The review discusses experimental findings and theoretical studies of photoreversible transformations of retinal-containing proteins (rhodopsins). Understanding these processes is essential for elucidating the mechanisms of photochemical and conformational transformations of these proteins. Particular emphasis is placed on visual rhodopsins, as photoreversibility plays a crucial role in its physiological regeneration in the rhabdoms of invertebrates and the cone photoreceptors of the vertebrate retina. In recent years, interest in the mechanisms of photoreversible reactions in retinal-containing proteins has grown substantially, driven by their potential applications as active components in optoelectronic devices and biosensors.
The EF-hand motif is one of the most widespread calcium-binding protein motifs in nature, mediating calcium signaling across a variety of biological processes. While structural and functional properties of the individual proteins within the EF-hand superfamily are well studied, general principles governing organization and function of these proteins are only now being elucidated. This review examines some of these emerging patterns, including recently discovered structural elements of the EF-hand motif (EF-hand zone, one-residue and three-residue units, local “Clamp” units, and “black” and “gray” clusters). Additionally, we discuss the ability of certain EF-hand proteins to recognize a broad spectrum of protein targets, as well as their zinc-binding properties. New structural data on the EF-hand domain allow proposing a structural-functional classification for the proteins in this family. Furthermore, tendency of some EF-hand proteins toward promiscuity and zinc binding significantly expands their functional importance.
The vertebrate visual system contains a sophisticated physiological assembly of highly-specialized proteins that mediate light stimuli to be processed by the brain. Two types of cells, the rod and cone cells, are involved in detecting dim and bright light, respectively. The rod cell contains rhodopsin whose structure and function has been extensively studied and the molecular interactions involving rhodopsin and the other proteins of the phototransduction cascade have been dissected in great detail, although several mechanistic features remain to be determined. The cone phototransduction process has been less studied, particularly at the cone opsin structural level, and some conformational and mechanistic information has been inferred from analogy to the rod system. In spite of this, current efforts of investigation are focused on the study of the structure and function of cone opsins and other proteins of the cone phototransduction system. In this review we cover the current knowledge on the genetic and molecular aspects of cone opsins and the consequences of cone opsin mutations on the structure and function of these photoreceptor proteins causing vision disorders ranging from mild color vision abnormalities to severe cone-mediated retinal degeneration. A second part of the review is dedicated to the analysis of the molecular interactions involving calcium-binding proteins (such as recoverin and guanylate cyclase-activating proteins) in zebrafish which has emerged as a very useful model organism for the study of cone opsin phototransduction particularly in the deactivation steps of the visual phototransduction cascade.
Cultured astrocytes were incubated with CuCl2, which caused dose-dependent cell death (25-200 μM, 24 h). Immunocytochemical detection of the nucleolar protein nucleophosmin/B23 (NPM/B23) demonstrated that exposure to Cu2+ (100 μM, 24 h) caused a significant increase in the surface area of NPM/B23 clusters, which was accompanied by the changes in the nucleolar ultrastructure characteristic of nucleolar stress. Longer incubation of astrocytes with Cu2+ (100 μM, 48 h) led to accumulation of the endoplasmic reticulum (ER) stress marker GRP78, which was accompanied by the increase in nucleolar size and migration of the nucleolar material into the nucleoplasm.
Acute liver failure (ALF) is a severe pathological condition with high mortality, arising from diverse etiologies and involving multiple pathogenetic mechanisms. ALF is often accompanied by multi-organ failure due to infectious complications. Because of disease’s rapid progression, clinical studies of ALF remain limited, making the search for novel therapeutic strategies targeting specific molecular pathways and biochemical cascades a pressing task. Preclinical in vivo models are unique tools for reproducing individual aspects of ALF development. However, there is still no consensus on the gold-standard experimental animal model that fully captures ALF pathogenesis. This review systematically categorizes existing mouse models of ALF, outlining their advantages and with respect to the translational applicability of the data obtained. Among these models, we highlight the LPS/D‑GalN model as the most reliable in reproducing key stages of ALF development complicated by bacterial infection. Using this model as an example, we describe the known stages of pathogenesis, supported by the experiments with pharmacologically active substances and the use of various genetically modified mouse lines. Finally, special attention is given to the intestine as an organ that sustains significant damage during ALF progression in both clinical cases and most animal models.
Lipofuscin granules (LGs) in retinal pigment epithelium (RPE) cells contain bis-retinoids and their oxidation and degradation products, rendering them photo- and cytotoxic to intracellular structures. LGs are implicated in the pathogenesis of multiple visual pathologies, including age-related macular degeneration (AMD). They exhibit strong autofluorescence, which has led to the development of fundus autofluorescence (FAF) imaging as a non-invasive diagnostic method in ophthalmology. Spectral analysis of autofluorescence can expand the capabilities of this method, including for preclinical diagnostics, as pathological conditions are often associated with increased proportions of oxidized bis-retinoid derivatives that alter LG autofluorescence parameters. However, limited knowledge of age-dependent changes in LG bis-retinoid composition remains a key limitation. In this study, we combined fluorescence spectroscopy, confocal fluorescence microscopy, and fluorescence lifetime imaging (time-correlated single-photon counting) to demonstrate that, under physiological conditions, aging is accompanied by a progressive increase in the relative abundance of oxidation and degradation products of bis-retinoids in LGs. These findings provide an age-dependent baseline for distinguishing physiological and pathological states, thereby improving the potential of FAF imaging for early (preclinical) diagnosis.
Aging is the major risk factor for age-related macular degeneration (AMD), a leading cause of vision loss in aging populations. Increasing evidence suggests that alterations of neurotransmitter systems contribute to the pathogenesis of AMD. Although biogenic amines in the retina were first detected over 50 years ago, their age-related dynamics and role in AMD development are still poorly understood. Here, we compare age-related changes in the concentrations of norepinephrine, serotonin, dopamine, and their main metabolites in the retinas of senescence-accelerated OXYS rats that develop an AMD-like retinopathy and Wistar rats. We also assessed activities of monoamine oxidases (MAOs) and tyrosine hydroxylase and compared changes in these activities with transcriptome data on genes associated with biogenic amine pathways. The results revealed that in OXYS rats, retinal aging and progression of AMD-like retinopathy are primarily associated with alterations in the dopaminergic system. Specifically, dopamine and its main metabolite DOPAC were present in the retinas at higher levels than serotonin and 5-hydroxyindoleacetic acid. Furthermore, the development of retinopathy in OXYS rats was accompanied by elevated dopamine and DOPAC levels, decreased MAO activity, and changes in the expression of genes associated with the dopaminergic synapse signaling.
Spontaneous isomerization of aspartate and deamidation of asparagine residues into isoaspartate (isoAsp) constitute major non-enzymatic post-translational modifications that alter protein structure, stability, and turnover. The repair enzyme protein L-isoaspartate O-methyltransferase (PCMT1) catalyzes methylation of isoAsp residues, thereby preventing their accumulation and preserving proteome integrity. Although PCMT1 has been studied extensively in cytoplasm and nucleus, its relationship to endoplasmic reticulum (ER) proteostasis remains poorly understood. Here, we investigated dynamics of aspartate isomerization within the cell, focusing on isoAsp accumulation and the regulation of PCMT1 localization under physiological and stress conditions. Using immunofluorescence, subcellular fractionation, and in vitro methylation assays, we detected isoAsp-modified proteins within the ER-enriched fractions of HeLa cells. We found that ER stress induction enhanced formation of isoAsp-containing proteins, with MG132 treatment producing the highest accumulation. PCMT1 expression increased under both stress conditions, accompanied by distinct subcellular redistribution between the cytoplasmic and nuclear compartments. These observations indicate that ER-folded proteins are susceptible to spontaneous aspartate isomerization, and that PCMT1 activity dynamically responds to proteostatic stress. Our findings provide the first experimental evidence linking isoAsp formation within the ER to PCMT1-mediated protein repair, thereby integrating chemical instability with cellular quality-control pathways. This study establishes a structural and cellular framework for understanding the dynamics of aspartate isomerization in the cell and underscores significance of PCMT1 in maintaining proteostasis under stress conditions.
Polycaprolactone (PCL) is a biodegradable polyester widely used in industry, but it degrades slowly in the environment. Enzymatic hydrolysis catalyzed by cutinases represents a promising approach for PCL waste utilization. Through screening, we selected the Aureobasidium pullulans VKM 1116 strain capable of degrading PCL. We amplified the ApCUT1 cutinase gene from the A. pullulans VKM 1116 genomic DNA. Heterologous expression in Komagataella phaffii yielded 60 mg/L of the target protein. The recombinant enzyme exhibited maximum activity at pH 6.0-6.5 and 30-40°C. Using site-directed mutagenesis, we generated single mutants (Y58W and L186F) and a double mutant (Y58W/L186F). The double mutant demonstrated the highest catalytic efficiency toward the model substrate 4-nitrophenyl 16-methylsulfonyl hexadecanoate (4-NP-(16-MS-C16)), exhibiting a two-fold increase in the hydrolysis rate compared to the wild-type enzyme. During PCL hydrolysis, the Y58W mutant showed the highest activity, providing a 1.8-fold increase in the suspension degradation rate and complete polymer granule degradation at a rate of 1 mg/h per 1 mg of protein. Our molecular modeling suggests that the hydrophobic bridge at the entrance to the active site functions as a “molecular trap.” For the low-molecular-weight substrate (4-NP-(16-MS-C16)), enzymatic activity is limited by the kinetics of the substrate retention step, which is consistent with the maximum catalytic efficiency of the Y58W/L186F mutant with a closing trap. Conversely, activity toward PCL is limited by the initial adsorption stage, for which an open conformation of the free enzyme – as observed in the Y58W variant – is critically important. These results demonstrate the potential of the ApCUT1_Y58W cutinase for developing advanced PCL recycling technologies.
S-acylation is post-translational attachment of fatty acid residues, mostly palmitoyl-groups to cysteines. This lipid modification could influence protein oligomerization, localization, and topology within the membrane, and could play a regulatory role in cellular signaling. Improvements in the experimental techniques, specifically, MALDI-TOF MS, have elevated the study of S-acylation to a level of refined detail. It was possible to conclusively show that, beyond palmitate, other types of fatty acids, such as stearate and oleate could be attached to the specific binding sites. For example, fusion proteins of the enveloped viruses have been shown to attach stearates exclusively to the cysteine located at the C-terminal end of the transmembrane domain, while cysteines in the cytoplasmic domain have been modified with palmitates. In addition, emerging evidence suggests that the covalently linked fatty acids in the viral fusion proteins could recruit cholesterol into the viral membrane. We termed this preferential attachment of fatty acid residues of different types to the specific sites, depending on their localization, as “differential” S-acylation. Furthermore, MALDI-TOF MS detected attachment of unsaturated fatty acids (oleates) to the GNAI protein in response to stearate supplementation, shifting this regulatory protein out of the detergent-resistant membranes (lipid rafts) and, consequently, exerting antitumor effect. Solving the first 3D-structures of DHHC acyltransferases allowed proposing a mechanism of different fatty acids selection. However, it remains unclear whether preference for the acyl chain length is determined by the structure of the enzyme, protein substrate, or by the lipid composition of the membrane. In this review, we summarize the latest advances in the study of protein S-acylation with different types of fatty acids and substantiate significance of this post-translational modification from both fundamental and practical perspectives.
Genome editing using the CRISPR/Cas9 system has become a staple of modern genome manipulation. In its original form, editing involved introducing double-strand breaks into DNA, which can cause genomic instability. The appearance of the first base editors in 2016 expanded the range of editing technologies and enabled single-nucleotide changes to be introduced into the genome through deamination of nucleobases, bypassing the double-strand break stage. Further development of base editors involves the incorporation of additional modules, DNA glycosylases, that can remove modified or even normal nucleobases and create non-instructive apurinic/apyrimidinic sites in DNA, significantly expanding the range of available single-nucleotide substitutions. This review examines the operating principles of the glycosylase base editors, the main limitations of these genome manipulation tools, and promising areas for the development of this technology.
Fate of monocytes that migrate into tissues is determined by various mediators at the site of inflammation. The microenvironment governs monocyte differentiation and polarization into macrophages, which acquire different surface markers and opposite functions. Secreted cyclophilin A (CypA) is a pro-inflammatory factor and a component of the monocyte humoral microenvironment. In these studies, effects of recombinant human CypA (rhCypA) on differentiation of human monocytic cells were evaluated in the THP-1 in vitro model. RhCypA induced polarization of M1 macrophages in the early stages of THP-1 ontogenesis without additional stimuli, whereas it promoted M2 polarization only in the cells differentiated to M0 monocytes and in the presence of IL-4 and IL-13. Under conditions of M2 induction, rhCypA significantly upregulated expression of the genes of characteristic M2 markers CD163 and IL-10 in the THP-1 cells, and decreased the level of p38 MAP kinase activation, suggesting diminished pro-inflammatory activity of the generated cells. The RhCypA-induced M1 and M2 macrophages inhibited proliferation of activated CD4+ T cells via direct cell-cell contact. These findings suggested that secreted CypA could induce generation of suppressor M2 macrophages and/or myeloid-derived suppressor cells at the site of inflammation during infection or cancer progression. This new immunoregulatory effect of rhCypA could be of particular clinical relevance for the development of strategies for controlling chronic inflammation and reprogramming the tumor microenvironment.
Ligation of nucleic acids by DNA and RNA ligases is a routine method in molecular biology. Enzymatic ligation is increasingly used in the development of novel methods for detecting specific nucleotide sequences, as well as in non-biological DNA application. In this study, we investigated how the type of 5′- and 3′-terminal nucleotides affects the efficiency of single-stranded DNA cyclization. Model oligodeoxyribonucleotides (ODNs) 23, 39, and 45 nucleotides in length, representing all possible combinations of the donor and acceptor terminal nucleotides, were used as substrates. The substrate specificity of T4 RNA ligase 1 was assessed by analyzing the primary structure of amplicons generated through rolling circle amplification (RCA) of ligation products. For longer ODNs (39 and 45 nt), T4 RNA ligase 1 most efficiently cyclized substrates containing 5′-CC or 5′-TT donor dinucleotides and 3′-AA acceptor dinucleotide. Among the 23-nt ODNs, efficient cyclization was observed predominantly for a single substrate containing 5′-TT and 3′-AA terminal dinucleotides, likely reflecting a greater flexibility of AT-rich DNA. Substrates containing 5′-dGG and 3′-dGG dinucleotides showed the lowest propensity for cyclization. These findings provide guidance for selecting substrate sequences that maximize the yield of circular DNA products.
Using kinetic and structural methods – differential scanning calorimetry, dynamic laser light scattering, turbidimetry, and mathematical modeling – a comprehensive study of the process of thermal inactivation of penicillin acylase (PA) from Escherichia coli was conducted. The first step of the process is denaturation of the protein globule. As denatured protein accumulates, start aggregates are formed, and after reaching a critical amount, rapid clustering of aggregates (formation of clusters) occurs, which proceeds in the kinetic regime of diffusion-limited cluster–cluster aggregation. A distinctive feature of the process of PA thermal aggregation is absence of enzyme dissociation into subunits and very prolonged accumulation of start aggregates. The rate-limiting step of the PA thermal inactivation process is denaturation of the protein globule and pronounced tendency of the denatured enzyme to aggregate makes this process irreversible. When searching for the ways to stabilize PA, primary attention should be paid not to prevention of protein aggregation but to preservation its three-dimensional structure.
Inflammaging, a chronic, low-grade systemic inflammatory state that develops with advancing age, is one of the key drivers of age-associated pathology and promotes the emergence of the senescence-associated secretory phenotype (SASP). Timely quantitative monitoring of pro-inflammatory and SASP-associated biomarkers therefore demands highly sensitive analytical platforms capable of reliable performance in complex biological matrices. Aptamer-based biosensors (aptasensors), which employ short single-stranded oligonucleotides offering high affinity and specificity for molecular recognition, have emerged as promising alternatives and complements to conventional antibody-based assays. This review provides a comprehensive overview of aptasensor platforms developed for biomarkers of age-associated inflammation and identifies molecular targets of relevance to aging biology that remain unexplored. Following PRISMA guidelines, a systematic literature search of the PubMed database was conducted for studies published between 2020 and 2026. A total of 221 original studies met the inclusion criteria and were qualitatively analyzed, encompassing 47 biomarkers and seven classes of analytical platforms. The analysis revealed a pronounced research bias: 73
Ischemic stroke is one of the most prevalent neurological disorders, with primary injury typically localized to the neocortex. However, secondary damage often extends to anatomically distant regions, particularly the hippocampus. One of the early neuroendocrine responses to cerebral ischemia is activation of the hypothalamic–pituitary–adrenal (HPA) axis. Increased release of corticosterone (CORT) by the adrenal glands into the bloodstream leads to its accumulation in the hippocampus. We hypothesize that this process may contribute to the hippocampal damage and underlie delayed cognitive and affective impairments following stroke. To test this, we investigated the levels of neurotrophic factors reflecting the neuroprotective potential of the hippocampus using two established middle cerebral artery occlusion (MCAO) models that differ in their effects on CORT production: the Koizumi et al. (1986) model (MCAO1) and the Longa et al. (1989) model (MCAO2). Although both models produced comparable neurological deficits, mortality rates, and infarct volumes, they differed markedly in endocrine and hippocampal responses. Significant increases in circulating CORT and its accumulation in both ipsilateral and contralateral hippocampus were observed only in MCAO1. Moreover, correlations between the CORT levels (in the blood and hippocampus) and both rat body weight and neurological deficit were stronger in MCAO1 than in MCAO2. In both acute models, brain-derived neurotrophic factor (BDNF) levels were elevated in the ipsilateral hippocampus, whereas the content of glial cell line-derived neurotrophic factor (GDNF) remained unchanged. In contrast, ciliary neurotrophic factor (CNTF) increased in the ipsilateral hippocampus only in MCAO2. These findings suggest that differential activation of the HPA axis and consequent variations in corticosteroid signaling in the hippocampus selectively modulate the neurotrophic response during the acute phase following MCAO.
Extrachromosomal DNA (ecDNA) refers to circular DNA molecules without centromeres that are often found in the nuclei of cancer cells. The presence of ecDNA is associated with the development of drug resistance and poor disease prognosis. Although ecDNA was discovered 60 years ago, technological advances of the past decade have dramatically expanded our understanding of its biological significance and revealed a far greater role in cancer development than previously recognized, thus driving a rapid increase in research interest. This review describes the milestones in the history of ecDNA research and summarizes the current state of knowledge as of 2025. It provides a comprehensive overview of the mechanisms underlying ecDNA formation, organization, and functions, describes the principal methods used for ecDNA detection and analysis, and discusses its clinical relevance, as well as future directions in ecDNA research.
Primary cilia are solitary, antenna-like organelles that project from the surface of most vertebrate cells. They consist of a microtubule-based axoneme extending from a modified centriole (basal body) and enclosed by a lipid bilayer membrane. For several decades after their discovery, the functions of primary cilia had remained speculative; they were even considered vestigial structures. Currently, primary cilium is recognized as essential sensory and signaling structure involved in both chemo- and mechanosensation. Its anchoring at the centrosome surrounded by a radially organized network of microtubules and ability to detect extracellular signals through the axoneme protruding beyond the cell surface and enveloped by a receptor-rich membrane, make primary cilium a unique signaling hub of the cell. The functional activity of primary cilia is critical for numerous biological processes, including embryonic development and cellular differentiation, whereas defects in ciliogenesis result in severe somatic disorders. Prominent neurological abnormalities observed in several ciliopathies have prompted investigations into the structure and function of primary cilia in other brain disorders. It has become evident that defects of neuronal primary cilia are characteristic of several monogenic neurological diseases that have not traditionally been classified as ciliopathies. A growing body of evidence indicates that many severe neurodegenerative disorders, particularly polyglutamine diseases such as Huntington’s disease, are associated with specific alterations and dysfunction of primary cilia. In this review, we summarize current knowledge on the detrimental effects of disease-causing mutant proteins on the function of primary cilium, with particular emphasis on the disruption of PCM1 (pericentriolar material 1) trafficking by mutant huntingtin, leading to ciliary elongation and alterations in signaling pathways. We further discuss the consequences of mutant protein-induced dysfunction of this cellular antenna and analyze associated signaling pathways that may represent promising therapeutic targets. Finally, we describe potential approaches for investigating dysfunction of the neuronal signaling hub with primary cilium as an antenna.
Neutrophils, key effector cells of the innate immune system, infiltrate infected tissues, adhere to them, and destroy pathogens by releasing the bactericidal content of intracellular granules and producing reactive oxygen species (ROS) and nitric oxide (NO). However, these aggressive antimicrobial products of neutrophil secretion can damage surrounding tissues and initiate inflammation. Adenosine, which is produced in excess by damaged tissues, is known to limit the neutrophil immune response and suppress inflammation, acting primarily through adenosine receptors. We compared the effects of ATP, adenosine, and iodoacetic acid (IAA), an inhibitor of glycolysis, on neutrophil adhesion to fibronectin and accompanying secretion of free amino acids, proteins, ROS, and NO. Our results indicate that the immunosuppressive effect of adenosine is unlikely to be due to the inhibition of oxidative stress or protein secretion. Neither ATP nor adenosine altered ROS and NO production during neutrophil adhesion, whereas IAA suppressed it. ATP did not affect the composition of secreted proteins, while adenosine stimulated the release of aggressive components of primary granules. We also investigated previously undescribed dynamics of secretion of the amino acid hydroxylysine by neutrophils during adhesion: ATP stimulated, while adenosine and IAA selectively and strongly inhibited hydroxylysine release. Hydroxylysine is a product of lysyl hydroxylase (LH), whose expression and activity positively correlate with cell invasiveness and metastatic potential of various types of tumor cells. Given the similarities between neutrophils and tumor cells in energy metabolism and invasive capacity, we hypothesize that adenosine and IAA may suppress immune responses by inhibiting neutrophil infiltration into tissues through selective blocking of LH activity/hydroxylysine release. Further studies are required to directly test this hypothesis.
