The Role of Non-Specific Interactions in Canonical and ALT-Associated PML-Bodies Formation and Dynamics

In this work, we put forward a hypothesis about the decisive role of multivalent nonspecific interactions in the early stages of PML body formation. Our analysis of the PML isoform sequences showed that some of the PML isoforms, primarily PML-II, are prone to phase separation due to their polyampholytic properties and the disordered structure of their C-terminal domains. The similarity of the charge properties of the C-terminal domains of PML-II and PML-VI isoforms made it possible for the first time to detect migration of PML-VI from PML bodies to the periphery of the cell nucleus, similar to the migration of PML-II isoforms. We found a population of “small” (area less than 1 µm2) spherical PML bodies with high dynamics of PML isoforms exchange with nucleoplasm and a low fraction of immobilized proteins, which indicates their liquid state properties. Such structures can act as “seeds” of functionally active PML bodies, providing the necessary concentration of PML isoforms for the formation of intermolecular disulfide bonds between PML monomers. FRAP analysis of larger bodies of toroidal topology showed the existence of an insoluble scaffold in their structure. The hypothesis about the role of nonspecific multiple weak interactions in the formation of PML bodies is further supported by the change in the composition of the scaffold proteins of PML bodies, but not their solidification, under conditions of induction of dimerization of PML isoforms under oxidative stress. Using the colocalization of ALT-associated PML bodies (APBs) with TRF1, we identified APBs and showed the difference in the dynamic properties of APBs and canonical PML bodies.

A Comprehensive Analysis of Novel Disulfide Bond Introduction Site into the Constant Domain of Human Fab

Generally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bonds can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the H: F130C-L: Q124C, H: F174C-L: S176C, H: V177C-L: Q160C, H: F174C-L: S162C, H: F130C-L: S121C, and H: A145C-L: F116C mutants mostly formed intermolecular disulfide bonds. All these mutants showed increased thermal stabilities compared to those without intermolecular disulfide bonds. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C-F118C mutant showed only a slight decrease in binding activity and β-helix content, owing to adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals the introduction of intermolecular disulfide bonds in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

The comparative analysis of the methods for keratin extraction from sheep wool and human hair

Nowadays, biopolymers such as keratins are widely used in biomedicine due to their low toxicity, biocompatibility, and biodegradability. At the molecular level, keratins differ from other structural proteins by a high content of disulfide bonds, which provide the formation of a compact three-dimensional structure resistant to biological and chemical degradation. Native keratins are highly ordered, whereas, recovered keratins are characterized by a flexible structure with more accessible functional groups. A characteristic feature of solubilized keratins is their ability to polymerize; therefore, they are widely used to create biomaterials. The extraction of keratins from natural fibers is an important step to the development of functional biomaterials. However, this process is complicated by the presence of a large number of intramolecular and intermolecular disulfide bonds in keratins. That is why keratin extraction by breaking the intermolecular disulfide bonds while preserving the covalent bonds of the polypeptide chain is necessary. The goal of our study was to estimate the different methods of solubilized keratin obtaining. In the experiments, samples of different types of wool and human hair were used. Various methods of keratin extraction were applied. The yield of solubilized keratin (%) was calculated from the ratio of the weight of the lyophilized keratin extract and the initial weight of fibers. The molecular mass of recovered keratins was evaluated by SDS-PAAG electrophoresis in the Laemmli buffer system. An analysis of the efficiency of keratin extraction has shown that solubilized keratin yield ranged from 32% to 51% and depended on the composition of the extraction mixture. Electrophoretic analysis of all keratin extracts obtained by various methods confirmed the presence of two bands, which according to the molecular weight corresponding to I and II types of proteins of intermediate filaments. The presence of these proteins provides self-assembly into complex structures.

A unique adhesive motif of protein disulfide isomerase P5 supports its function via dimerization

SUMMARYP5, also known as PDIA6, is a PDI-family member that plays an important role in the ER quality control. Herein, we revealed that P5 dimerizes via a unique adhesive motif contained in the N-terminal thioredoxin-like domain. This motif is apparently similar to, but radically different from conventional leucine-zipper motifs, in that the former includes a periodic repeat of leucine or valine residues at the third or fourth position spanning five helical turns on 15-residue anti-parallel α-helices, unlike the latter of which the leucine residues appear every two helical turns on ∼30-residue parallel α-helices at dimer interfaces. A monomeric P5 mutant with the impaired adhesive motif showed structural instability and local unfolding, and behaved as an aberrant protein that induces the ER stress response. Disassembly of P5 to monomers compromised its ability to inactivate IRE1α via reduction of intermolecular disulfide bonds and its Ca2+-dependent regulation of chaperone function in vitro. Thus, the leucine-valine adhesive motif supports structure and physiological function of P5.

Oxicam-type nonsteroidal anti-inflammatory drugs inhibit NPR1-mediated salicylic acid pathway

Salicylic acid (SA) and its structural analogs are nonsteroidal anti-inflammatory drugs (NSAIDs) that target mammalian cyclooxygenases. In plants, SA acts as a defense hormone that regulates NON-EXPRESSOR OF PATHOGENESIS RELATED GENES 1 (NPR1), the master transcriptional regulator of immunity-related genes. We identified a number of NSAIDs that enhance bacterial effector-induced cell death. Among them, the oxicam-type NSAIDs tenoxicam (TNX), meloxicam, and piroxicam, but not other types of NSAIDs, exhibit an inhibitory effect on immunity to bacteria and SA-dependent immune responses in plants. TNX treatment reduces NPR1 levels, independently from the proposed SA receptors NPR3 and NPR4. Instead, TNX induces oxidation of cytosolic redox status, which is also affected by SA and regulates NPR1 homeostasis. Surprisingly, however, cysteine modification associated with NPR1 oligomerization via intermolecular disulfide bonds is not affected by either SA or TNX. Therefore, oxicam-type NSAIDs highlight importance of SA effects on the cytosolic redox status, but not on cysteine modification or oligomerization of NPR1.

Deorphanizing FAM19A proteins as pan-neurexin ligands with an unusual biosynthetic binding mechanism

Neurexins are presynaptic adhesion molecules that organize synapses by binding to diverse trans-synaptic ligands, but how neurexins are regulated is incompletely understood. Here we identify FAM19A/TAFA proteins, “orphan" cytokines, as neurexin regulators that interact with all neurexins, except for neurexin-1γ, via an unusual mechanism. Specifically, we show that FAM19A1-A4 bind to the cysteine-loop domain of neurexins by forming intermolecular disulfide bonds during transport through the secretory pathway. FAM19A-binding required both the cysteines of the cysteine-loop domain and an adjacent sequence of neurexins. Genetic deletion of neurexins suppressed FAM19A1 expression, demonstrating that FAM19As physiologically interact with neurexins. In hippocampal cultures, expression of exogenous FAM19A1 decreased neurexin O-glycosylation and suppressed its heparan sulfate modification, suggesting that FAM19As regulate the post-translational modification of neurexins. Given the selective expression of FAM19As in specific subtypes of neurons and their activity-dependent regulation, these results suggest that FAM19As serve as cell type–specific regulators of neurexin modifications.

Glutenin and Gliadin, a Piece in the Puzzle of their Structural Properties in the Cell Described through Monte Carlo Simulations

Gluten protein crosslinking is a predetermined process where specific intra- and intermolecular disulfide bonds differ depending on the protein and cysteine motif. In this article, all-atom Monte Carlo simulations were used to understand the formation of disulfide bonds in gliadins and low molecular weight glutenin subunits (LMW-GS). The two intrinsically disordered proteins appeared to contain mostly turns and loops and showed “self-avoiding walk” behavior in water. Cysteine residues involved in intramolecular disulfide bonds were located next to hydrophobic peptide sections in the primary sequence. Hydrophobicity of neighboring peptide sections, synthesis chronology, and amino acid chain flexibility were identified as important factors in securing the specificity of intramolecular disulfide bonds formed directly after synthesis. The two LMW-GS cysteine residues that form intermolecular disulfide bonds were positioned next to peptide sections of lower hydrophobicity, and these cysteine residues are more exposed to the cytosolic conditions, which influence the crosslinking behavior. In addition, coarse-grained Monte Carlo simulations revealed that the protein folding is independent of ionic strength. The potential molecular behavior associated with disulfide bonds, as reported here, increases the biological understanding of seed storage protein function and provides opportunities to tailor their functional properties for different applications.