Biology subjects

Ivanova, L.

Publications and source records attributed to Ivanova, L..

10 recordsLinked to original sources

bioRxiv2026

From cellular stress to cytokine modulation: An in vitro study of intestinal effects of major Alternaria mycotoxins

Mycotoxins are frequently detected in food, making the gastrointestinal tract the first organ exposed. Here, we used various in vitro intestinal cell models to explore the barrier and immune responses to the Alternaria mycotoxins alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT), altertoxin I (ATX-I), tentoxin (TEN), and tenuazonic acid (TeA). The mycotoxins exhibited low toxicity in undifferentiated Caco-2 cells, but AOH, AME, and ATX-I affected the cell cycle and reactive oxygen species (ROS) levels. None of the toxins alone or in combination with IL-1beta; had any effect on the barrier integrity or viability of the differentiated Caco-2/HT29-MTX-E12 co-culture. Furthermore, AOH and AME reduced IL-1beta-induced IL-8 secretion, whereas TeA increased the secretion, demonstrating immunomodulatory effects. For verification, we used the more in vivo-like EpiIntestinal TM model, which is based on human primary cells. AOH, AME, and TeA had minor effects on viability without compromising barrier integrity; also, cytokine secretion was less affected than in the co-culture model. Studies on uptake confirmed low absorption of AOH, AME, and TeA. Overall, the studies indicate a low absorption and moderate effects of the toxins on the gastrointestinal tract.

cell biology↗

bioRxiv2026

Evaluation of immunotoxic effects and metabolism of enniatins and beauvericin using in vitro models of the human intestinal epithelium

Enniatins (ENNs) and beauvericin (BEA) are mycotoxins produced by Fusarium species. They are frequently detected in cereal-based foods such as maize and wheat. Consequently, the gastrointestinal tract is a primary site of exposure for consumers of grain-containing foods. This study examined intestinal effects of four major ENNs: ENNB, ENNB1, ENNA, and ENNA1, and the structurally related mycotoxin BEA, using the primary human intestinal model EpiIntestinal (TM) tissues and a Caco-2/TH29-MTX-E12 co-culture. None of the compounds induced cytotoxicity or impaired the barrier integrity at concentrations ranging from 1 to 10 microM. Furthermore, all ENNs and BEA stimulated IL-6 secretion, with ENNA and ENNA1 inducing the strongest responses, while IL-8 secretion was only triggered by ENNA and ENNA1. Transport studies revealed that approximately 32% of ENNB and 25% of ENNB1 crossed from the apical to the basolateral side of the intestinal tissue within 4 h, whereas the other ENNs and BEA had transport ratios below 10%. Further findings suggested that uptake and intracellular accumulation of ENNs and BEA were influenced by their affinity to the efflux transporter P-glycoprotein (P-gp). In addition, several ENNB and ENNB1 metabolites were generated in the EpiIntestinal (TM) tissues and transported. Lastly, it was demonstrated that ENNB can disturb the metabolism of the sex hormone estradiol in human liver microsomes. Overall, ENNs and BEA showed limited acute toxicity in human intestinal models but elicited distinct inflammatory responses and differed in transport and metabolism. These findings may be of relevance when evaluating health risks from chronic, low-level dietary exposure

cell biology↗

bioRxiv2026

Common ISAV haemagglutinin esterase variants at residues 229 and 230 influence the efficiency of receptor destruction and erythrocyte release in Atlantic salmon

Sialic acid-binding viruses often encode receptor-destroying enzymes that modulate infection through host glycan alteration. Isavirus salaris (ISAV), the aetiological agent of infectious salmon anaemia, encodes a haemagglutinin esterase with 4-O-acetylesterase activity. The ISAV esterase removes the virus-targeted epitope and causes homologous attachment interference, but its broader biological roles remain poorly understood. Several aspects of ISAV receptor engagement and release remain unresolved: Although ISAV agglutinates erythrocytes from both rainbow trout and Atlantic salmon, only rainbow trout erythrocytes elute from this interaction. By contrast, Atlantic salmon erythrocytes remain persistently bound, despite the presence of an active esterase. Here, we used ISAV-erythrocyte interactions to dissect receptor destruction and dissociation across viral genotypes and strains. We uncovered substantial functional diversity in viral permissiveness for Atlantic salmon erythrocyte elution. Common variants of residues 229 and 230 of the haemagglutinin-esterase, located at the distal rim of the P1 esterase pocket, were key determinants of elution permissiveness. Elution further required an active esterase catalytic triad. In vivo, Atlantic salmon infected with ISAV carrying the elution-permissive 229N variant showed earlier loss of erythrocyte Neu4,5Ac2 than fish infected with an elution-restrictive strain. The loss of Neu4,5Ac2 preceded the reduction in ISAV-bound erythrocytes by several days, indicating that receptor removal alone was insufficient to trigger virion release. Targeted sialic acid analyses revealed similar levels of Neu4,5Ac2 on Atlantic salmon and rainbow trout erythrocytes. Atlantic salmon erythrocytes additionally expressed di-O-acetylated Neu4,5,9Ac3. By contrast, brown trout erythrocytes expressed little or no Neu4,5Ac2, consistent with their reported inability to support ISAV haemagglutination. Together, our findings give insight into viral and host determinants of ISAV receptor interactions. We demonstrate functional diversity within the ISAV esterase and link virion binding and release to salmonid erythrocyte sialic acid composition. The delay between receptor loss and virion dissociation further supports the existence of additional erythrocyte attachment factors.

microbiology↗

bioRxiv2024

CDNF rescues human iPSCs-derived dopamine neurons through direct binding to unfolded protein response sensors PERK and IRE1α

Cerebral dopamine neurotrophic factor (CDNF) is an unconventional trophic factor that protects dopamine neurons in cellular and animal models of Parkinsons disease (PD). CDNF was safe and well tolerated in phase 1 clinical trials for PD treatment, and currently, its peptide analogue is under investigation in phase 1 clinical trials for PD. Despite prominent neuroprotective and neurorestorative activity, the receptors and exact mechanism of CDNF functioning have been obscure. Intracellularly acting CDNF exerts cytoprotection by attenuating endoplasmic reticulum (ER) stress and unfolded protein response (UPR). We demonstrated that this activity occurs through the direct binding of CDNF to ER transmembrane UPR sensors PERK and IRE1 for purified proteins and in cells. We identified CDNF mutants deficient for binding to UPR sensors. CDNF binding to PERK and IRE1 appeared to be crucial for the survival of mouse dopamine neurons in culture. Importantly for clinical translation, CDNF rescues human induced pluripotent stem cell-derived dopamine neurons and promotes their regeneration. CDNF binding to UPR sensors alleviated terminal UPR and promoted neurite outgrowth of human dopamine neurons through direct binding to PERK and IRE1. CDNF binding to BiP was dispensable for the neuroprotective and neurorestorative activity of CDNF. Therefore, CDNF, or small molecules mimicking its binding to UPR sensors and acting selectively on dopamine neurons with activated UPR, are promising drug candidates for PD treatment.

neuroscience↗

bioRxiv2024

USP14 regulates pS129 α-synuclein levels and oxidative stress in human SH-SY5Y dopaminergic cells

Ubiquitin specific protease-14 (USP14) is critical for controlling protein homeostasis disturbed in human disorders like Parkinso[n]s disease (PD). Here we investigated the role of USP14 in regulating proteasome and autophagy pathways, and their influence on -synuclein (-syn) degradation. Data showed that -syn and phosphorylated serine129 -syn (pS129 -syn) were elevated in USP14 gene-deleted SH-SY5Y dopaminergic cells with concomitant reduction in proteasome activity. Inhibition of proteasomes using MG132 particularly elevated pS129 -syn in these cells, but the levels were not influenced by inhibiting autophagy using chloroquine. In contrast, autophagy and the CLEAR (Coordinated Lysosomal Expression and Regulation) pathways were elevated in USP14 lacking cells with an upregulation of the transcription factor TFEB. USP14-ablated cells also exhibited increases in reactive oxidative species (ROS) and elongation of mitochondria. The addition of N-Acetylcysteine amide (NACA) to counteract oxidative stress, reduced pS129 -syn and -syn levels in USP14 deficient cells. Phospho-proteomic analyses revealed that USP14 is phosphorylated at S143 affecting its function and structure as shown by molecular modeling, and protein interaction studies. Re-expression of wild-type and the phospho-mimetic S143D-USP14 mutant decreased ROS, pS129 -syn, and -syn in USP14 lacking cells. These results demonstrate that pS129 -syn levels are sensitive to oxidative stress in SH-SY5Y dopaminergic cells. USP14 by stimulating the proteasome activity and reducing oxidative stress is a promising factor for targeting -syn and its pathogenic variants in PD.

neuroscience↗

bioRxiv2023

Small molecule activation of m6A mRNA methylation as a novel approach for neuroprotection

N6-Methyladenosine (m6A) is the most common mRNA base modification in eukaryotes. Methylation of adenosine residues to m6A contributes to the regulation of splicing, transport, stability, and translation of mRNA and two main classes of enzymes regulate it. The formation of m6A is catalysed by a methyltransferase complex containing methyltransferase-like 3 (METTL3), METTL14, and Wilms tumour 1-associated protein (WTAP) as well as monomeric METTL16. Demethylation of m6A is catalysed by the fat mass and obesity-associated protein FTO and the RNA demethylase AlkB homolog 5 (ALKBH5). The m6A mRNA methylation dysregulation occurs in the nervous system and in Parkinsons disease (PD), but it remains poorly studied. Moreover, the role of m6A mRNA methylation in neuronal survival, neuroprotection, and neuroregeneration is unclear. We have earlier used high-throughput virtual screening of large compound libraries and identified four unique small-molecule ligands that activate m6A mRNA methylation by binding to the METTL3/14/WTAP complex and enhancing the binding of the methylation substrate SAM to nanomolar concentrations. Following this, we now discovered that two methyltransferase activators at 10 nM concentrations supported the survival and protected dopamine (DA) neurons in culture in growth factor deprivation and 6-hydroxydopamine (6-OHDA) neurotoxin models. In contrast, METTL3/14 inhibitor STM2457 triggered death of DA neurons. For clinical translation we also tested the most efficient compound C4 on induced pluripotent stem cell-derived human DA neurons and in animal model of Parkinsons disease (PD). C4 compound protected human DA neurons from 6-OHDA-induced cell death and increased neurite outgrowth and the number of processes demonstrating that it has both neuroprotective and neurorestorative properties. METTL3/14 activator C4 improved motor behaviour and protected DA neurons and their fibres faster and much more efficiently than GDNF in the rat 6-OHDA model of PD. These are the first specific activators of METTL3/14/WTAP and first demonstration that m6A regulators can protect and regenerate neurons. These data demonstrate that m6A mRNA methylation is a novel pathway regulating neuronal survival and regeneration.

neuroscience↗

bioRxiv2023

Novel RNA m6A methyltransferase METTL16 inhibitors

The overexpression of RNA 6-N-methyladenosine (m6A) methyltransferase METTL16 has oncogenic role in the case of several cancer types, including leukemia, but efficient small-molecule inhibitors are not available. Initially identified by high-throughput virtual screening of the ZINC15 database in vivo subset, but then confirmed by measuring catalytic activity, two nanomolar-active METTL16 inhibitors, compounds 1 (IC50 = 25.82 {+/-} 17.19 nM) and 2 (IC50 = 60.91 {+/-} 2.75 nM) were found. The inhibitory activity of the compounds was measured using the m6A antibody-based ELISA assay. We also present the results on the effect of these inhibitors on the viability of promyeloblast HL-60 and lymphoblast CCRF-CEM leukemia cell lines. In unstressed growth conditions, both identified METTL16 inhibitors reduced the viability of HL-60 cells by up to 40%. The effect on the viability of CCRF-CEM cells was smaller with no dose dependency observed. In parallel, the level of the m6A as compared to unmodified adenosine in the HL-60 cell mRNAs was significantly reduced by the inhibitor 1. Collectively, we herein demonstrate novel METTL16 inhibitors that exert tumor cell-lineage-selective antiproliferative effects.

pharmacology and toxicology↗

bioRxiv2020

Maximum Antigen Diversification in a Lyme Bacterial Population and Evolutionary Strategies to Overcome Pathogen Diversity

Natural populations of microbes and their hosts are engaged in an arms race in which microbes diversify to escape host immunity while hosts evolve novel immunity. This co-evolutionary process, known as the "Red Queen" hypothesis, poses a fundamental challenge to the development of broadly effective vaccines and diagnostics against a diversifying pathogen. Based on surveys of natural allele frequencies and experimental immunization of mice, we show minimal antigenic cross-reactivity among natural variants of the outer surface protein C (OspC), a dominant antigen of a Lyme Disease-causing bacterium (Borrelia burgdorferi). To overcome the challenge of OspC antigenic diversity to clinical development of preventive measures, we implemented a number of evolution-based strategies to broaden OspC immunological cross-reactivity. In particular, the centroid algorithm - a genetic algorithm to minimize sequence differences with natural variants - generated synthetic OspC analogs with the greatest promise as diagnostic and vaccine candidates against diverse Lyme pathogen strains coexisting in the Northeast United States. Mechanistically, we propose a model of runaway maximum antigen di-versification (MAD) mediated by amino-acid variations distributed across hypervariable regions on the OspC molecule. Under the MAD model, evolutionary centroids display high cross-reactivity by occupying the central void in the antigenic space excavated by diversifying natural variants. In contrast to the vaccine design based on concatenated epitopes, the centroid algorithm generates analogs of native antigens and is automated. The MAD model and evolution-inspired antigen designs have broad implications for combating diversifying pathogens driven by pathogen-host coevolution. ImportanceMicrobial pathogens rely on molecular diversity of cell surface antigens to escape host immunity. Vaccines based on one antigen variant often fail to protect the host against pathogens carrying other variants. Here we show evolution-based designs of synthetic antigens that are broadly reactive to all natural variants. The evolutionary analogs of a major surface antigen of a Lyme disease bacterium (Borrelia burgdorferi) showed promise as vaccine candidates against diverse pathogen strains coexisting in the endemic areas of Lyme disease in Northeast United States. Our evolution-based computational design is automated, generates molecular analogs of natural antigens, and opens a novel path to combating fast-evolving microbial pathogens.

microbiology↗

bioRxiv2020

MANF regulates unfolded protein response and neuronal survival through its ER-located receptor IRE1α

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an endoplasmic reticulum (ER)-located protein with cytoprotective effects in numerous cell types in vitro and in models of neurodegeneration and diabetes in vivo. So far, the exact mode of its action has remained elusive and plasma membrane or ER-located receptors of MANF have not been identified. We have found that MANF can directly interact with transmembrane unfolded protein response (UPR) receptor IRE1 and compete with the major ER chaperone BiP (GRP78) for the interaction with IRE1. With lower affinities MANF can also interact with other UPR receptors, PERK and ATF6. Using molecular modeling and mutagenesis analysis, we have identified the exact structural MANF regions involved in its binding to the luminal domain of IRE1. MANF attenuates UPR signaling by decreasing IRE1 oligomerization and IRE1 phosphorylation. MANF mutant deficient in IRE1 binding cannot regulate IRE1 oligomerization and fails to protect neurons from ER stress induced death. Importantly, we found that MANF-IRE1 interaction is also crucial for the survival promoting action of MANF for dopamine neurons in an animal model of Parkinsons disease. Our data reveal a novel mechanism of IRE1 regulation during ER stress and demonstrate the intracellular mode of action of MANF as a modulator of UPR and neuronal cell survival through the direct interaction with IRE1 and regulation of its activity. Furthermore, our data explain why MANF in contrast to other growth factors has no effects on naive cells and rescues only ER stressed or injured cells.

cell biology↗

bioRxiv2019

Structure of herpes simplex virus pUL7:pUL51, a conserved complex required for efficient herpesvirus assembly

Herpesviruses are an ancient family of highly-prevalent human and animal pathogens that acquire their membrane envelopes in the cytoplasm of infected cells. While multiple conserved viral proteins are known to be required for efficient herpesvirus production, many of these proteins lack identifiable structural homologues and the molecular details of herpesvirus assembly remain unclear. We have characterized the complex of assembly proteins pUL7 and pUL51 from herpes simplex virus (HSV)-1, an -herpesvirus, using multi-angle light scattering and small-angle X-ray scattering with chemical crosslinking. HSV-1 pUL7 and pUL51 form a stable 1:2 complex that is capable of higher-order oligomerization in solution. We solved the crystal structure of this complex, revealing a core heterodimer comprising pUL7 bound to residues 41-125 of pUL51. While pUL7 adopts a previously-unseen compact fold, the extended helix-turn-helix conformation of pUL51 resembles the cellular endosomal complex required for transport (ESCRT)-III component CHMP4B, suggesting a direct role for pUL51 in promoting membrane scission during virus assembly. We demonstrate that the interaction between pUL7 and pUL51 homologues is conserved across human -, {beta}- and {gamma}-herpesviruses, as is their association with trans-Golgi membranes in cultured cells. However, pUL7 and pUL51 homologues do not form complexes with their respective partners from different virus families, suggesting that the molecular details of the interaction interface have diverged. Our results demonstrate that the pUL7:pUL51 complex is conserved across the herpesviruses and provide a structural framework for understanding its role in herpesvirus assembly.\n\nSignificance StatementHerpesviruses are extremely common human pathogens that cause diseases ranging from cold sores to cancer. Herpesvirus acquire their membrane envelope in the cytoplasm via a conserved pathway, the molecular details of which remain unclear. We have solved the structure of a complex between herpes simplex virus (HSV)-1 proteins pUL7 and pUL51, two proteins that are required for efficient HSV-1 assembly. We show that formation of this complex is conserved across distantly-related human herpesviruses, as is the association of these homologues with cellular membranes that are used for virion assembly. While pUL7 adopts a previously-unseen fold, pUL51 resembles key cellular membrane-remodeling complex components, suggesting that the pUL7:pUL51 complex may play a direct role in deforming membranes to promote virion assembly.

microbiology↗