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.

Amyloids are fibrillar protein aggregates with a cross-β structure. More than two hundred different proteins with amyloid or amyloid-like properties are already known. Functional amyloids with conservative amyloidogenic regions were found in different organisms. Protein aggregation appears to be beneficial for the organism in these cases. Therefore, this property might be conservative for orthologous proteins. The amyloid aggregates of the CPEB protein were suggested to play an important role in the long-term memory formation in Aplysia californica, Drosophila melanogaster, and Mus musculus. Moreover, the FXR1 protein demonstrates amyloid properties among the Vertebrates. A few nucleoporins (e.g., yeast Nup49, Nup100, Nup116, and human Nup153 and Nup58), are supposed or proved to form amyloid fibrils. In this study, we performed wide-scale bioinformatic analysis of nucleoporins with FG-repeats (phenylalanine–glycine repeats). We demonstrated that most of the barrier nucleoporins possess potential amyloidogenic properties. Furthermore, the aggregation-prone properties of several Nsp1 and Nup100 orthologs in bacteria and yeast cells were analyzed. Only two new nucleoporins, Drosophila melanogaster Nup98 and Schizosaccharomyces pombe Nup98, aggregated in different experiments. At the same time, Taeniopygia guttata Nup58 only formed amyloids in bacterial cells. These results rather contradict the hypothesis about the functional aggregation of nucleoporins.

The NOS1AP gene encodes the nitric oxide synthase 1 adaptor protein (NOS1AP), which binds to neuronal nitric oxide synthase (nNOS) and regulates nitric oxide (NO) production by dissociating nNOS from NMDA receptors (NMDARs). Notably, NOS1AP expression is upregulated upon NMDAR activation; however, there is no available data regarding its production under the receptor inhibition. The NOS1AP gene is also 1 among more than 1000 genes that are presumed to be associated with the development of schizophrenia. Various animal models of this disorder have been developed, some of which are based on the use of NMDAR antagonists such as dizocilpine (MK-801). In this study, we investigated the expression and production of NOS1AP in rats injected with a low dose of dizocilpine (0.1 mg/kg), as well as in SH-SY5Y and HEK293T cell lines treated with varying concentrations of the same compound (10–200 µM). According to our results, neither the expression of the NOS1AP gene nor the production of NOS1AP protein was affected by dizocilpine treatment.

Background: Endometriosis (EM) is a benign gynecological disorder characterized by the hyperproliferation of endometrial cells outside the uterus. This abnormal proliferation followed by neoangiogenesis leads to the formation of EM lesions, which often require surgical removal. Contemporary pharmacological treatment of EM involves hormonal therapies, which are frequently accompanied by adverse effects and a high rate of symptom recurrence following cessation of treatment. Consequently, the development of innovative pharmaceuticals is crucial for the effective management of EM. Small interfering RNA (siRNA) therapeutics are evolving from a niche modality into a major pharmaceutical platform applicable for the therapy of inherited and multifactorial diseases. However, a major obstacle hindering broader clinical use is the challenge of delivering siRNA to tissues beyond the liver. Peptide-based nanoparticles mediating siRNA delivery hold potential to overcome this limitation through receptor targeting and by shielding excessive positive charges. Methods: In the current study, we developed ternary polyplexes composed of anti-VEGFA siRNA, a cationic arginine-rich polymer, and an anionic glutamate-rich polymer modified with a cyclic RGD (cRGD) moiety for αVβ3-integrin binding. The polyplexes were characterized physicochemically, and their toxicity and GFP marker gene silencing properties were studied in vitro. Anti-angiogenic effects were evaluated in vivo using a surgically induced EM model in female Wistar rats. Results: A significant reduction in the volume of modeled EM lesions, along with decreased VEGFA gene expression and apparent reduction in endothelial marker CD34 immunostaining in representative sections, was observed following intralesional administration of anti-VEGFA siRNA-loaded peptide-based nanoparticles decorated with the cRGD ligand, compared to control ligand-free nanoparticles. Conclusions: The results provide evidence that electrostatically stabilized siRNA-peptide nanoparticles can exert anti-angiogenic effects in EM lesions following intralesional administration, warranting further investigation of their targeting properties, safety, and therapeutic potential.

BACKGROUND: Molecular chaperones regulate the proper folding of proteins in the cell. Members of the Hsp70 family, including the Ssa1 protein, are molecular chaperones that prevent protein aggregation, promote their proper folding and degradation, and are the most common among the various chaperones, highly conserved, and present in a variety of organisms. AIM: The aim of the work was to optimize methods for the production, extraction and purification of Ssa1 protein from cells of Saccharomyces cerevisiae. MATERIALS AND METHODS: The SSA1-4 gene sequences were cloned into a vector under the control of the TEF1 promoter and fused with a sequence encoding His6-tag. Yeast strains with different genetic backgrounds were transformed with the obtained constructs, and the production of Ssa1-4 proteins was assessed under different cultivation conditions. Affinity and ion-exchange chromatography were used to purify the Ssa1 protein. Fluorescence microscopy was used to confirm the localization of recombinant Ssa proteins fused with TagRFP-T in the cytosol. RESULTS AND CONCLUSIONS: Methods for the production, extraction and purification of Ssa1 protein from yeast cells have been optimized. The same approach can be further used to purify other Hsp70 proteins and adapted to obtain various proteins from eukaryotic cells.

The essential SUP35 gene encodes yeast translation termination factor Sup35/eRF3. The N-terminal domain of Sup35 is also responsible for Sup35 prionization that leads to generation of the [PSI⁺] prion. Previously we isolated different types of sup35 mutations (missense and nonsense) and demonstrated that sup35 nonsense mutations (sup35-n) are incompatible with the [PSI⁺] prion, leading to lethality of sup35-n [PSI⁺] haploid cells. Here, we show that sup35 missense mutations (sup35-m) within conservative regions of the Sup35 C-domain result in lethality of [PSI⁺] cells because of weak activity of Sup35/eRF3 as a translation termination factor. Mutant Sup35 maintain their ability to be incorporated into pre-existing [PSI⁺] aggregates and to form amyloid aggregates in vitro, while sup35-m mutations do not influence the [PSI⁺] prion induction and stability. All these mutations (D363N, R372K, T378I) are located in the conservative GTPase region of Sup35, decreasing the GTPase activity of mutated proteins. We propose that such low activity of mutant Sup35 combined with aggregation of Sup35 constituting the [PSI⁺] prion is not sufficient to maintain the viability of yeast cells.