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The experimental results available in the ProteomeXchange database (accession code PXD016538) (Simats et al. (2020) Molecular and Cellular Proteomics, 19(12), 1921-1936) obtained using a comprehensive multi-omics approach were analyzed in mouse blood to identify potential biomarkers of ischemic stroke. Acetylation, methylation, and ubiquitination were considered as post-translational modifications. The analysis of the significance of changes in the level of protein modification was evaluated for ischemic tissue in comparison with tissue undamaged by stroke and control taken from mice after sham operation. At the level of statistically significant differences according to the Mann-Whitney test (p < 0.05), 2 proteins were found (Q02248 and Q8BL66); for additional 7 proteins, the differences were at the level of a statistical trend (p < 0.1). For 7 of 9 selected proteins there are reports in the literature, for their association with cerebral ischemia.

Colorectal cancer (CRC) is a malignancy featured by a poor overall survival and a high recurrence rate, whereas the biomarkers for CRC remain to be investigated. Herein, it was found that lysophosphatidylcholine acyltransferase 1 (LPCAT1) was highly expressed in CRC, and LPCAT1 overexpression significantly promoted CRC cell proliferation, while it was reversed by LPCAT1 depletion. In addition, HECT domain-containing 2 (HECTD2) protein was determined as a post-translational mediator of LPCAT1 because HECTD2 co-immunoprecipitated with high ubiquitinated LPCAT1. Furthermore, upregulated LPCAT1 rescued the impairment of CRC cell proliferation caused by HECTD2 overexpression. In conclusion, our findings supported HECTD2/LPCAT1 axis as a potential prognostic biomarker in CRC.

One of the most informative methods to study the roles of individual proteins in cell functions is based on changing their intracellular concentrations. Genetic knockouts or knockdowns are most commonly used for the purpose. However, acting directly at the level of an expressed protein is more informative or convenient to perform in some cases. This action should ideally be controlled in time and reversible. The review analyzes the current data on systems developed to achieve controlled degradation of proteins via their ubiquitination with subsequent proteasome-mediated degradation or other mechanisms.

The ubiquitin-proteasome system (UPS) is an important regulator of the main cellular processes. The components of the UPS are involved in the regulation of the cell cycle, signal transduction, the cell response to DNA damage, metabolism, and transcription control. E3 ubiquitin ligases (the enzymes that covalently attaches ubiquitin to target proteins) play a key role in the functioning of the UPS. The Drosophila tumor suppressor Hyd (hyperplastic discs) is one of the most interesting E3 ligases; it is required for the regulation of proliferation, growth, and cell differentiation. The study of hyd mutations in different tissues of Drosophila demonstrated that depending on the cellular context, Hyd can not only perform proteolytic functions associated with protein degradation, but can also, interacting with other proteins and/or nucleic acids, act as an important regulator of cellular processes.

It becomes increasingly clear that ubiquitination of cellular proteins is not an indispensable prerequisite of their degradation in proteasomes. There are a number of proteins to be eliminated which are not pre-ubiquitinated for their recognition by regulatory subcomplex of 26S proteasome, but which directly interact with the 20S proteasome core particle (20S proteasome). The obligatory precondition for such interaction consists in existence of disordered (hydrophobic) fragments in the target protein. In this study we have investigated the interaction of a number of multifunctional (moonlighting) proteins (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase) and neurodegeneration-related proteins (a-synuclein, myelin basic protein) with 20S proteasome immobilized on the SPR-biosensor chip and stabilized by means of a bifunctional agent dimethyl pimelimidate (in order to prevent possible dissociation of this subcomplex). Only two of all investigated proteins (aldolase and pyruvate kinase) interacted with the immobilized 20S proteasome (Kd of 8.17´10-7 M and 5.56´10-7 M, respectively). In addition to earlier detected GAPDH ubiquitination, mass spectrometric analysis of the studied proteins revealed the presence of the ubiquitin signature (Lys-e-Gly-Gly) only in aldolase. Oxidation of aldolase and pyruvate kinase, which promotes elimination of proteins via their direct interaction with 20S proteasome, caused a 2-3-fold decrease in their Kd values as comparison with this parameter obtained for the intact proteins. The results of this study provide further evidence for direct interaction of both ubiquitinated proteins (aldolase), and non-ubiquitinated proteins (pyruvate kinase) with the 20S proteasome core particle (20S proteasome). The effectiveness of this interaction is basically equal for the ubiquitinated proteins and non-ubiquitinated proteins.

The assembly of LIM-homeodomain (LIM-HD) transcriptional complex plays important roles in early neuronal development. The stability of LIM-HD is controlled by single-strand binding protein 3 (SSBP3) via a cascade mechanism protecting it from proteasomal degradation. The expression level of SSBP3 has to be precisely regulated. Although a decrease of SSBP3 level is associated with several diseases, the mechanism of SSBP3 downregulation and whether SSBP3 itself is subject to proteasomal degradation remain largely unknown. Two strongly conserved transcripts of the SSBP3 gene, SSBP3a and SSBP3c, were cloned from a human brain cDNA library. By RT-PCR, we show that Ssbp3c is continuously expressed in both embryonic and adult mouse brain, whereas Ssbp3a is restricted to embryonic brain tissue. By co-IP and GST pulldown assays, we identified SIVA1 as a novel SSBP3-binding factor. In a ubiquitination assay, we show that SIVA1 enhances the ubiquitination of SSBP3 and regulates its abundance. Our findings reveal the proteasomal degradation of SSBP3 for the first time and provide a rationale for an SIVAl-SSBP3-dependent mechanism for the disassembly of LIM-HD multiprotein complexes.

Proteasomes are large supramolecular protein complexes present in all prokaryotic and eukaryotic cells, where they perform targeted degradation of intracellular proteins. Until recently, it was generally accepted that prior proteolytic degradation in proteasomes the proteins had to be targeted by ubiquitination: the ATP-dependent addition of (typically four sequential) residues of the low-molecular ubiquitin protein, involving the ubiquitin-activating enzyme, ubiquitin-conjugating enzyme and ubiquitin ligase. The cytoplasm and nucleoplasm proteins labeled in this way are then digested in 26S proteasomes. However, in recent years it has become increasingly clear that using this route the cell eliminates only a part of unwanted proteins. Many proteins can be cleaved by the 20S proteasome in an ATP-independent manner and without previous ubiquitination. Ubiquitin-independent protein degradation in proteasomes is a relatively new area of studies of the role of the ubiquitin-proteasome system. However, recent data obtained in this direction already correct existing concepts about proteasomal degradation of proteins and its regulation. Ubiquitin-independent proteasome degradation needs the main structural precondition in proteins: the presence of unstructured regions in the amino acid sequences that provide interaction with the proteasome. Taking into consideration that in humans almost half of all genes encode proteins that contain a certain proportion of intrinsically disordered regions, it appears that the list of proteins undergoing ubiquitin-independent degradation will demonstrate further increase. Since 26S of proteasomes account for only 30% of the total proteasome content in mammalian cells, most of the proteasomes exist in the form of 20S complexes. The latter suggests that ubiquitin-independent proteolysis performed by the 20S proteasome is a natural process of removing damaged proteins from the cell and maintaining a constant level of intrinsically disordered proteins. In this case, the functional overload of proteasomes in aging and/or other types of pathological processes, if it is not accompanied by triggering more radical mechanisms for the elimination of damaged proteins, organelles and whole cells, has the most serious consequences for the whole organism.

Zinc and vitamin C supplementation of the body is important for CNS functioning. Zinc ions are involved in the neurotransmission (signal transmission from acetylcholine, catecholamine, serotonin, prostaglandin receptors) and in ubiquitin-related protein degradation. Zinc deficits are associated with Alzheimer's disease and depression. Zinc supplementation (10-30 mg daily) improves neurologic recovery rate in patients with stroke and brain injury, has a positive impact on memory and reduces hyperactivity in children. Vitamin C, a zinc synergist, maintains antioxidant resources of the brain, synaptic activity and detoxification. Vitamin C in dose 130-500 mg daily should be used to prevent dementia and neurodegenerative pathology.

Transcription factor p63 is a member of the p53 protein family. Due to the high degree of structural similarity p53, p63, and p73 are known to have overlapping functions relating to cell cycle regulation, apoptosis and tumor transformation. Furthermore, p63 plays crucial role in epidermal tissue development and differentiation. Pirh2 (product of RCHY1 gene) is an E3 ubiquitin ligase modifying all three members of the p53 family resulting in their subsequent proteasomal degradation. Our results demonstrate that p63, similar to p53, is able to regulate expression levels of Pirh2. Importantly, Pirh2 expression is activated only by transcriptionally active isoform of p63--TAp63, but not the N-terminally truncated ΔNp63.

Protein ubiquitination is considered as an important mechanism that is responsible not only for specific labeling of proteins for their subsequent degradation but also for localization of proteins in the cell and regulation of protein-protein interactions. In the context of protein-protein interactions binding of (mono/poly)ubiquitinated molecules to proteins containing specific ubiquitin binding domains appear to play the decisive role. Although formation of the ubiquitin interactome has been demonstrated for cytosol, involvement of mitochondria and associated extramitochondrial proteins into such interactions still requires detailed investigation. In this study using an optical biosensor we have demonstrated binding of proteins of mouse brain mitochondrial lysates to immobilized monomeric ubiquitin. Model purified proteins, which are known to be associated with the outer mitochondrial compartment (glyceraldehyde-3-phosphate dehydorgenase, creatine phosphokinase), interacted with immobilized ubiquitin as well as with each other. This suggests that (poly)ubiquitinated chains may be involved in protein-protein interactions between ubiquitinated and non-ubiquitinated proteins and thus may contribute to formation of (mitochondrial) ubiquitin subinteractome.

In the present study we have used a transgenic mice overexpressing an amyloidogenic protein, gamma-synuclein, in the nervous system to address the effect of dimebon on proteinopathy progression. Neuroprotective effect of chronic dimebon administration in these mice at organismal level was confirmed by the increased lifespan. Using histological and biochemical approaches we have demonstrated that dimebon reduced the number of amyloid inclusions in spinal cord of transgenic animals and decreased the content of ubiquitinated proteins in detergent-insoluble fractions. These effects are likely to occur at the level of aggregated protein species, since transgene expression was not altered. Thus, pathological protein aggregation serves as one of dimebon targets in neurodegeneration.

26S proteasome is a highly specialized evolutionarily conserved ribonucleoprotein machine to instantly restore order in the intracellular proteome. It is composed of the 20S core particle and attached there to regulatory complexes. In addition to a comprehensively studied ubiquitin-proteasome pathway of protein degradation, proteasomes are involved (directly or indirectly) in most stages of the regulation of gene expression (at the levels of transcription, splicing, mRNA stability control, etc.). In the present review an attempt to systematize the recent literature on proteasomes' role in the regulation of transcription at all stages, including the modulation of the activity and stability of transcription factors, chromatin remodulation, elongation and termination of RNA synthesis is made. In addition, very interesting but controversial feature of the proteasome: their ability to bind and hydrolyze certain types of RNA, is observed. Obviously, universal role of proteasomes in gene regulation is determined by complicated composition of these protein complexes possessing a unique set of different enzymatic activities: ATPase/helicase, proteolytic and ribonuclease, which, depending on the case, can be used together or separately.

In this paper, we present a comparative analysis of different methods of purification of proteasomes from the culture medium in which proerithroleukemia human K562 cells were grown. The results obtained allowed us to purify proteasomes from samples of conditioned cell culture medium and control the quality of the proteasome preparations at all stages of their separation. Extracellular proteasomes purified via different approaches possess all the three types of peptidase activity described for intracellular counterparts.

Eukaryotic gene expression is known as a multistep process of high complexity. Transcription is one of cardinal and tightly regulated phase during gene expression. To provide accurate and precise work of gene regulation apparatus including a plethora of modification of chromatin structure and nucleosome dynamic turnover must be occurred. All transcription steps are under control of large multiprotein coactivator complexes. In this review we discuss an evolutionary conservative SAGA complex, which acetylates and deubiquitinates histones during transcription activation and furthermore is involved in subsequent stages of mRNP biogenesis and export.

Ubiquitin-dependent proteasomal degradation is one of the major pathways of non-lysosomal protein degradation in the cell. The ubiquitination process involves several enzymatic reactions and includes the following enzymes: E1--activating, E2--conjugating, and the third--E3--ligating enzymes. E3 ligases determine the specificity of ubiquitination reaction, i. e. what target protein will be subjected to the covalent modification by ubiquitins. The p53b tumor suppressor protein is one of the most intensively studied over the past several decades. Regulation of its activity is a complex and multi-level process that involves many factors. Ubiquitination is one of the major post-translational modifications of p53, and plays a fundamental role in the control of p53 function, its amount, activity and subcellular localization. This review is focused on p53-specific E3 ubiquitin ligases that are potential targets for cancer therapy using small molecule inhibitors.

The analysis of the extracellular proteasomes by isobaric tagging for relative and absolute quantifications (iTRAQ) mass spectrometry has been carried out. Here we show a standard set of 26S proteasomal subunits in the composition of the extracellular proteasomes. Moreover, extracellular proteasomes have a number of PA200 activators, which, as previously thought, are localized in the cell nucleus. Posttranslational modifications (PTMs) of subunits of the extracellular proteasomes were revealed by iTRAQ mass spectrometry. For the first time we have identified several ubiquitination and acetylation sites on subunits alpha2 (K196), alpha4 (K189 and K234), alpha6 (K217), and Rpn6 (A2). We have revealed a large number of proteasome-interacting proteins that are involved in various cell processes, such as transcription, DNA repair, translation, cytoskeletal proteins and the proteins of the ubiquitin-proteasome system (UPS). Immunoblot analysis has confirmed the interactions between purified extracellular proteasomes and nine proteins which were randomly selected from the set of interacting proteins.

The crucial predictor of renal cell carcinoma (RCC) is a clinical stage, although the significance of this parameter isn 't confirmed for all histological types of carcinomas. It remains unknown what factors ensure the sensitivity of the tumor to chemotherapy and why metastasis is accompanied by the development of resistance to therapy. The data about the prognosis of RCC rare variants are extremely scarce. Ubiquitin-dependent proteolysis plays an important role in the development of RCC, but the prognostic significance of this signal way's proteins is poorly understood. In order to find out significant factors for prognosis of RCC clinical course it's necessary to use the complex spectrum of tumor growth regulation factors and genetic tests.

Analysis of ubiquitination of EGF receptor carrying different mutations of C-terminal domain was done. The mutants differed both by the set of major autophosphorylation sites that determine the way of interaction with ubiquitin-ligase c-Cbl, and by the presence of lysine residues which can be possible acceptor sites for ubiquitin. It was found that the receptor lacking tyrosine kinase activity due to lysine for phenylalanine substitution at ATP-binding site of tyrosine kinase (TK) domain (K721) failed to be ubiquitinated as well as the receptor without all binding sites for c-Cbl (CD165), while dynamics and pattern of ubiquitination of other deletion mutants was significantly different. The mutant lacking Grb2 binding sites but able to bind c-Cbl directly (CD123) was minimally ubiquitinated and only at early stages upon EGF endocytosis stimulation. At the same time, the receptor possessing all binding sites for Cbl but lacking C-terminal domain of 63 aminoacid residues (CD63) which contains two autophosphorylation sites (Y1148 and 1173) and 4 lysines, was less ubiquitinated and had more low-ubiquitinated forms comparing to the WT one. However, these lysines are not acceptor sites for ubiquitin since the full-size receptor lacking like CD63 the same major autophosphorylation sites underwent ubiquitination similar to the deletion mutant. Thus, C-terminal region of the EGF receptor, being not a substrate for ubiquitination per se, is involved in its regulation. It was also found that ubiquitination pattern at fast endocytosis differed from those at slow one. In the first case the total level of EGFR decreased dramatically as a result of efficient lysosomal degradation. The level of receptor-associated c-Cbl was practically the same, while the total intracellular c-Cbl dropped. Treatment of cells with proteasomal inhibition MG132 blocked the loss of Cbl only partially. In the second case, total amount of both EGF receptor and c-Cbl did not notably change that suggested recycling pathway for receptors even despite them beeng ubiquitinated.