[A Test System for Assessment of the Activity of Mutant Cas9 Variants in Saccharomyces cerevisiae].

The key component of the revolutionary Streptococcus pyogenes CRISPR/Cas genome editing technology is the multidomain protein Cas9. However, the specificity of wild type Cas9 is not sufficiently high for editing large genomes of higher eukaryotes, which limits the realization of the potential of genomic editing both in fundamental investigations and in the therapy of genetic diseases. The main way to obtain more specific variants of Cas9 is through mutagenesis followed by characterization of mutant proteins in in vitro or in vivo test systems. The in vitro and some in vivo test systems described in the literature are often labor-intensive and have scaling limitations, which makes it challenging to screen SpCas9 mutant variant libraries. In order to develop a simple method for high-throughput screening of Cas9 mutants in vivo, we characterized three test systems using CRISPR/Cas9-mediated inactivation of the reporter genes, tsPurple, ADE2, and URA3, in the Saccharomyces cerevisiae yeast as a model subject. We measured the activities of high-precision forms of Cas9, evoCas9, and HiFiCas9, and compared them with the wild-type form. ADE2 gene inactivation was found to be the most valid method for the evaluation of Cas9 activity. In the test-system developed, the sensitivity to chromatin structure was demonstrated for the high-fidelity variant of Cas9, HiFiCas9. The proposed test-system can be used for the development of new generation genome editors.

[Rpn4p without the DNA-Binding Domain Provides Saccharomyces cerevisiae Resistance to Oxidative Stress and Cycloheximide].

The ubiquitin-proteasome system is involved in the control of all essential molecular processes under normal conditions and the response of cells to stress. Rpn4p serves as a key transcriptional regulator of the proteasome in Saccharomycetes yeast and is also involved in the cellular response to various stresses. In addition to proteasomal genes, Rpn4 affects the expression of several hundred other genes, including genes involved in DNA repair and oxidative stress response. At the same time, the molecular mechanisms used by Rpn4 in controlling target genes and its functioning as a regulator of the cellular response to stress remain largely unclear. The aim of this work was to determine the Rpn4 domains required to ensure cell resistance to stress. It was shown that the N-terminal and central regions of the protein contain sites required for resistance to all types of stresses. The putative nuclear localization signal does not affect the functioning of Rpn4. Unexpectedly, a protein with the deletion of both zinc finger motifs that form the DNA-binding domain provides yeast resistance to oxidative stress and cycloheximide. Moreover, we showed that Rpn4 can be recruited to the promoter regions of the regulated genes even if they do not contain its binding sites. Based on these data, it can be assumed that Rpn4 is involved in gene regulation and the cellular response to stress due to protein-protein interactions.

[Structures of the Mouse Central Nervous System Contain Different Quantities of Proteasome Gene Transcripts].

Proteasomes are multisubunit complexes that degrade most intracellular proteins. Three of the 14 subunits of the 20S proteasome, specifically ß1, ß2, and ß5, demonstrate catalytic activity and hydrolyze peptide bonds after acidic, basic, and hydrophobic amino acids, respectively. Within proteasome, the constitutive catalytic subunits ß1, ß2, and ß5 can be substituted by the immune ßli, ß2i, and ß5i subunits, respectively. However, proteasomes do not always contain all the immune subunits at once; some proteasomes contain both immune and constitutive catalytic subunits simultaneously. Incorporation of immune subunits modifies the pattern of peptides produced by proteasomes. This is essential for antigen presentation and cellular response to stress as well as for a number of intracellular signaling pathways. We have developed a quantitative PCR-based system for the determination of the absolute levels of murine constitutive and immune proteasome subunits gene expression. Using the obtained system, we have estimated the expression levels of genes encoding proteasome subunits in the mouse central nervous system (CNS) tissues. We have shown that the quantity of transcripts of proteasome catalytic subunits in different CNS structures differed significantly. These data allow us to assume that the studied brain regions can be divided into two groups, with relatively "high" (cerebral cortex and spinal cord) and "low" (hippocampus and cerebellum) levels of proteasome subunit genes expression. Moreover, it was possible to distinguish structures with similar and significantly different gene expression profiles of proteasome catalytic subunits. Thus, the gene expression profiles in the cortex, spinal cord, and cerebellum were similar, but different from the expression profile in the hippocampus. Based on the obtained data, we suggest that there are differences in the proteasome pool, as well as in the functional load on the ubiquitin-proteasome system in different parts of the CNS.

[Dynamics of the Functional Activity and Expression of Proteasome Subunits during Cellular Adaptation to Heat Shock].

The ubiquitin-proteasome system (UPS) performs proteolysis of most intracellular proteins. The key components of the UPS are the proteasomes, multi-subunit protein complexes, playing an important role in cellular adaptation to various types of stress. We analyzed the dynamics of the proteasome activity, the content of proteasome subunits, and the expression levels of genes encoding catalytic subunits of proteasomes in the human histiocytic lymphoma U937 cell line immediately, 2, 4, 6, 9, 24, and 48 h after a heat shock (HS). The initial decrease (up to 62%) in the proteasome activity in cellular lysates was revealed, then 10 h after HS the activity began to recover. The amount of proteasomal α-subunits in the cells decreased 2 h after HS, and was restored to 24-48 h after HS. Fluctuations in the levels of mRNAs encoding proteasome catalytic subunits with the maximum expression 2 h after HS and a gradual decrease to 48 h after HS were observed. The average estimated number of mRNA copies per cell ranged from 10 for weakly to 150 for highly expressed proteasome genes. Thus, the recovery efficiency of UPS functionality after HS, which reflects the important role of proteasomes in maintaining cell homeostasis, was evaluated.

[Candida glabrata Rpn4-like Protein Complements the RPN4 Deletion in Saccharomyces cerevisiae].

Expression of Saccharomyces cerevisiae proteasomal genes is regulated in a coordinated manner by a system that includes the ScRpn4 transcription factor and its binding site known as PACE. Earlier we showed that, Rpn4-like proteins from the biotechnologically important yeast species Komagataella pfaffii (Pichia pastoris), Yarrowia lipolytica, and Debaryomyces hansenii are capable of complementing the RPN4 deletion in S. cerevisiae in spite of their low structural similarity to ScRpn4. The opportunistic yeast pathogen Candida glabrata has a gene coding for a Rpn4-like protein, which has not been characterized experimentally yet. The С. glabrata ortholog ScRpn4 was expressed heterologously and found to restore the stress resistance and expression of proteasomal genes in a mutant S. cerevisiae strain with a RPN4 deletion. This complementation required the unique N-terminal region of CgRpn4. The results indicate that CgRpn4 acts as a transcriptional activator of proteasomal genes. The S. cerevisiae model can be used for further structural and functional analyses of CgRpn4.

The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells.

Despite high interest in the cellular degradation machinery and protein degradation signals (degrons), few degrons with universal activity along species have been identified. It has been shown that fusion of a target protein with a degradation signal from mammalian ornithine decarboxylase (ODC) induces fast proteasomal degradation of the chimera in both mammalian and yeast cells. However, no degrons from yeast-encoded proteins capable to function in mammalian cells were identified so far. Here, we demonstrate that the yeast transcription factor Rpn4 undergoes fast proteasomal degradation and its central domain can destabilize green fluorescent protein and Alpha-fetoprotein in human HEK 293T cells. Furthermore, we confirm the activity of this degron in yeast. Thus, the Rpn4 central domain is an effective interspecies degradation signal.