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Everolimus, an mTORC1 inhibitor, may also affect proteasome activity in a manner similar to bortezomib, necessitating further investigation. In this study, we employed ultrafast expression proteomics in combination with cell viability and proteasome activity assays to identify potential secondary targets of everolimus and to obtain a more comprehensive understanding of its mechanism of action across the proteomes of multiple cancer cell lines. The results were compared with those obtained for bortezomib and lonidamine, which were used as positive and negative controls for proteasome inhibition, respectively. Our findings reveal that everolimus inhibits 20S proteasome in lung (A549) and colon (HCT116) cancer cells, while having no detectable effect in breast cancer cells (MCF-7). An in silico model of everolimus interaction with 20S proteasome was built suggesting an allosteric mechanism of inhibition.
Systemic blockade of proinflammatory cytokines such as IL-1, TNF, and IL-6 using therapeutic antibodies has proven effective in treating a wide range of autoimmune and other chronic inflammatory diseases. However, such blockade also suppresses non-redundant protective and homeostatic functions of cytokines, leading to a number of undesirable side effects. In this study, a novel bispecific mini-antibody featuring modules targeting human TNF and CD14 demonstrated efficacy in controlling TNF secretion from human peripheral blood monocytes. Administration of this antibody protected humanized TNF mice from lethal hepatotoxicity induced by a combination of LPS and D-galactosamine.
In recent years, targeted proteolysis systems have emerged as powerful tools for directed degradation of pathogenic proteins, offering novel therapeutic strategies for cancer, neurodegenerative disorders, and infectious diseases. This review systematizes key mechanisms and recent advances in inducible targeted proteolysis, including targeted proteasomal degradation (PROTACs, AbTACs, molecular glues), lysosome-mediated degradation (LYTACs, AUTACs, ATTECs) via endocytosis or autophagy, and targeted proteolysis in bacteria (BacPROTACs), which extends degradation technologies to prokaryotic systems. The structural features, advantages, and limitations of each platform are discussed in detail, along with key publications demonstrating their preclinical and clinical efficacy. Special attention is given to the prospects for translating these technologies into therapeutics, including overcoming challenges such as selectivity and in vivo delivery.
Mutations in the N-terminal peptide (Ser-Thr to Ala-Gly substitution) of the coat protein (CP) of potato virus X (PVX-ST) render its genomic RNA translationally competent, unlike in the wild-type PVX virions. Consequently, RNA within the PVX-ST virions can be translated without additional triggers (such as phosphorylation or interaction with the triple gene block 1 protein), unlike the encapsidated RNA of the wild-type virus. Comprehensive structural analysis using molecular dynamics (MD), small-angle X-ray scattering (SAXS), and tritium planigraphy revealed differences in the virion organization. The mutations were shown to increase hydrophobicity and induce partial folding of the N-terminal peptides. This triggers structural rearrangement in the PVX-ST virion: packing density of the coat proteins within the helical capsid is altered. This conclusion is supported by the SAXS data, increased accessibility for tritium labeling of the key CP domains (including the RNA-binding region), and reduced stability against the action of the sodium dodecyl sulfate detergent. The obtained results provide explanation for the mechanism by which the encapsidated RNA of the PVX-ST mutant becomes accessible to ribosomes. This mechanism is associated with structural rearrangement of the N-terminal coat protein peptide and change in the packing density of the helical capsid.
Hepatitis B virus (HBV) infects human hepatocytes, causing acute or chronic liver infection. Chronic HBV infection leads to progressive liver damage, potentially resulting in cirrhosis or hepatocellular carcinoma. One promising antiviral strategy involves activating cytidine deaminases of the APOBEC/AID family, which could induce mutational degradation of HBV. Using a CRISPRa-based transcriptional activation system with modified sgRNAs, we investigated antiviral and oncogenic effects of the activating genes encoding APOBEC3C, APOBEC3D, and APOBEC3H.
Schizophrenia is a severe mental disorder whose molecular mechanisms remain poorly understood. Investigating brain-derived neurotrophic factor (BDNF)-dependent signaling pathways and their contribution to schizophrenia pathogenesis is a promising research direction in schizophrenia research. BDNF activates multiple intracellular cascades, among which the MAPK/ERK pathway plays a central role. In this study, expression levels of key regulatory proteins of the MAPK/ERK signaling pathway (ERK1/2, STAT3, STAT5, NF-κB, IGF1R, IRS1, IR, TSC2, and CREB1) were examined in lysates of peripheral blood mononuclear cells (PBMCs) from schizophrenia patients using multiplex analysis. The study group included 58 patients diagnosed with schizophrenia (F20); the control group included 60 healthy individuals. The results revealed significantly increased expression of ERK1/2 and STAT3, along with decreased NF-κB levels, in PBMCs from schizophrenia patients compared to controls. Moreover, patients with leading positive symptoms exhibited elevated expression of CREB1 and ERK1/2. These findings suggest that dysregulation of the MAPK/ERK signaling may play a significant role in the pathogenesis schizophrenia. BDNF-dependent signaling pathways may therefore represent promising targets for diagnostics and therapy of this disorder.
Pathological aggregation of α-synuclein is a key event in the development of synucleinopathies, such as Parkinson's disease and Lewy body dementia. Currently, no effective disease-modifying therapy is available, necessitating the search for new therapeutic agents. One promising strategy involves the use of low-molecular-weight compounds capable of inhibiting the formation of toxic protein aggregates. This study evaluates the anti-aggregation properties of EC3222x, a conjugate of pharmacophoric fragments of amantadine and a fluorinated derivative of tetrahydro-γ-carboline. α-Synucleinopathy was modeled in the SH-SY5Y neuroblastoma cell line by transfection with a plasmid vector encoding the mutant human α-synuclein A53T protein. EC3222x at a concentration of 1 µM reduced the number of cells with α-synuclein A53T aggregates. Its efficacy was comparable to that of SynuClean-D and Buntanetap, known inhibitors of α-synuclein aggregation. Treatment with EC3222x reduced both the level of diffusely distributed intracellular α-synuclein and the formation of mature fibrillar aggregates and large aggresomes. Importantly, EC3222x did not affect the accumulation of another aggregation-prone protein, TDP-43, in a similar cellular model, indicating its specificity for α-synuclein. These findings suggest that EC3222x may represent a promising candidate for the development of therapeutic agents targeting synucleinopathies.
Insulin exerts a complex effect on metabolism, cell growth, and differentiation interacting with its receptor. Adipose tissue is one of the key targets for insulin; in this tissue insulin regulates the processes of energy storage, as well as tissue renewal and emergence of new adipocytes. Insulin activates conversion of glucose into fatty acids, inhibits lipolysis, and induces adipogenic differentiation of adipose tissue stem cells. The insulin receptor is a classic tyrosine kinase receptor that activate phosphoinositide-3-kinase and mitogen-activated protein kinase signaling cascades. At the same time, insulin receptor activates several non-canonical signaling cascades that determine features of the receptor functioning. For example, insulin can affect phosphoinositide metabolism, as well as calcium and redox-dependent signaling. In addition, the insulin receptor can also interact with the trimeric G proteins-coupled receptors (GPCRs). Here, we review canonical and non-canonical signaling cascades activated by the insulin receptor and molecular mechanisms of their involvement in regulating the human adipose tissue renewal.
Ecological adaptations of a species can be shaped by its repertoire of gene variants. The black garden ant Lasius niger shows high level of CYP9E duplication. In contrast to its congener, the jet ant L. fuliginosus, it exhibits tolerance toward fungus-infected aphids. In these two species, we compared expression of a subset of CYP9E genes, potentially involved in mycotoxin metabolism. No significant differences in expression were found. Similarly to L. niger, the jet ant has six copies of these genes, grouping pairwise on the phylogenetic tree with their L. niger counterparts. Beyond the gene subset targeted in the expression study, we found multiple CYP9E genes in the genomes of L. niger, L. fuliginosus, and - fewer by a third - in the outgroup Formica rufa, suggesting CYP9E amplification as an ancestral trait of the genus Lasius or a more basal clade.
The TPO gene belongs to the group of genes responsible for the biosynthesis of thyroid hormones and encodes thyroid peroxidase, a key enzyme involved in this process. Mutations in these genes can result in thyroid dysfunction characterized by reduced levels of thyroid hormones. Hypothyroidism caused by TPO pathogenic variants typically presents as permanent hypothyroidism and is frequently associated with endemic goiter. This analytical review summarizes and systematizes data from the studies conducted in different regions of the world on mutations identified in the TPO gene in patients with hypothyroidism. Particular attention is given to mutations within structural and functional domains of thyroid peroxidase, which has a unique molecular architecture within its family.
Mitochondria are semi-autonomous, multifunctional organelles that supply cells with energy. They are highly dynamic structures, capable of moving, fusing, dividing, and forming branched networks. The number, density, and complexity of mitochondrial network are unique to each cell type and reflect cellular demands for ATP and other mitochondria-dependent metabolites. Mitochondrial dysfunction is a hallmark of many neurodegenerative diseases; however, the relationships between neurodegeneration and mitochondrial morphogenesis, intracellular localization, and dynamics remain incompletely understood. Interpretation and comparison of published data are complicated by the diversity of analytical approaches used to study mitochondrial behavior. In this research, we investigated the effects of a pathogenic mutation in the huntingtin protein (HTT), which causes Huntington's disease (HD), on mitochondrial morphology and motility, with particular emphasis on associated disruptions in the cytoskeletal organization. We performed a systematic evaluation of automated mitochondrial analysis tools and selected MiNA, TrackMate, and JACoP as the optimal platforms for quantitative assessment of the effects of mutant HTT (mHTT) on the mitochondrial morphology, motility, and interaction with cytoskeletal components and identification of specific disruptions directly related to HD pathogenesis. Our analysis revealed that mitochondria in mHTT-expressing cells are significantly shorter, more branched, and less motile than in control cells. Moreover, their interactions with microtubules and vimentin intermediate filaments are markedly altered. Together, these findings establish a link between HD and specific defects in the mitochondrial network, thus contributing to understanding cellular mechanisms of HD development, and suggest that mHTT disrupts the interaction of mitochondria with cytoskeletal components responsible for their movement and distribution in the cell, thereby negatively affecting mitochondrial motility and morphology.
Brain-derived neurotrophic factor (BDNF) is widely recognized as a critical molecule for the survival, growth, and maintenance of neurons in both the central and peripheral nervous systems, as well as for the development of cognitive abilities and emotions. However, recent studies have shown that, in addition to its role as a universal brain "fertilizer", BDNF acts as a metabotrophin linking neuronal signaling with systemic metabolism. BDNF serves as a key factor that integrates the body's response to stress, physical activity, and food intake with cellular mechanisms underlying neural plasticity and normal brain function. The review presents evidence supporting BDNF as a bidirectionally metabolic "bridge": body metabolism controls BDNF production in the brain, while brain BDNF regulates body metabolism. Disruption of this regulatory axis is associated with a broad range of neurological and somatic disorders, as well as their comorbidities. Cellular mechanisms associated with disruptions in BDNF functions are explored in detail through the example of alcohol dependence, a condition characterized by both impaired brain signaling and somatic pathologies accompanied by metabolic changes.
The pathophysiology of type 2 diabetes (T2D) remains poorly understood, largely because multiple early changes are obscure as they evolve during prolonged period of prediabetes. These changes are interconnected, involve feedback loops, and gradually develop in tissue-specific manner, ultimately leading to manifestation as overt diabetes. Insulin resistance (IR) and pancreatic β-cell dysfunction are regarded as central events driven by lipotoxicity and glucotoxicity. Understanding molecular mechanisms of their causes and consequences is essential for developing effective preventive and therapeutic strategies for T2D. This review describes the evolution of current perspectives on T2D pathophysiology, examines the mechanistic roles of lipotoxicity and glucotoxicity, and integrates current concepts on the molecular basis of IR. The hypotheses on the early events in prediabetes and potential role of IR in their progression toward overt T2D are discussed. A deeper understanding of T2D as a metabolic disease of biochemical origin may provide new insights into T2D prevention and major associated mortality risks, including cardiovascular complications and cancer.
Pyridoxal-5'-phosphate (PLP)-dependent D-amino acid transaminases (DATAs) catalyze stereoselective transfer of an amino group from a D-amino acid to an α-keto acid to form new D-amino acid and α-keto acid. These enzymes are found in bacteria and plants; they are responsible for the synthesis of D-amino acids and are incorporated into the nitrogen cycle. In general, the mechanism of D-transamination is similar to the known mechanism of transamination for aspartate aminotransferase: D-transamination reaction consists of two half-reactions with intermediate transfer of the amino group to the cofactor and formation of its reduced form, pyridoxamine-5'-phosphate. DATAs are characterized by broad substrate specificity and an open active site, which, however, does not affect their high stereoselectivity: no side L-products is detected in the DATA-catalyzed D-transamination. As in other PLP-dependent fold type IV transaminases, the functional unit of DATAs is a dimer. The active site is formed by amino acid residues of both subunits and binding of α-carboxylate group is crucial for proper substrate coordination. DATAs with promiscuous activity towards substrates without an α-carboxylate group, primary (R)-amines, have also been discovered and characterized. The promiscuous activity is achieved through the mobility of certain residues in the active site of DATAs. High stereoselectivity and stability of DATAs make then promising candidates for multienzyme cascade processes as biocatalysts of the (R)-stereoselective amination stage. Open configuration of active site makes binding and conversion of bulk non-natural substrates possible. The review describes in detail properties, structure, and relationships of DATAs from two currently known groups differing in organization of their active sites. The prospects for biotechnological applications of DATAs are discussed as well.
Widespread interest and broad application of alginic acid and its salts in tissue engineering, regenerative medicine, biotechnology, and pharmaceutical industry is due to the several unique properties: biomechanical compatibility with living tissue, lack of toxicity, and bioabsorption capacity. This literature review analyzes effects of the anionic structure of the binary alginate copolymer on physicochemical properties of the resulting solutions and gels, as well as characteristics and conditions of their processing to obtain functional products for medical and biological applications. Dependence of functionality of the products on the ratio of M(β-D-mannuronate)/G(α-L-guluronate) blocks in the chain and on the source of alginate are also considered. Influence of the quantitative content of guluronic (G) acid blocks in the chain of linear alginate on its susceptibility to H+-induced gelation is described. A review of the mechanisms of gelling in the alginate solutions caused by formation of ionic, hydrogen, and covalent bonds is provided. In particular, attention is paid to the rate of dissolution of alginate salts, viscosity properties of the solutions based on them, and their dependence on ionic strength and pH. The mechanisms of interaction between both native and chemically modified alginates with various biologically active substances, drugs, and surfactants are considered. A detailed study of these processes opens new possibilities not only for obtaining dimensionally stable gels for tissue engineering structures, but also for obtaining systems designed for the controlled release of drugs.
Hydrophobic weakly basic drugs, such as doxorubicin and sunitinib, are currently key components of cancer chemotherapy. It has been shown that several of these compounds induce increase in the total lysosomal volume in tumor cells. Moreover, hypoxia, a hallmark of solid tumors in vivo, promotes chemoresistance by sequestering doxorubicin within lysosomes. To enhance efficacy of chemotherapy, various strategies have been proposed, including those aimed at lysosome destabilization. Inhibition of autophagy is widely recognized as a means to reduce chemoresistance. However, it remains unclear whether doxorubicin itself directly influences lysosomal physiology. In the present study, using the human colorectal carcinoma cell line HCT116, we demonstrate that doxorubicin accumulates substantially in lysosomes even under normoxic conditions. Under normoxia, doxorubicin induces a marked increase in the total lysosomal volume, whereas this effect is weaker under hypoxia. Co-treatment with doxorubicin and chloroquine, a well-established lysosomotropic agent, results in the increased lysosomal volume under both normoxic and hypoxic conditions. Notably, under normoxia, doxorubicin activates TFEB (Transcription Factor EB), a master regulator of lysosomal biogenesis, which likely accounts for the observed expansion of the lysosomal compartment. Furthermore, the lysosomes retain their functional degradative activity in the presence of doxorubicin. A similar effect, lysosomal volume expansion and enhanced degradative capacity in response to doxorubicin, was also observed in the human fibrosarcoma cell line HT1080. In summary, this study provides the first evidence that doxorubicin directly modulates lysosomal parameters in the tumor cell lines under varying oxygen concentrations.
Cytokines play a critical role in brain functioning by modulating neurotransmitter and energy metabolism, neuroplasticity, and neuronal activity. Dysregulated or excessive cytokine production can disrupt neuronal metabolic processes and contribute to brain dysfunction. Among the proposed mechanisms underlying the development and progression of affective disorders (ADs), the cytokine hypothesis emphasizes the role of inflammatory markers as key factors in the development of depressive pathologies. The aim of this study was to investigate molecular characteristics of selected immunoinflammatory markers in patients with AD. The study included 239 patients diagnosed with AD and 205 healthy controls. Polymorphic variants of the immunoinflammatory genes IL1B (rs16944, rs1143627), IL13 (rs1295686), TNFB (rs2229094), and TGFA (rs2166975) were analyzed, and cytokine levels in the blood serum and peripheral blood mononuclear cells were measured. As association was identified between the rs2229094 polymorphism of the TNFB gene and AD: the carriage of the A allele and the AA genotype of this variant was associated with an increased risk of AD. Furthermore, the levels of TGF-α and IL-13 in peripheral blood mononuclear cells and the serum content of TNF-β were significantly elevated in patients with AD compared with healthy controls. These pilot findings suggest that the studied cytokines may contribute to the pathogenetic mechanisms underlying development of ADs.
Parkinson's disease is associated with amyloid aggregation of alpha-synuclein, which could be affected by the proteins of the SARS-CoV-2 coronavirus, possibly accelerating and provoking neurodegeneration. The purpose of this work was to compare the effects of the N-protein and the receptor binding domain (RBD) of the S protein on fibrillization of the alpha-synuclein preparation produced using an original technique that excludes presence of non-native forms of alpha-synuclein that alter kinetics of the process. Presence of an elongated form of alpha-synuclein in the previously studied protein preparations is associated with the erroneous reading of the rare for E. coli TGA stop codon in the pET33b(+) expression plasmid as tryptophan, which led to the continued translation to the next stop codon. To prevent this effect, a new plasmid design was suggested with replacement of the original stop codon with a double stop codon TAA, which made it possible to obtain a homogeneous protein preparation without the admixture of alpha-synuclein with increased molecular weight. It has been shown that the N-protein is able to accelerate alpha-synuclein fibrillization, while the RBD of the S protein inhibits aggregation. According to the electron microscopy data, structure of the fibrils formed in the presence of viral proteins is also different. The obtained data are important for understanding the mechanisms of development of post-covid synucleinopathies, as well as consequences of vaccination with the viral proteins.
Patients with coronary and cerebral atherosclerosis are characterized by increased levels of total serum calcium, ionized calcium, and phosphate, against a background of reduced levels of total serum protein and albumin. Here we aimed to develop a rapid diagnostic assay for mineral homeostasis disorders, based on assessing capacity of the acidic plasma proteins to bind excess calcium and phosphate ions. Plasma from bony fish, amphibians, reptiles, birds, mice, and patients with myocardial infarction was incubated with excess concentrations of calcium and phosphate at 37°C for varying time periods. The following assay readouts were defined: (i) plasma optical density after supersaturation with calcium and phosphate ions, reflecting excessive formation of calciprotein particles (CPPs); and (ii) CPP concentration in plasma. CPPs were formed in all vertebrates. The most pronounced plasma calcification propensity was observed in the human and mouse plasma, suggesting an evolutionary significance of CPP formation as a mechanism for clearance of excess circulating calcium and phosphate ions in mammals. Among the 11 protocols of supersaturation with calcium and phosphate ions, stable increase in plasma optical density at 620 nm wavelength (normalized OD620, a measure of plasma calcification propensity) was achieved by adding solutions of CaCl2 (+2 mmol/L, +50 µL), Na2HPO4·12H2O (+2 mmol/L, +50 µL), and NaCl (+15.4 mmol/L, +20 µL) to plasma (80 µL). Increase in the normalized OD620 was consistently detected within 10 min from the reaction onset during incubation in a microplate shaker (37°C), with mild-to-moderate variability across the parallel or sequential measurements and between the different operators. These results support relevance of validating the developed diagnostic assay for assessing mineral homeostasis disorders in the expanded cohorts of patients with myocardial infarction and ischemic stroke.
Macrophages are a heterogeneous cell population whose functional diversity is formed during their maturation and depends on factors of the microenvironment after their migration into the bloodstream or tissues. One such factor is the pro-inflammatory protein cyclophilin A (CypA, 18 kDa). Using a model of early human monocytic THP-1 cells, it was shown that recombinant human CypA (rhCypA) exerts a differentiating effect on these cells, inducing their maturation, adhesion, and spreading. Under the effect of rhCypA, the THP-1 cells developed an actin cytoskeleton characteristic of motile cells with numerous pseudopodia and podosomes, which ensure tight adhesion of the cells to the substrate and determine their migratory capabilities. Combination of low concentrations of rhCypA and other activators (phorbol myristate acetate) showed an additive effect and ensured effective monocyte differentiation. It was shown that rhCypA, along with other pro-inflammatory factors (IFNγ, TNFα), promotes cell fusion and induces formation of multinucleated macrophages, which are formed during osteoclast maturation under normal conditions as well as during granuloma formation in chronic inflammation (tuberculosis, Crohn's disease). Multinucleated giant cells have significantly higher functional activity (phagocytosis, bactericidal, and pro-inflammatory activity) compared to the mononuclear forms. The study showed that rhCypA enhances expression of the CD147 molecule, an integral functional regulator of CD29 and CD98 molecules involved in the processes of cell adhesion and fusion. Elevated doses of CypA cause deterioration in macrophages, inducing their apoptosis, which may play a role in regulation of the immune response. The findings of this study determined the mechanisms by which secreted CypA mediates monocyte differentiation and maturation, as well as it showed functional role of macrophages in the development of the immune response, which could facilitate further development of therapeutic approaches for the treatment of infectious, autoimmune, and other diseases.