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.
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.
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.
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.
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.
