•We showed proteasomal hydrolysis of yeast transcription factor Rpn4 in human cells•N-terminal degron of the yRpn4 cannot serve as an effective degron in human cells•We have shown that central domain ...of the yRpn4 contains efficient interspecies degron•Discovered degron can be used to induce degradation of target proteins 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.
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.
•The patient with an SLC6A1 mutation, typically linked to disorders like epilepsy and autism, exhibited symptoms consistent with schizophrenia and bipolar disorder.•The patient's polygenic risk score ...was aligned with both schizophrenia and bipolar disorder, despite no family history of psychiatric disorders.•The study contributed to ongoing discussion of the importance of GABAergic processes in schizophrenia's etiology.
The 26S proteasome is a multisubunit ATP-dependent protease complex and is necessary for the normal function of the eukaryotic cell and its survival in stress. Twenty years ago, we, in collaboration ...with German researchers, were the first to experimentally describe a system for coordinated regulation of proteasomal gene expression in the yeast Saccharomyces cerevisiae. This system consists of the ScRpn4 transcription factor and its binding site, called PACE. Based on the results of a bioinformatics search in the first sequenced yeast genomes, Rpn4-like proteins and PACE-like elements were postulated for other species of the class Saccharomycetes. We experimentally characterized Rpn4-like proteins in the biotechnologically significant yeast species Komagataella pfaffii (Pichia pastoris), Yarrowia lipolytica, and Debaryomyces hansenii and the opportunistic yeast Candida glabrata. As ample information accumulates for the genome sequences of new yeast species and strains, the question arises as to how diverse the regulatory system of proteasomal genes is in terms of structure and likely mechanisms of function. In this work, a bioinformatics search for Rpn4-like proteins and PACE-like elements was conducted in 3111 strains belonging to 427 yeast species of the class Saccharomycetes. It was shown that only the DNA-binding domain is conserved among Rpn4-like proteins, in accordance with conservation of PACE elements. Certain systems were found to contain more than one Rpn4-like protein with structural differences in the DNA-binding domain or to include an autoregulation of the genes for Rpn4-like proteins. Given that Rpn4-like proteins and proteasomes play a role in the cell response to stress, the diversity of systems for the regulation of proteasomal genes was assumed to corresponds to adaptation of organisms to their living environments.
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.
Methods for processing optical emission spectroscopy data in the study of the region of interaction between helium plasma with a density of the main gas He ≈ 10
12
–10
14
cm
–3
and electrons
n
e
≈ 10
...11
–10
13
cm
–3
and a tungsten sample in a PLM facility designed for testing materials with a plasma load are presented. A method for measuring the electron temperature using the coronal approximation is proposed. For the calculation, it is necessary to choose the ratio of the intensities of spectral lines that is most sensitive to the electron temperature; in this case, this is the ratio of the intensity of ionic lines to atomic lines. Comparison of the ratio of the experimental intensities of the He II 468.5 nm ion line and a number of He I atomic lines with well-known electron excitation constants with the calculated dependence of this ratio on the mean electron energy is a reliable method for the spectral determination of the electron temperature of a magnetized rarefied helium plasma. In experiment, the concentration of atomic helium is He ≈ 10
14
cm
–3
; the discharge current is 2–10 A; the voltage drop is 160–180 V; the radius and length of the discharge are 16 and 370 mm, respectively. The electron temperature measured using two singlet and two triplet lines of He I is
T
e
= 2.4 ± 0.2 eV. With consideration of the nonlocality of the electron energy distribution function (EEDF), the complex nature of the drift and diffusion of charges in crossed and inhomogeneous electric and magnetic fields, etc., the mean electron energy of
= (3/2)
kT
e
≈ 3.6 eV corresponding to this temperature can be considered a lower estimate for the energy of the Maxwellian section of the EEDF of the plasma.
The results of studies of materials under extreme thermal and plasma-beam loads, carried out to justify existing technological solutions in the design of thermonuclear reactors, are presented. The ...study of materials under plasma and beam loads and the intensifying heat transfer of in-vessel cooled components of fusion devices is reported. Tests of limiter and divertor mock-ups irradiated with high heat fluxes in electron beam and plasma facilities are described. Methods for cooling in-vessel components using advanced approaches with dispersed gas-liquid flow for heat removal, including at loads of up to 15 MW/m2 have been developed and tested.
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
C. 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.
Expression of the Saccharomyces cerevisiae proteasomal genes is regulated coordinately by ScRpn4 transcription factor and its binding site known as PACE. Genomic analysis demonstrates that only ...Saccharomycetes yeasts carry ScRpn4-orthologous genes and PACE-like elements in the promoters of proteasomal genes. This taxonomic group includes species of biotechnological importance such as Komagataella (Pichia) pastoris and Yarrowia lipolytica. A literature search has failed to identify studies focused on ScRpn4 orthologs of these yeast species. In the present work, using the genetic background of S. cerevisiae, we showed that ScRpn4 orthologs from K. pastoris and Y. lipolytica restore the mRNA levels of proteasomal genes and confer stress tolerance in a S. cerevisiae strain with knockout of the SCRPN4 gene. In other words, these proteins are functionally similar to ScRpn4 of S. cerevisiae.
The 26S proteasome is a multisubunit ATP-dependent protease complex and is necessary for the normal function of the eukaryotic cell and its survival in stress. Twenty years ago, we, in collaboration ...with German researchers, were the first to experimentally describe a system for coordinated regulation of proteasomal gene expression in the yeast
Saccharomyces cerevisiae
. This system consists of the ScRpn4 transcription factor and its binding site, called PACE. Based on the results of a bioinformatics search in the first sequenced yeast genomes, Rpn4-like proteins and PACE-like elements were postulated for other species of the class Saccharomycetes. We experimentally characterized Rpn4-like proteins in the biotechnologically significant yeast species
Komagataella pfaffii
(
Pichia pastoris
),
Yarrowia lipolytica,
and
Debaryomyces hansenii
and the opportunistic yeast
Candida glabrata
. As ample information accumulates for the genome sequences of new yeast species and strains, the question arises as to how diverse the regulatory system of proteasomal genes is in terms of structure and likely mechanisms of function. In this work, a bioinformatics search for Rpn4-like proteins and PACE-like elements was conducted in 3111 strains belonging to 427 yeast species of the class Saccharomycetes. It was shown that only the DNA-binding domain is conserved among Rpn4-like proteins, in accordance with conservation of PACE elements. Certain systems were found to contain more than one Rpn4-like protein with structural differences in the DNA-binding domain or to include an autoregulation of the genes for Rpn4-like proteins. Given that Rpn4-like proteins and proteasomes play a role in the cell response to stress, the diversity of systems for the regulation of proteasomal genes was assumed to corresponds to adaptation of organisms to their living environments.
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