Amyloids are fibrillar proteins with a cross-β structure. Pathological amyloids are associated with the development of a number of incurable diseases, while functional amyloids regulate vital processes. The detection of unknown amyloids in living objects is a difficult task, and therefore the question of the prevalence and biological significance of amyloids remains open. We present a description of two methods, the combination of which makes it possible to find and identify amyloid proteins in the proteome of various organisms. The method of proteomic screening for amyloids allows the detection of the proteins that form SDS-resistant aggregates. SDS resistance is a general feature of amyloid fibrils. Protein aggregates resistant to SDS treatment can be collected by ultracentrifugation and further identified by mass spectrometry. However, in addition to amyloids, SDS-resistant aggregates contain some non-amyloid proteins. To test the amyloid properties of proteins identified by proteomic screening, we developed the method of fibril immunoprecipitation followed by Congo red staining and birefringence analysis. The methods of proteomic screening and immunoprecipitation of fibrillar proteins have been successfully tested and applied for the identification of amyloid proteins in yeast and vertebrates.

CRISPR/Cas-based systems are widely used as genome editing systems, nucleic acid detection systems and molecular visualization instruments [1]. In our laboratory using CRISPR/Cas9 technology we have obtained several KO mouse lines harboring deletions ranging from 2 to 20 base pairs. While 20 bp deletions are easily PCR-detected, when it comes to 2 bp deletions it is essential to genotype numerous mice using time-consuming Sanger sequencing. We propose a microdeletion/microinsertion detection system based on Cas12a nuclease fromLachnospiraceaebacterium (LbCas12a). Its active complex consists of the Cas12a enzyme and one crisprRNA [2]. A special sequence called protospacer adjacent motif (PAM) is required for target recognition by LbCas12a. In our laboratory we have discovered new PAM TTAA recognized by LbCas12a [3]. Via agarose electrophoresis and fluorescent analysis using FAM-labeled probes we have shown that new PAM allowed detection of 1 bp substitutions in target DNAin vitro. Also we have tested different FAM-labeled probes and have shown that AT-rich probes longer than 10 bp are cleaved most effectively. Finally we have used our system for detecting 2 bp deletions in Pde6b-KO mice and Grin3A-KO mice and successfully distinguished these mice from wild type mice. In conclusion, new PAM TTAA greatly increases specificity of DNA cleavage allowing to use this system as an instrument for rapid detection if microdeletions in mice. This work was supported by a Saint Petersburg State University grant for the development of scientific research (ID 94030690) and the Genome Research Centre development program “Kurchatov Genome Centre–PNPI” (agreement No. 075-15-2019-1663)

The formation of self-perpetuating protein aggregates such as amyloids is associated with various diseases and provides a basis for transmissible (infectious or heritable) protein isoforms (prions). Many human proteins involved in the regulation of transcription contain potentially amyloidogenic regions. Here, it is shown that short N-terminal isoforms of the human protein PHC3, a component of the chromatin-modifying complex PRC1 (Polycomb repressive complex 1), can form prion-like aggregates in yeast assays, exhibit amyloid properties in the E. coli-based C-DAG assay, and produce detergent-resistant aggregates when ectopically expressed in cultured human cells. Moreover, aggregates of short isoforms can sequester the full-length PHC3 protein, causing its accumulation in the cytosol instead of the nucleus. The introduction of an aggregating short PHC3 isoform alters the transcriptional profile of cultured human cells. It is proposed that the aggregation of short isoforms is involved in the feedback downregulation of PRC1 activity, leading to more open chromatin configuration.

Amyloids are protein aggregates characterized by their insolubility in detergents and ability to form fibrils. They are often associated with various diseases, including neurodegenerative disorders, type 2 diabetes and certain forms of cancer. Amyloids also play important roles in bacteria and different physiological processes in both lower and higher eukaryotes. Together with the laboratory of Prof. Y.O. Chernoff we have developed a comprehensive approach for screening new potentially amyloidogenic proteins. This involves using bioinformatics algorithms to predict protein amyloidogenicity and further verifying using a yeast model. We have created a yeast test system specifically designed to study changes in phenotype in genetically modified Saccharomyces cerevisiae strains [1]. This system involves the production of recombinant amyloidogenic proteins fused with reporter proteins Sup35N or YFP. Using yeast assay, we have investigated 22 human proteins that were predicted to be amyloidogenic by ArchCandy algorithm [2]. Currently, additional in vitro biochemical tests are underway with proteins that have shown the potential to form amyloids in yeast models. There are also plans to evaluate the amyloid-forming ability of specific human proteins in mammalian cell cultures. These various approaches appear to be enhancing our comprehension of the impact of amyloid formation in health and disease.

SARS-CoV-2 main protease (M^pro) is essential for viral polyprotein processing and represents a prime target for antiviral drug discovery. However, most available screening strategies rely on computational predictions or cell-free biochemical approaches that provide limited functional context and often require specialized instrumentation, while mammalian cell-based models remain costly and require high biosafety levels. Accordingly, there remains a shortage of simple, rapid, and biosafe functional screening tools suitable for early-stage prioritization of potential M^pro inhibitors, particularly those derived from natural sources and in urgent situations such as the COVID-19 pandemic. In this study, a bacterial colorimetric reporter assay was developed that directly links SARS-CoV-2 M^pro activity to β-galactosidase function in Escherichia coli. To the best of our knowledge, the developed assay represents the first bacterial colorimetric model for functional detection of SARS-CoV-2 M^pro inhibition based on a phenotypic readout. The assay enables the rapid visual detection of protease inhibition on X-gal-containing medium and provides a cost-effective and biosafe platform for prioritizing candidate inhibitors, under standard laboratory conditions, prior to further validation. Due to its bacterial expression context, this assay is intended for functional screening to provide the most promising candidate compounds and/or extracts for subsequent biochemical or mammalian cell-based validation; it is not intended to determine quantitative potency or to replace further validation approaches. It should be noted that the selective compound uptake in E. coli restricts the range of chemical compositions that can be evaluated using this method. Therefore, proof-of-concept application was demonstrated using pomegranate juice, a representative natural inhibitor source, rather than most currently known specific M^pro inhibitors. In addition, other plant-derived preparations, including rhubarb, grape, and red/black currant juices, were tested demonstrating the assay’s applicability to diverse natural matrices.