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Vol 89, No 9 (2024)
Articles
Universal adapter protein Bag3 and small heat shock proteins
Abstract
Bag3 (Bcl-2 associated athanogene) protein contains several functional domains and interacts with a number of different protein-partners among which are small heat shock proteins (sHsp) and heat shock proteins of Hsp70 family. Triple complex Bag3-sHsp-Hsp70 binds and transports to phagosomes denatured proteins and thus plays key role in chaperone assisted selective autophagy (CASA). In addition, this complex regulates formation and pathway of stress-granules (granulostasis) and participates in cytoskeleton regulation. Since Bag3 and small heat shock proteins participate in many cellular processes mutations of these proteins are often correlate with neurodegenerative diseases and cardiomyopathies. Probable role of sHsp in Bag3 dependent processes is analyzed.
1479-1491
The role of filamin C in muscle cells
Abstract
Filamin C (FLNC) is a member of a high-molecular weight protein family, which bind actin filaments in the cytoskeleton of various cells. In human genome FLNC is encoded by the FLNC gene located on chromosome 7 and is expressed predominantly in striated skeletal and cardiac muscle cells. Filamin C is involved in organization and stabilization of thin actin filaments three-dimensional network in sarcomeres, and is supposed to play a role of mechanosensor transferring mechanical signals to different protein targets. Under mechanical stress FLNC can undergo unfolding, that increases the risk of its aggregation. FLNC molecules with an impaired native structure can be eliminated by BAG3-mediated chaperone-assisted selective autophagy. Mutations of the FLNC gene can be accompanied by changes in FLNC interaction with its protein partners and can lead to the formation of aggregates, which overload autophagy and proteasome protein degradation systems, thus resulting in the development of various pathological processes. The molecular mechanisms of FLNC-associated congenital disorders, called filaminopathies, remain poorly understood. This review is devoted to the analysis of structure and mechanisms of filamin C function in muscle and heart cells in normal state and in FLNC-associated pathologies. The presented data summarize the results of research at the molecular, cellular and tissue levels and allow us to outline promising ways for further investigation of filaminopathies pathogenetic mechanisms.
1492-1505
Diversity of molecular functions of RNA-binding ubiquitin ligases from the MKRN protein family
Abstract
Makorin RING finger protein family includes four members (MKRN1, MKRN2, MKRN3, and MKRN4) that belong to E3 ubiquitin ligases and play a key role in various biological processes, such as cell survival, cell differentiation, and innate and adaptive immunity. MKRN1 contributes to the tumor growth suppression, energy metabolism, anti-pathogen defense, and apoptosis and has a broad variety of targets, including hTERT, APC, FADD, p21, and various viral proteins. MKRN2 regulates cell proliferation, inflammatory response; its targets are p65, PKM2, STAT1, and other proteins. MKRN3 is a master regulator of puberty timing; it controls the levels of gonadotropin-releasing hormone in the arcuate nucleus neurons. MKRN4 is the least studied member of the MKRN protein family, however, it is known to contribute to the T cell activation by ubiquitination of serine/threonine kinase MAP4K3. Proteins of the MKRN family are associated with the development of numerous diseases, for example, systemic lupus erythematosus, central precocious puberty, Prader–Willi syndrome, degenerative lumbar spinal stenosis, inflammation, and cancer. In this review, we discuss the functional roles of all members of the MKRN protein family and their involvement in the development of diseases.
1506-1522
Anomalous temperature dependence of the triplet-triplet energy transfer in Cereibacter sphaeroides I(L177)H mutant reaction centers
Abstract
In photosynthetic reaction centers, quenching of the primary donor triplet state by energy transfer to the carotenoid molecule provides efficient suppression of singlet-excited oxygen generation, the potent chemical oxidant. This process in the Cereibacter sphaeroides reaction centers is thermoactivated, and discontinues at temperatures below 40 K. In these reaction centers, substitution of amino acid residue isoleucine for hystidine at the 177 position of the L-subunit results in a sharp decrease of the activation energy, so the carotenoid triplets are populated even at 10 K. Activation energy of the T-T energy transfer was estimated as 7.5 cm−1, which is more than 10-fold lower than the activation energy of the original reaction centers. At certain temperatures the energy transfer in the mutant is decelerated, which is related to the increase of the effective distance of the triplet-triplet transfer. To our knowledge, the described mutation presents the first reaction center modification leading to a significant decrease in activation energy of T-T energy transfer to the carotenoid molecule. The I(L177)H mutant reaction centers present a considerable interest for further studies of the triplet state quenching mechanisms, and of other photophysical and photochemical processes in reaction centers of bacterial photosynthesis.
1523-1531
The influence of long-term housing in overcrowding on the social behavior of rats and the expression of genes associated with neuroinflammation
Abstract
The effect of long-term housing in overcrowding on the social behavior of adult male Wistar rats was studied. From 30 to 180 postnatal day (PND) the rats lived in standard conditions for 5 individuals (360 cm2 per rat, group of STD), or in crowded conditions for 15 individuals in cages (120 cm2 per rat, group of CROW). Starting from 100 PND, the behavior of rats was studied in social preference test, social dominance tube test and in the resident intruder test for aggressive behavior. After decapitation of rats by 180 PND, brain samples from the amygdala, dorsal hippocampus, ventromedial hypothalamus and medial prefrontal cortex were taken for subsequent analysis of the expression of IL-1β, TNF, TGF-β1, IL-6 mRNA genes using a real-time polymerase chain reaction. Compared with the STD group, rats of CROW group had a shorter interaction time with a social object in the social preference test. Males of the CROW group had more wins in the tube test compared to the STD group, as well as more attacks in the resident intruder test. The expression of IL-1β in the hippocampus and medial prefrontal cortex was significantly increased in the rats of the CROW group, as well as the expression of TGF-β1 in the hippocampus, amygdala and prefrontal cortex was increased. The social stress of overcrowding led to an increase in social dominance, aggressiveness and decreased sociability. Changes in social behavior in the CROW rats were accompanied by an increase in the expression of the proinflammatory cytokine IL-1β and the anti-inflammatory cytokine TGF-β1 in a number of brain structures, which can be considered, respectively, as a manifestation of neuroinflammation and compensatory processes.
1532-1545
Serum exosome protein composition in ischemic stroke patients is associated with cognitive impairment in the post-stroke period
Abstract
Ischemic stroke (IS) and subsequent neuropsychiatric disorders are among the leading causes of disability worldwide. Several strategies have been previously proposed to utilize exosomes to assess the risk of IS-related diseases. The aim of this work was to evaluate serum exosome proteins of IS patients in the chronic post-stroke period and to search for their associations with the development of post-stroke moderate cognitive impairment (MCI). Comparative quantitative proteomic analysis of serum exosomes of patients without poststroke MCI (19 patients mean age 52.0 ± 8.1 years) and patients with post-stroke MCI (11 patients, mean age 64.8 ± 5.6 years) revealed significant differences in the levels of 62 proteins out of 186 identified. Increased levels of proteins associated with the immune system and decreased levels of proteins involved in lipid metabolism were observed in patients with MCI compared to patients without MCI in the chronic post-stroke period. The obtained data suggest that a higher level of immune system activation in patients in the distant period after IS may be one of the risk factors for the development of post-stroke cognitive disorders and suggest the participation of exosomal transport in these processes.
1546-1561
A new approach for studying poly(ADP-ribose) polymerase inhibitors using permeabilized adherent cells
Abstract
Poly(ADP-ribose) polymerase (PARP) inhibitors have been proposed as pharmacological agents in the treatment of various diseases. Recently, factors and mechanisms responsible for regulating PARP catalytic activity have been identified, some of which can significantly influence the effectiveness of inhibitors of this enzyme. In this regard, it is important to develop new models and methods that would reflect the cellular context in which PARP functions. We proposed to use digitonin-permeabilized adherent cells to study poly(ADP-ribosyl)ation reaction (PARylation) in order to maintain the nuclear localization of PARP and to control the concentrations of its substrate (NAD+) and tested compounds in the cell. A specific feature of the approach is that before permeabilization, cellular PARP is converted to the DNA-bound state under conditions preventing premature initiation of the PARylation reaction. Experiments were carried out in rat H9c2 cardiomyoblasts. The activity of PARP in permeabilized cells was analyzed by measuring the immunofluorescence of the reaction product poly(ADP-ribose). The method was verified in the studies of PARP inhibition by the classic inhibitor 3-aminobenzamide and a number of new 7-methylguanine derivatives. One of them, 7,8-dimethylguanine, was found to be a stronger inhibitor compared to 7-methylguanine, due to a formation of additional hydrophobic contact with the protein. The proposed approach opens up new prospects for studying the mechanisms of PARP activity regulation in cells and can be used in high-throughput screening of PARP inhibitors.
1562-1575
T-cell receptor cross-reactive to SPR homologous epitopes of coronaviruses
Abstract
The COVID-19 pandemic, initiated by the rapid spread of the novel SARS-CoV-2 coronavirus, has increased interest in the study of the T-cell immune response. A polyclonal and cross-reactive T-cell response to seasonal coronaviruses and other SARS-CoV-2 strains have been shown to reduce disease severity. In this study, we investigated the immunodominant T-cell epitope SPRWYFYYYL from the nucleocapsid protein of SARS-CoV-2 virus. The immune response to this epitope is characterized by the formation of highly homologous (convergent) receptors found in the T-cell receptor (TCR) repertoires of different individuals. This epitope belongs to highly conserved peptides that are rarely mutated in new strains of SARS-CoV-2 and are homologous to epitopes from seasonal coronaviruses. There is a suggestion that the presence of cross-reactive responses to homologous peptides contributes to reduced COVID-19 disease severity. However, other investigators have questioned this, suggesting that low affinity receptors reduce the strength of the immune response. The aim of this study was to evaluate the effect of amino acid substitutions in the SPR epitope on the affinity of binding to a specific T-cell receptor. To this end, antigen-dependent cellular expansions were performed on samples from four COVID-19-transfected donors and TCR repertoires were sequenced. The resulting SPR-specific β-chain TCRs had greater sequence diversity than that of α-chains, but public receptors were also present in all four donor repertoires, three of which were cloned and used to generate a Jurkat E6-1 TPR cell line. Only for one of the three receptors we demonstrated the ability to be activated by the SPR peptide and recognized the mutant LPRWYFYYY homologue from seasonal coronaviruses with the same affinity as the wild-type peptide. This indicates that the presence of the mutation did not affect the strength of the immune response, which may explain why the cross-reactive response to the SPR epitope is so frequent and contributes positively to COVID-19 infection.
1576-1589
Changes of transcriptomic activity in rat brain cells under the influence of synthetic adrenocorticotropic hormone-like peptides
Abstract
Synthetic peptides have a wide range of clinical effects. Of particular interest are peptides based on adrenocorticotropic hormone (ACTH) both as already used and as potential drugs for preventing the consequences of cerebral ischemia. However, it is necessary to study the peptide influence on brain cells under normal physiological conditions, including understanding the risks of their use. Here, we used high-throughput RNA sequencing (RNA-Seq) to identify differentially expressed genes (DEGs) in the frontal cortex of rats receiving intraperitoneal administration of ACTH-like peptides ACTH(4-7)PGP (Semax) and ACTH(6–9)PGP or saline. We identified 258 and 228 DEGs, respectively, with a threshold of > 1.5 and Padj < 0.05 at 22.5 hours after the first administration of Semax and ACTH(6-9)PGP. Metabolic pathways, characterizing both the general and specific effects of peptides on the transcriptome were identified. Both peptides predominantly caused a decrease in the expression of genes associated with the immune system. At the same time, when comparing the effects of ACTH(6-9)PGP relative to Semax, DEGs were identified that characterized the main differences in the effects of the peptides. These genes were mostly downregulated and associated with neurosignaling systems and regulation of ion channels and characterized differences in the effects of peptides. Our data show how differences in the structure of ACTH derivatives are associated with changes in the brain cell transcriptome following exposure to these related peptides. Furthermore, our results evident that when studying the influence of regulatory peptides on the transcriptome in pathological conditions, it is necessary to take into account their actions under normal physiological conditions.
1590-1604