Half-life extension via ABD-fusion leads to higher tumor uptake of an affibody-drug conjugate compared to PAS- and XTENylation.

A critical parameter during the development of protein therapeutics is to endow them with suitable pharmacokinetic and pharmacodynamic properties. Small protein drugs are quickly eliminated by kidney filtration, and in vivo half-life extension is therefore often desired. Here, different half-life extension technologies were studied where PAS polypeptides (PAS300, PAS600), XTEN polypeptides (XTEN288, XTEN576), and an albumin binding domain (ABD) were compared for half-life extension of an anti-human epidermal growth factor receptor 2 (HER2) affibody-drug conjugate. The results showed that extension with the PAS or XTEN polypeptides or the addition of the ABD lowered the affinity for HER2 to some extent but did not negatively affect the cytotoxic potential. The half-lives in mice ranged from 7.3 h for the construct including PAS300 to 11.6 h for the construct including PAS600. The highest absolute tumor uptake was found for the construct including the ABD, which was 60 to 160% higher than the PASylated or XTENylated constructs, even though it did not have the longest half-life (9.0 h). A comparison of the tumor-to-normal-organ ratios showed the best overall performance of the ABD-fused construct. In conclusion, PASylation, XTENylation, and the addition of an ABD are viable strategies for half-life extension of affibody-drug conjugates, with the best performance observed for the construct including the ABD.

Coal-Derived Humic Substances: Insight into Chemical Structure Parameters and Biomedical Properties.

An investigation was carried out on humic substances (HSs) isolated from the coal of the Kansk-Achinsk basin (Krasnoyarsk Territory, Russia). The coal HSs demonstrate the main parameters of molecular structure inherent to this class of natural compounds. An assessment was performed for the chemical, microbiological, and pharmacological safety parameters, as well as the biological efficacy. The HS sample meets the safety requirements in microbiological purity, toxic metals content (lead, cadmium, mercury, arsenic), and radionuclides. The presence of 11 essential elements was determined. The absence of general, systemic toxicity, cytotoxicity, and allergenic properties was demonstrated. The coal HS sample was classified as a Class V hazard (low danger substances). High antioxidant and antiradical activities and immunotropic and cytoprotective properties were identified. The ability of the HS to inhibit hydroxyl radicals and superoxide anion radicals was revealed. Pronounced actoprotective and nootropic activities were also demonstrated in vivo. Intragastric administration of the HS sample resulted in the improvement of physical parameters in mice as assessed by the "swim exhaustion" test. Furthermore, intragastric administration in mice with cholinergic dysfunction led to a higher ability of animals with scopolamine-induced amnesia to form conditioned reflexes. These findings suggest that the studied HS sample is a safe and effective natural substance, making it suitable for use as a dietary bioactive supplement.

Dynamic properties of the layers of cupin-1.1 aggregates at the air/water interface.

Spread layers of amorphous aggregates of the structural domain of plant protein vicilin, cupin-1.1, at the water - air interface were studied by the surface tensiometry, dilational surface rheology, Brewster angle and atomic force microscopy. The layer properties differed strongly from the results for the layers of previously studied proteins. The dependency of the dynamic elasticity of the layer on surface pressure had two local maxima with the second peak being four times higher than the first one. In the region of the first maximum the obtained results are similar to those for dispersions of polymer microgels with a hairy corona. At the beginning of surface compression separate threads of the corona are stretched along the surface and the surface elasticity increases. The further compression results in the formation of loops and tails leading to a decrease of the elasticity. The second local maximum of the dynamic surface elasticity is presumably caused by the interactions of the rigid cores of the aggregates leading finally to the formation of multilayer structures at high surface pressures. In this case, the surface elasticity starts to decrease as a result of the segment exchange between different layers at the interface.

OmpC and OmpF Outer Membrane Proteins of Escherichia coli and Salmonella enterica Form Bona Fide Amyloids.

Outer membrane proteins (Omps) of Gram-negative bacteria represent porins involved in a wide range of virulence- and pathogenesis-related cellular processes, including transport, adhesion, penetration, and the colonization of host tissues. Most outer membrane porins share a specific spatial structure called the ß-barrel that provides their structural integrity within the membrane lipid bilayer. Recent data suggest that outer membrane proteins from several bacterial species are able to adopt the amyloid state alternative to their ß-barrel structure. Amyloids are protein fibrils with a specific spatial structure called the cross-ß that gives them an unusual resistance to different physicochemical influences. Various bacterial amyloids are known to be involved in host-pathogen and host-symbiont interactions and contribute to colonization of host tissues. Such an ability of outer membrane porins to adopt amyloid state might represent an important mechanism of bacterial virulence. In this work, we investigated the amyloid properties of the OmpC and OmpF porins from two species belonging to Enterobacteriaceae family, Escherichia coli, and Salmonella enterica. We demonstrated that OmpC and OmpF of E. coli and S. enterica form toxic fibrillar aggregates in vitro. These aggregates exhibit birefringence upon binding Congo Red dye and show characteristic reflections under X-ray diffraction. Thus, we confirmed amyloid properties for OmpC of E. coli and demonstrated bona fide amyloid properties for three novel proteins: OmpC of S. enterica and OmpF of E. coli and S. enterica in vitro. All four studied porins were shown to form amyloid fibrils at the surface of E. coli cells in the curli-dependent amyloid generator system. Moreover, we found that overexpression of recombinant OmpC and OmpF in the E. coli BL21 strain leads to the formation of detergent- and protease-resistant amyloid-like aggregates and enhances the birefringence of bacterial cultures stained with Congo Red. We also detected detergent- and protease-resistant aggregates comprising OmpC and OmpF in S. enterica culture. These data are important in the context of understanding the structural dualism of Omps and its relation to pathogenesis.

Immunomodulatory Activity of Polysaccharides Isolated from Saussurea salicifolia L. and Saussurea frolovii Ledeb.

The genus Saussurea has been used in the preparation of therapies for a number of medical problems, yet not much is known about the therapeutic high-molecular-weight compounds present in extracts from these plants. Since polysaccharides are important in immune modulation, we investigated the chemical composition and immunomodulatory activity of Saussurea salicifolia L. and Saussurea frolovii Ledeb polysaccharides. Water-soluble polysaccharides from the aerial parts of these plants were extracted using water at pHs of 2 and 6 and subsequently precipitated in ethanol to obtain fractions SSP2 and SSP6 from S. salicifolia and fractions SSF2 and SSF6 from S. frolovii. The molecular weights of fractions SSP2, SSP6, SFP2, and SFP6 were estimated to be 143.7, 113.2, 75.3, and 64.3 kDa, respectively. The polysaccharides from S. frolovii contained xylose (67.1-71.7%) and glucose (28.3-32.9%), whereas the polysaccharides from S. frolovii contained xylose (63.1-76.7%), glucose (11.8-19.2%), galactose (4.7-8.3%), and rhamnose (6.8-9.4%). Fractions SSP2, SSP6, and SFP2 stimulated nitric oxide (NO) production by murine macrophages, and NO production induced by SSP2, SSP6, and SFP2 was not inhibited by polymyxin B treatment of the fractions, whereaspolymyxin B treatment diminished the effects of SFP6, suggesting that SFP6 could contain lipopolysaccharide (LPS). The LPS-free fractions SSP2, SSP6, and SFP2 had potent immunomodulatory activity, induced NO production, and activated transcription factors NF-κB/AP-1 in human monocytic THP-1 cells and cytokine production by human MonoMac-6 monocytic cells, including interleukin (IL)-1α, IL-1ß, IL-6, granulocyte macrophage colony-stimulating factor (GM-CSF), interferon-γ, monocyte chemotactic protein 1 (MCP-1), and tumor necrosis factor (TNF). These data suggest that at least part of the beneficial therapeutic effects reported for water extracts of the Saussurea species are due to the modulation of leukocyte functions by polysaccharides.

Amyloid Fibrils of Pisum sativum L. Vicilin Inhibit Pathological Aggregation of Mammalian Proteins.

Although incurable pathologies associated with the formation of highly ordered fibrillar protein aggregates called amyloids have been known for about two centuries, functional roles of amyloids have been studied for only two decades. Recently, we identified functional amyloids in plants. These amyloids formed using garden pea Pisum sativum L. storage globulin and vicilin, accumulated during the seed maturation and resisted treatment with gastric enzymes and canning. Thus, vicilin amyloids ingested with food could interact with mammalian proteins. In this work, we analyzed the effects of vicilin amyloids on the fibril formation of proteins that form pathological amyloids. We found that vicilin amyloids inhibit the fibrillogenesis of these proteins. In particular, vicilin amyloids decrease the number and length of lysozyme amyloid fibrils; the length and width of ß-2-microglobulin fibrils; the number, length and the degree of clustering of ß-amyloid fibrils; and, finally, they change the structure and decrease the length of insulin fibrils. Such drastic influences of vicilin amyloids on the pathological amyloids' formation cause the alteration of their toxicity for mammalian cells, which decreases for all tested amyloids with the exception of insulin. Taken together, our study, for the first time, demonstrates the anti-amyloid effect of vicilin fibrils and suggests the mechanisms underlying this phenomenon.

Enhanced Antioxidant Activity and Reduced Cytotoxicity of Silver Nanoparticles Stabilized by Different Humic Materials.

The current article describes the biological activity of new biomaterials combining the "green" properties of humic substances (HSs) and silver nanoparticles. The aim is to investigate the antioxidant activity (AOA) of HS matrices (macroligands) and AgNPs stabilized with humic macroligands (HS-AgNPs). The unique chemical feature of HSs makes them very promising ligands (matrices) for AgNP stabilization. HSs have previously been shown to exert many pharmacological effects mediated by their AOA. AgNPs stabilized with HS showed a pronounced ability to bind to reactive oxygen species (ROS) in the test with ABTS. Also, higher AOA was observed for HS-AgNPs as compared to the HS matrices. In vitro cytotoxicity studies have shown that the stabilization of AgNPs with the HS matrices reduces the cytotoxicity of AgNPs. As a result of in vitro experiments with the use of 2,7-dichlorodihydrofluorescein diacetate (DCFDA), it was found that all HS materials tested and the HS-AgNPs did not exhibit prooxidant effects. Moreover, more pronounced AOA was shown for HS-AgNP samples as compared to the original HS matrices. Two putative mechanisms of the pronounced AOA of the tested compositions are proposed: firstly, the pronounced ability of HSs to inactivate ROS and, secondly, the large surface area and surface-to-volume ratio of HS-AgNPs, which facilitate electron transfer and mitigate kinetic barriers to the reduction reaction. As a result, the antioxidant properties of the tested HS-AgNPs might be of particular interest for biomedical applications aimed at inhibiting the growth of bacteria and viruses and the healing of purulent wounds.

Comparison of HER2-targeted affibody conjugates loaded with auristatin- and maytansine-derived drugs.

Treatment with antibody drug conjugates targeting receptors over-expressed on cancer cells is well established for clinical use in several types of cancer, however, resistance often occurs motivating the development of novel drugs. We have recently investigated a drug conjugate consisting of an affibody molecule targeting the human epidermal growth factor receptor 2 (HER2), fused to an albumin-binding domain (ABD) for half-life extension, loaded with the cytotoxic maytansine derivative DM1. In this study, we investigated the impact of the cytotoxic payload on binding properties, cytotoxicity and biodistribution by comparing DM1 with the auristatins MMAE and MMAF, as part of the drug conjugate. All constructs had specific and high affinity binding to HER2, human and mouse albumins with values in the low- to sub-nM range. ZHER2-ABD-mcMMAF demonstrated the most potent cytotoxic effect on several HER2-over-expressing cell lines. In an experimental therapy study, the MMAF-based conjugate provided complete tumor regression in 50% of BALB/c nu/nu mice bearing HER2-over-expressing SKOV3 tumors at a 2.9 mg/kg dose, while the same dose of ZHER2-ABD-mcDM1 provided only a moderate anti-tumor effect. A comparison with the non-targeting ZTaq-ABD-mcMMAF control demonstrated HER2-targeting specificity. In conclusion, a combination of potent cytotoxicity in vitro, with minimal uptake in normal organs in vivo, and efficient delivery to tumors provided a superior anti-tumor effect of ZHER2-ABD-mcMMAF, while maintaining a favorable toxicity profile with no observed adverse effects.

Salmonella-Based Biorodenticides: Past Applications and Current Contradictions.

The idea of using pathogens to control pests has existed since the end of the 19th century. Enterobacteria from the genus Salmonella, discovered at that time, are the causative agents of many serious diseases in mammals often leading to death. Mostly, the strains of Salmonella are able to infect a wide spectrum of hosts belonging to vertebrates, but some of them show host restriction. Several strains of these bacteria have been used as biorodenticides due to the host restriction until they were banned in many countries in the second part of the 20th century. The main reason for the ban was their potential pathogenicity for some domestic animals and poultry and the outbreaks of gastroenteritis in humans. Since that time, a lot of data regarding the host specificity and host restriction of different strains of Salmonella have been accumulated, and the complexity of the molecular mechanisms affecting it has been uncovered. In this review, we summarize the data regarding the history of studying and application of Salmonella-based rodenticides, discuss molecular systems controlling the specificity of Salmonella interactions within its multicellular hosts at different stages of infection, and attempt to reconstruct the network of genes and their allelic variants which might affect the host-restriction mechanisms.

RopB protein of Rhizobium leguminosarum bv. viciae adopts amyloid state during symbiotic interactions with pea (Pisum sativum L.).

Amyloids represent protein aggregates with highly ordered fibrillar structure associated with the development of various disorders in humans and animals and involved in implementation of different vital functions in all three domains of life. In prokaryotes, amyloids perform a wide repertoire of functions mostly attributed to their interactions with other organisms including interspecies interactions within bacterial communities and host-pathogen interactions. Recently, we demonstrated that free-living cells of Rhizobium leguminosarum, a nitrogen-fixing symbiont of legumes, produce RopA and RopB which form amyloid fibrils at cell surface during the stationary growth phase thus connecting amyloid formation and host-symbiont interactions. Here we focused on a more detailed analysis of the RopB amyloid state in vitro and in vivo, during the symbiotic interaction between R. leguminosarum bv. viciae with its macrosymbiont, garden pea (Pisum sativum L.). We confirmed that RopB is the bona fide amyloid protein since its fibrils exhibit circular x-ray reflections indicating its cross-ß structure specific for amyloids. We found that fibrils containing RopB and exhibiting amyloid properties are formed in vivo at the surface of bacteroids of R. leguminosarum extracted from pea nodules. Moreover, using pea sym31 mutant we demonstrated that formation of extracellular RopB amyloid state occurs at different stages of bacteroid development but is enhanced in juvenile symbiosomes. Proteomic screening of potentially amyloidogenic proteins in the nodules revealed the presence of detergent-resistant aggregates of different plant and bacterial proteins including pea amyloid vicilin. We demonstrated that preformed vicilin amyloids can cross-seed RopB amyloid formation suggesting for probable interaction between bacterial and plant amyloidogenic proteins in the nodules. Taken together, we demonstrate that R. leguminosarum bacteroids produce extracellular RopB amyloids in pea nodules in vivo and these nodules also contain aggregates of pea vicilin amyloid protein, which is able to cross-seed RopB fibrillogenesis in vitro. Thus, we hypothesize that plant nodules contain a complex amyloid network consisting of plant and bacterial amyloids and probably modulating host-symbiont interactions.

Quantitative Structure-Activity Relationship, Ontology-Based Model of the Antioxidant and Cell Protective Activity of Peat Humic Acids.

Peat humic acids are well known for their wide range of biological effects which can be attributed to the complex chemical structure of naturally occurring humic substances. One of the promising tools is an ontology-based quantitative analysis of the relationship between physical and chemical parameters describing a chemical structure of peat humic acids and their biological activity. This article demonstrates the feasibility of such an approach to estimate the antioxidant and cell protective properties of the peat humic acids. The structural parameters of the peat humic acids were studied by electronic, fluorescence, infrared, 13C-NMR spectroscopy, titrimetric analysis, elemental C,H,N, and O- analysis, and gel chromatography. Antioxidant and antiradical activities were assessed by physicochemical methods of analysis: electronic paramagnetic resonance, cathodic voltammetry, ABTS•+ scavenging, assay of DPPH radical-scavenging activity, assay of superoxide radical-scavenging activity, iron chelating activity, and scavenging of hydroxyl radicals. Cytoprotective activity was evaluated by the neutral red-based cytotoxicity test in 3T3-L1 cell culture in a wide range of concentrations. Assessment of intracellular ROS production was carried out using a 2,7-dichlorodihydrofluoresceindiacetate (DCFDA) fluorescent probe. Intracellular ROS production was induced using two common prooxidants (tert-butyl hydroperoxide, Fe2+ ions). We suggested an ontology-based model for the antioxidant and cytoprotective activity of humic acids based on experimental data and numerical models. This model establishes the way to further research on the biological effects of humic acids and provides a useful tool for numerical simulation of these effects. Remarkable antioxidant and cell protective activity of humic acids makes them a promising natural source of new pharmaceutical substances that feature a wide range of biological effects.

Amyloid fibril length distribution from dynamic light scattering data.

The study of the aggregation of amyloid proteins is challenging. A new approach to processing dynamic light scattering data was developed and tested using aggregates of the well-known model Sup35NM amyloid. After filtering and calculating the moving averages of autocorrelation functions to reduce impacts of noise, each averaged autocorrelation function is converted to the fibril length distribution via numerical modeling. The processing results were verified using atomic force and scanning electron microscopy data. Analysis of fibril length distribution changes over time gives valuable information about the aggregation process.

Effect of Inter-Domain Linker Composition on Biodistribution of ABD-Fused Affibody-Drug Conjugates Targeting HER2.

Targeted drug conjugates based on Affibody molecules fused to an albumin-binding domain (ABD) for half-life extension have demonstrated potent anti-tumor activity in preclinical therapeutic studies. Furthermore, optimization of their molecular design might increase the cytotoxic effect on tumors and minimize systemic toxicity. This study aimed to investigate the influence of length and composition of a linker between the human epidermal growth factor receptor 2 (HER2)-targeted affibody molecule (ZHER2:2891) and the ABD domain on functionality and biodistribution of affibody-drug conjugates containing a microtubulin inhibitor mertansin (mcDM1) (AffiDCs). Two conjugates, having a trimeric (S3G)3 linker or a trimeric (G3S)3 linker were produced, radiolabeled with 99mTc(CO)3, and compared side-by-side in vitro and in vivo with the original ZHER2:2891-G4S-ABD-mcDM1 conjugate having a monomeric G4S linker. Both conjugates with longer linkers had a decreased affinity to HER2 and mouse and human serum albumin in vitro, however, no differences in blood retention were observed in NMRI mice up to 24 h post injection. The use of both (S3G)3 and (G3S)3 linkers reduced liver uptake of AffiDCs by approximately 1.2-fold compared with the use of a G4S linker. This finding provides important insights into the molecular design for the development of targeted drug conjugates with reduced hepatic uptake.

ß-Barrels and Amyloids: Structural Transitions, Biological Functions, and Pathogenesis.

Insoluble protein aggregates with fibrillar morphology called amyloids and ß-barrel proteins both share a ß-sheet-rich structure. Correctly folded ß-barrel proteins can not only function in monomeric (dimeric) form, but also tend to interact with one another-followed, in several cases, by formation of higher order oligomers or even aggregates. In recent years, findings proving that ß-barrel proteins can adopt cross-ß amyloid folds have emerged. Different ß-barrel proteins were shown to form amyloid fibrils in vitro. The formation of functional amyloids in vivo by ß-barrel proteins for which the amyloid state is native was also discovered. In particular, several prokaryotic and eukaryotic proteins with ß-barrel domains were demonstrated to form amyloids in vivo, where they participate in interspecies interactions and nutrient storage, respectively. According to recent observations, despite the variety of primary structures of amyloid-forming proteins, most of them can adopt a conformational state with the ß-barrel topology. This state can be intermediate on the pathway of fibrillogenesis ("on-pathway state"), or can be formed as a result of an alternative assembly of partially unfolded monomers ("off-pathway state"). The ß-barrel oligomers formed by amyloid proteins possess toxicity, and are likely to be involved in the development of amyloidoses, thus representing promising targets for potential therapy of these incurable diseases. Considering rapidly growing discoveries of the amyloid-forming ß-barrels, we may suggest that their real number and diversity of functions are significantly higher than identified to date, and represent only "the tip of the iceberg". Here, we summarize the data on the amyloid-forming ß-barrel proteins, their physicochemical properties, and their biological functions, and discuss probable means and consequences of the amyloidogenesis of these proteins, along with structural relationships between these two widespread types of ß-folds.

The Human NUP58 Nucleoporin Can Form Amyloids In Vitro and In Vivo.

Amyloids are fibrillar protein aggregates with a cross-ß structure and unusual features, including high resistance to detergent or protease treatment. More than two hundred different proteins with amyloid or amyloid-like properties are already known. Several examples of nucleoporins (e.g., yeast Nup49, Nup100, Nup116, and human NUP153) are supposed to form amyloid fibrils. In this study, we demonstrated an ability of the human NUP58 nucleoporin to form amyloid aggregates in vivo and in vitro. Moreover, we found two forms of NUP58 aggregates: oligomers and polymers stabilized by disulfide bonds. Bioinformatic analysis revealed that all known orthologs of this protein are potential amyloids which possess several regions with conserved ability to aggregation. The biological role of nucleoporin amyloid formation is debatable. We suggest that it is a rather abnormal process, which is characteristic for many proteins implicated in phase separation.

The Role of Rotational Motion in Diffusion NMR Experiments on Supramolecular Assemblies: Application to Sup35NM Fibrils.

Pulsed-field gradient (PFG) NMR is an important tool for characterization of biomolecules and supramolecular assemblies. However, for micrometer-sized objects, such as amyloid fibrils, these experiments become difficult to interpret because in addition to translational diffusion they are also sensitive to rotational diffusion. We have constructed a mathematical theory describing the outcome of PFG NMR experiments on rod-like fibrils. To test its validity, we have studied the fibrils formed by Sup35NM segment of the prion protein Sup35. The interpretation of the PFG NMR data in this system is fully consistent with the evidence from electron microscopy. Contrary to some previously expressed views, the signals originating from disordered regions in the fibrils can be readily differentiated from the similar signals representing small soluble species (e.g. proteolytic fragments). This paves the way for diffusion-sorted NMR experiments on complex amyloidogenic samples.

Biological Functions of Prokaryotic Amyloids in Interspecies Interactions: Facts and Assumptions.

Amyloids are fibrillar protein aggregates with an ordered spatial structure called "cross-ß". While some amyloids are associated with development of approximately 50 incurable diseases of humans and animals, the others perform various crucial physiological functions. The greatest diversity of amyloids functions is identified within prokaryotic species where they, being the components of the biofilm matrix, function as adhesins, regulate the activity of toxins and virulence factors, and compose extracellular protein layers. Amyloid state is widely used by different pathogenic bacterial species in their interactions with eukaryotic organisms. These amyloids, being functional for bacteria that produce them, are associated with various bacterial infections in humans and animals. Thus, the repertoire of the disease-associated amyloids includes not only dozens of pathological amyloids of mammalian origin but also numerous microbial amyloids. Although the ability of symbiotic microorganisms to produce amyloids has recently been demonstrated, functional roles of prokaryotic amyloids in host-symbiont interactions as well as in the interspecies interactions within the prokaryotic communities remain poorly studied. Here, we summarize the current findings in the field of prokaryotic amyloids, classify different interspecies interactions where these amyloids are involved, and hypothesize about their real occurrence in nature as well as their roles in pathogenesis and symbiosis.

Accumulation of storage proteins in plant seeds is mediated by amyloid formation.

Amyloids are protein aggregates with a highly ordered spatial structure giving them unique physicochemical properties. Different amyloids not only participate in the development of numerous incurable diseases but control vital functions in archaea, bacteria and eukarya. Plants are a poorly studied systematic group in the field of amyloid biology. Amyloid properties have not yet been demonstrated for plant proteins under native conditions in vivo. Here we show that seeds of garden pea Pisum sativum L. contain amyloid-like aggregates of storage proteins, the most abundant one, 7S globulin Vicilin, forms bona fide amyloids in vivo and in vitro. Full-length Vicilin contains 2 evolutionary conserved ß-barrel domains, Cupin-1.1 and Cupin-1.2, that self-assemble in vitro into amyloid fibrils with similar physicochemical properties. However, Cupin-1.2 fibrils unlike Cupin-1.1 can seed Vicilin fibrillation. In vivo, Vicilin forms amyloids in the cotyledon cells that bind amyloid-specific dyes and possess resistance to detergents and proteases. The Vicilin amyloid accumulation increases during seed maturation and wanes at germination. Amyloids of Vicilin resist digestion by gastrointestinal enzymes, persist in canned peas, and exhibit toxicity for yeast and mammalian cells. Our finding for the first time reveals involvement of amyloid formation in the accumulation of storage proteins in plant seeds.

Estimation of amyloid aggregate sizes with semi-denaturing detergent agarose gel electrophoresis and its limitations.

Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was proposed by Vitaly V. Kushnirov in the Michael D. Ter-Avanesyan's laboratory as a method to compare sizes of amyloid aggregates. Currently, this method is widely used for amyloid investigation, but mostly as a qualitative approach. In this work, we assessed the possibilities and limitations of the quantitative analysis of amyloid aggregate size distribution using SDD-AGE results. For this purpose, we used aggregates of two well-characterized yeast amyloid-forming proteins, Sup35 and Rnq1, and developed a protocol to standardize image analysis and process the result. A detailed investigation of factors that may affect the results of SDD-AGE revealed that both the cell lysis method and electrophoresis conditions can substantially affect the estimation of aggregate size. Despite this, quantitative analysis of SDD-AGE results is possible when one needs to estimate and compare the size of aggregates on the same gel, or even in different experiments, if the experimental conditions are tightly controlled and additional standards are used.

Design of a New [PSI +]-No-More Mutation in SUP35 With Strong Inhibitory Effect on the [PSI +] Prion Propagation.

A number of [PSI +]-no-more (PNM) mutations, eliminating [PSI +] prion, were previously described in SUP35. In this study, we designed and analyzed a new PNM mutation based on the parallel in-register ß-structure of Sup35 prion fibrils suggested by the known experimental data. In such an arrangement, substitution of non-charged residues by charged ones may destabilize the fibril structure. We introduced Q33K/A34K amino acid substitutions into the Sup35 protein, corresponding allele was called sup35-M0. The mutagenized residues were chosen based on ArchCandy in silico prediction of high inhibitory effect on the amyloidogenic potential of Sup35. The experiments confirmed that Sup35-M0 leads to the elimination of [PSI +] with high efficiency. Our data suggested that the elimination of the [PSI +] prion is associated with the decreased aggregation properties of the protein. The new mutation can induce the prion with very low efficiency and is able to propagate only weak [PSI +] prion variants. We also showed that Sup35-M0 protein co-aggregates with the wild-type Sup35 in vivo. Moreover, our data confirmed the utility of the strategy of substitution of non-charged residues by charged ones to design new mutations to inhibit a prion formation.