-regulated gene expressions associated with Cd uptake/translocation.

Effect of Active Coatings Containing Lippa citriodora Kunth. Essential Oil on Bacterial Diversity and Myofibrillar Proteins Degradation in Refrigerated Large Yellow Croaker.

The research evaluated the effects of locust bean gum (LBG) and sodium alginate (SA) active coatings containing 0.15, 0.30 or 0.60% lemon verbena (Lippa citriodora Kunth.) essential oil (LVEO) on the bacterial diversity and myofibrillar proteins (MPs) of large yellow croaker during refrigerated storage at 4 °C for 18 days. Variability in the dominant bacterial community in different samples on the 0, 9th and 18th day was observed. Pseudomonas and Shewanella were the two major genera identified during refrigerated storage. At the beginning, the richness of Pseudomonas was about 37.31% and increased for control (CK) samples during refrigerated storage, however, the LVEO-treated samples increased sharply from day 0 to the 9th day and then decreased. LBG-SA coatings containing LVEO treatments significantly delayed MPs oxidation by retarding the formation of free carbonyl compounds and maintaining higher sulfhydryl content, higher Ca2+-ATPase activity, better organized secondary (higher contents of α-helix and ß-sheet) and tertiary structures during refrigerated storage. The transmission electron microscope (TEM) images showed that the integrity of the sarcomere was damaged; the boundaries of the H-, A-, and I-bands, Z-disk, and M-line were fuzzy in the CK samples at the end of storage. However, the LVEO-treated samples were still regular in appearance with distinct dark A-bands, light I-bands, and Z-disk. In brief, LBG-SA active coatings containing LVEO treatments suggested a feasible method for protecting the MPs of large yellow croaker during refrigerated storage.

Effects of Physical and Chemical Factors on the Structure of Gluten, Gliadins and Glutenins as Studied with Spectroscopic Methods.

This review presents applications of spectroscopic methods, infrared and Raman spectroscopies in the studies of the structure of gluten network and gluten proteins (gliadins and glutenins). Both methods provide complimentary information on the secondary and tertiary structure of the proteins including analysis of amide I and III bands, conformation of disulphide bridges, behaviour of tyrosine and tryptophan residues, and water populations. Changes in the gluten structure can be studied as an effect of dough mixing in different conditions (e.g., hydration level, temperature), dough freezing and frozen storage as well as addition of different compounds to the dough (e.g., dough improvers, dietary fibre preparations, polysaccharides and polyphenols). Additionally, effect of above mentioned factors can be determined in a common wheat dough, model dough (prepared from reconstituted flour containing only wheat starch and wheat gluten), gluten dough (lack of starch), and in gliadins and glutenins. The samples were studied in the hydrated state, in the form of powder, film or in solution. Analysis of the studies presented in this review indicates that an adequate amount of water is a critical factor affecting gluten structure.

Anatomy of a crosslinker.

Crosslinking mass spectrometry has become a core technology in structural biology and is expanding its reach towards systems biology. Its appeal lies in a rapid workflow, high sensitivity and the ability to provide data on proteins in complex systems, even in whole cells. The technology depends heavily on crosslinking reagents. The anatomy of crosslinkers can be modular, sometimes comprising combinations of functional groups. These groups are defined by concepts including: reaction selectivity to increase information density, enrichability to improve detection, cleavability to enhance the identification process and isotope-labelling for quantification. Here, we argue that both concepts and functional groups need more thorough experimental evaluation, so that we can show exactly how and where they are useful when applied to crosslinkers. Crosslinker design should be driven by data, not only concepts. We focus on two crosslinker concepts with large consequences for the technology, namely reactive group reaction kinetics and enrichment groups.

Deamidation in Moxetumomab Pasudotox Leading to Conformational Change and Immunotoxin Activity Loss.

Asparagine (Asn) deamidation is a common posttranslational modification in which Asn is converted to aspartic acid or isoaspartic acid. By introducing a negative charge, deamidation could potentially impact the binding interface and biological activities of protein therapeutics. We identified a deamidation variant in moxetumomab pasudotox, an immunotoxin Fv fusion protein drug derived from a 38-kDa truncated Pseudomonas exotoxin A (PE38) for the treatment of hairy-cell leukemia. Although the deamidation site, Asn-358, was outside of the binding interface, the modification had a significant impact on the biological activity of moxetumomab pasudotox. Surprisingly, the variant eluted earlier than its unmodified form on anion exchange chromatography, which often leads to the conclusion that it has a higher positive charge. Here we describe the characterization of the deamidation variant with differential scanning calorimetry and hydrogen-deuterium exchange mass spectrometry, which revealed that the Asn-358 deamidation caused the conformational changes in the catalytic domain of the PE38 region. These results provide an explanation for why the deamidation affected the biological activity of moxetumomab pasudotox and suggest the approach that can be used for process control to ensure product quality and process consistency.

Influence of moderate electric fields in ß-lactoglobulin thermal unfolding and interactions.

In this study, the effects of moderate electric fields during thermal denaturation of ß-lactoglobulin were examined through an in situ circular dichroism approach, complemented by intrinsic extrinsic fluorescence analysis. Results have shown that the effects of electric fields in protein unfolding were linearly dependent on the applied electric field intensity (V/cm) and increased by the use of low electric frequencies - i.e. 50 to 200 Hz. These electric effects caused significant changes on ß-lactoglobulin melting temperature, unfolded conformation and subsequent intermolecular interactions, revealed by the increase of surface hydrophobicity (ANS affinity) and higher conservation of retinol binding. The obtained data provides a clear evidence that moderate electric fields contribute to distinct folding/unfolding of ß-lactoglobulin, resulting in structural modifications. These findings are relevant for (bio)-technological applications involving electric fields processing, bringing new insights for the development of innovative strategies to control protein function and tune production of functional protein systems.

Ferroptosis - An iron- and disorder-dependent programmed cell death.

Programmed cell death (PCD) is an integral component of both developmental and pathological features of an organism. Recently, ferroptosis, a new form of PCD that is dependent on reactive oxygen species and iron, has been described. As with apoptosis, necroptosis, and autophagy, ferroptosis is associated with a large set of proteins assembled in protein-protein interaction (PPI) networks, interactability of which is driven by the presence of intrinsically disordered proteins (IDPs) and IDP regions (IDPRs). Previous investigations have identified the prevalence and functionality of IDPs/IDPRs in non-ferroptosis PCD. The intrinsic disorder in protein structures is used to increase the regulatory powers of these processes. As uncontrolled PCD is associated with the onset of various pathological traits, uncovering the association between intrinsic disorder and ferroptosis-related proteins is crucial. To understand this association, 31 human ferroptosis-related proteins were analyzed via a multi-dimensional array of bioinformatics and computational techniques. In addition, a disorder meta-predictor (PONDR® FIT) was implored to look at the evolutionary conservation of intrinsic disorder in these proteins. This study presents evidence that IDPs and IDPRs are prevalent in ferroptosis. The intrinsic disorder found in ferroptosis has far-reaching functional implications related to ferroptosis-related PPIs and molecular interactions.