Biochemistry (Moscow)最新文献

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.

The deleterious role of oxidative stress in liver damage is a growing problem, and effective therapeutic interventions are highly warranted. This study evaluated whether peroxisome proliferator-activated receptor gamma (PPARγ) activation protects against H₂O₂-induced oxidative stress and apoptosis in human L02 hepatocytes. Cells pretreated with rosiglitazone, a PPARγ agonist, were incubated with H₂O₂, and cell viability was assessed using CCK8 and LDH release assays 24 h after the treatment. The content of apoptotic cells was determined using Hoechst 33258 staining, and the levels of apoptosis-related proteins were determined by immunoblotting. In addition, several oxidative stress indicators were measured. Possible involvement of the nuclear factor erythroid 2-related factor (Nrf2) pathway was investigated using the Nrf2 inhibitor ML385. Rosiglitazone (20 μM) increased cell viability and improved nuclear morphology in H₂O₂-treated L02 cells, possibly by increasing the Bcl-2/Bax ratio and reducing caspase-3 activation. Rosiglitazone also decreased reactive oxygen species and malonaldehyde levels, as well as increased the activities of catalase, glutathione peroxidase, and superoxide dismutase. Rosiglitazone also promoted nuclear translocation of Nrf2 and increased the antioxidant levels in H₂O₂-treated L02 cells. Inhibition of the Nrf2 pathway by ML385 partially abolished the rosiglitazone-induced amelioration of oxidative stress and apoptosis. We conclude that activation of PPARγ protects liver cells against oxidative stress and apoptosis through the Nrf2 pathway.

DNA damage repair by the base excision repair (BER) mechanism is a complex, multistage process that requires precise coordination and regulation of activities of enzymes at each step of DNA repair. The central stage in this process is DNA synthesis in the gap formed by removal of the damaged nucleotide. DNA polymerases β (Pol β) and λ (Pol λ) of the X family possess all properties necessary for the DNA repair synthesis. Pol β is the major enzyme in DNA synthesis in BER, which may be due to the influence of other BER factors regulating its activity. The scaffold protein XRCC1 and poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) are essential for the formation of functional BER complexes. We investigated the effect of XRCC1, PARP1, PARP2, and poly(ADP-ribosyl)ation on the activity of Pol β and Pol λ in the single-nucleotide gap filling reaction in BER. XRCC1 stimulated the activity of Pol λ to a significantly greater extent than the Pol β activity. PARP1 and PARP2 inhibited both DNA polymerases; the inhibitory effect of PARP1 was more pronounced on DNA with a tetrahydrofuran-phosphate group at the 5′ end of the gap, while PARP2 effect was more significant on DNA containing a 5′ phosphate group. XRCC1 partially restored the DNA polymerase activity, especially that of Pol β. It can be assumed that the XRCC1–Pol β complex competes with PARP1/2 for DNA binding more strongly than the XRCC1–Pol λ complex. Addition of NAD⁺ to PARP2 led to a more efficient restoration of Pol β (but not Pol λ) activity. These differences may be one of the causes why Pol β is the main DNA polymerase in BER.

Antibody-dependent enhancement (ADE) of infection is a phenomenon observed during secondary infection caused by some viruses (primarily, from the Flaviviridae family), including dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), tick-borne encephalitis virus, yellow fever virus, and some others. There are two types of ADE mediated by (i) Fc receptors and (ii) receptors for complement system components. The first type involves 4 types of Fc receptors for IgG molecules (FcRI, FcRIIa, FcRIIb, and FcRIIIa) which ensure the binding of the virus-antibody complex to the target cell and its subsequent internalization. The Fc-mediated uptake of the virus is an active process that involves a number of signaling molecules and triggers branched cascades of activation events. The Fc-mediated pathway of viral entry increases the viral load and switches the cell from the antiviral response to the proinflammatory pathway, thus promoting more efficient viral replication. Unlike other Fc receptors, FcRIIb bound to IgG inhibits the capture of the virus–antibody complex and prevents the ADE. The development of ADE in response to vaccination is highly undesirable. Understanding the biochemical mechanisms underlying ADE is important for the development of new vaccines and therapeutic monoclonal antibodies for protecting against viral infections.

Human terminal deoxynucleotidyl transferase (TdT) belongs to the family X DNA polymerases responsible for increasing diversity of immunoglobulins and T-cell receptor genes during V(D)J recombination by means of template-independent addition of random nucleotides to the 3′-end of the rearranged V-D and J-D segments. According to the X-ray structural data, coordination of the incoming dNTP and 3′-terminal nucleotides of the single-stranded primer in the active site of TdT is accomplished through contacts with the hydrophobic (W449, F404, and L397) and hydrophilic (R336, H342, D395, E456, R453, and R457) residues that form the dNTP-binding pocket. The residues R336, H342, and D345 are directly involved in orientation of the phosphate groups of the incoming dNTP and stabilization of the 3′-terminal nucleotide of the DNA primer. In this work, we analyzed the consequences of replacing these residues using molecular dynamics methods and experimentally tested enzymatic properties of the mutant forms of the human TdT containing the R336Q, H342A, and D345E substitutions. The obtained data showed that the H342A substitution increases dissociation constant of the enzyme complex with dNTP and DNA primer and significantly reduces efficiency of the dNTP addition to the growing chain. Extension of the side chain at the D345 residue disrupts coordination of the cofactor metal ions and also affects efficiency of the catalytic reaction. Substitution of the R336 residue leads to significant destabilization of the protein globule and complete loss of the catalytic activity of the enzyme.

Internal tandem duplications in the gene encoding the membrane domain of FLT3 (FLT3-ITD) are the most common genetic alteration and an unfavorable prognostic factor in the patients with acute myeloid leukemia (AML). New-generation FLT3 inhibitors effectively induce cell death in the AML cells with the FLT3-ITD-positive phenotype (FLT3-ITD⁺) and potentially exhibit cytotoxic activity against the AML cells with the FLT3-ITD-negative phenotype (FLT3-ITD⁻), but at higher concentrations. However, potential impact of the new-generation FLT3 inhibitors on the cytotoxic activity of molecular effectors of antitumor immunity – particularly in the context of heterogeneity of the primary clonal composition of AML, which includes both FLT3-ITD⁺ and FLT3-ITD⁻ cells – remains unclear. This study demonstrated that the use of quizartinib, a new-generation FLT3 inhibitor, increased resistance of the FLT3-ITD⁻ AML cells, but not of the FLT3-ITD⁺ AML cells, to the cytotoxic action of the key molecular effector of antitumor immunity, the cytokine Apo2L/TRAIL. This effect was mediated by the changes in the expression of proapoptotic TRAIL receptors, content of the cFLIP protein, and expression of the genes encoding proteins of the IAP and BCL-2 families. Additionally, the quizartinib-induced changes in the intracellular signaling pathways that potentially regulate TRAIL resistance in the AML cells were identified. The identified quizartinib-induced transcriptional changes are of interest not only in the context of combination therapy with TRAIL but also have broader implications for understanding the mechanisms of drug resistance in the AML cells.

In recent years, understanding of the key role of epithelial cytokines (IL-25, IL-33, and TSLP) in the development of respiratory tract inflammatory diseases has significantly expanded. These molecules, released by airway epithelium in response to allergens, pathogens, and damaging agents, act as alarmins, initiating a cascade of the Th2-mediated inflammation that leads to the IgE-mediated reactions, eosinophilic infiltration, and tissue remodeling. Beyond classical allergic processes, epithelial cytokines play a crucial role in the virus-induced exacerbations of respiratory diseases and pathogenesis of chronic obstructive pulmonary disease (COPD). Monoclonal antibody therapeutics targeting these cytokines have demonstrated efficacy in treating severe asthma. This review summarizes current data on contribution of TSLP, IL-33, and IL-25 to the respiratory tract inflammation and discusses the prospects of their targeted inhibition to improve therapeutic efficacy.

Since the Mycobacterium tuberculosis genome was decoded in 1998, our understanding of this dangerous pathogen – which has become the leading cause of bacterial infection-related deaths in modern human history – has expanded significantly. This review examines genetic, physiological, and metabolic factors believed to play a key role in the adaptation strategies of M. tuberculosis to survival within the human host. These strategies underpin critical aspects of the bacterium life cycle, including infection, persistence, dissemination, and transmission to the new hosts. We also discuss how M. tuberculosis adapts to the primary factors of the host’s innate and adaptive immunity, including their roles in disease progression. Granulocyte migration, phagosome damage and repair, autophagy, and cell death are pivotal processes that determine outcome of the host–mycobacterium interactions. To date, experimental evidence has accumulated indicating that small bacterial RNAs play a crucial role in regulating numerous physiological processes and key stages of the pathogen life cycles. Our review explores the hypothesis that M. tuberculosis small RNAs may not only adapt the bacterial transcriptome to changing conditions but also interact with the transcriptome of the infected host, interfering with antibacterial defense processes. Given the growing recognition of the significance of asymptomatic infection and transmission of M. tuberculosis, we argue for stronger integration between the laboratory and clinical research in this field.

In the 1990s, Vladimir P. Skulachev, a proponent of the genetic program of aging, proposed extending the concept of programmed cell death (apoptosis) to the level of an entire organism, a phenomenon he termed phenoptosis. According to his terminology, rapid phenoptosis, is characteristic of species with a single reproductive cycle, such as pink salmon and mayflies, whereas slow phenoptosis is typical of species with multiple reproductive cycles, including humans. Interestingly, rapid phenoptosis resembles obligate apoptosis observed during development, such as the disappearance of pharyngeal slits, tail, and interdigital webbing in human embryo. Slow phenoptosis is more akin to non-obligate apoptosis, which is triggered by irreversible damage or functional cell redundancy. Just as non-obligate apoptosis is not inevitable, a similar non-inevitability should not be excluded for slow phenoptosis – that is, natural aging. This interpretation is supported by the plasticity of aging, the reversibility of age-associated traits, and the absence of the replicative (Hayflick) limit in tissue stem cells, a feature they share with immortalized cells. Additionally, human (and animal) mortality patterns resemble those of non-aging hydras and immortalized cells subjected to suboptimal conditions. It has been said that a “correctly posed” question endures indefinitely. In our view, the question “Is aging programmed or stochastic?” falls into the category of “correct” questions. Its apparent dichotomy excludes the obvious third option: in many species with repeated reproductive cycles, aging is associated with neither genetic program nor purely stochastic damage, but rather results from cumulative consequences of living under conditions that are pessimal for stable, non-aging functioning.

Pulsatilla vulgaris (pasque flower) is a rare and endangered plant species. Using ITS1-5.8S rDNA-ITS2 barcode sequencing and locus-specific NGS of the ITS1 spacer, we studied intraspecific and interspecific polymorphism of 35S rDNA loci and investigated the origin of tetraploid P. vulgaris from a unique Russian population. For the first time, the chromosome number in the karyotype of plants from this population was determined as 2n = 32. It was shown that one of the parental species of this tetraploid is the diploid Pulsatilla patens (2n = 16), while the identity of the second parental species remains unknown, although it appears to be closely related to the diploid Pulsatilla pratensis.