Eukaryotic Research Articles

A special issue of Essays in Biochemistry on AMPK and AMPK-related kinases

Abstract In eukaryotic cells, AMP-activated protein kinase (AMPK) plays a central role in responding to nutrient limitation by switching-off ATP-consuming (anabolic) pathways and switching-on ATP generating (catabolic) pathways. Over the last 30 years or so, a considerable body of research has been carried out that has provided us with a wealth of knowledge regarding the regulation and role of AMPK. Despite this, there is still much to learn about AMPK and the field remains highly active, with many groups around the world continuing to explore new roles for AMPK, providing insight into its biological function. This review series was inspired by recent AMPK-focused meetings in Scotland (2022) and Australia (2023) and draws on some of the research presented at those meetings.

Studies on the Oxidative Damage of the Wobble 5-Methylcarboxymethyl-2-Thiouridine in the tRNA of Eukaryotic Cells with Disturbed Homeostasis of the Antioxidant System

We have previously shown that 2-thiouridine (S2U), either as a single nucleoside or as an element of RNA chain, is effectively desulfurized under applied in vitro oxidative conditions. The chemically induced desulfuration of S2U resulted in two products: 4-pyrimidinone nucleoside (H2U) and uridine (U). Recently, we investigated whether the desulfuration of S2U is a natural process that also occurs in the cells exposed to oxidative stress or whether it only occurs in the test tube during chemical reactions with oxidants at high concentrations. Using different types of eukaryotic cells, such as baker’s yeast, human cancer cells, or modified HEK293 cells with an impaired antioxidant system, we confirmed that 5-substituted 2-thiouridines are oxidatively desulfurized in the wobble position of the anticodon of some tRNAs. The quantitative LC-MS/MS-MRMhr analysis of the nucleoside mixtures obtained from the hydrolyzed tRNA revealed the presence of the desulfuration products of mcm5S2U: mcm5H2U and mcm5U modifications. We also observed some amounts of immature cm5S2U, cm5H2U and cm5U products, which may have indicated a disruption of the enzymatic modification pathway at the C5 position of 2-thiouridine. The observed process, which was triggered by oxidative stress in the living cells, could impair the function of 2-thiouridine-containing tRNAs and alter the translation of genetic information.

The T4bSS of Legionella features a two-step secretion pathway with an inner membrane intermediate for secretion of transmembrane effectors.

To promote intracellular survival and infection, Legionella spp. translocate hundreds of effector proteins into eukaryotic host cells using a type IV b protein secretion system (T4bSS). T4bSS are well known to translocate soluble as well as transmembrane domain-containing effector proteins (TMD-effectors) but the mechanisms of secretion are still poorly understood. Herein we investigated the secretion of hydrophobic TMD-effectors, of which about 80 were previously reported to be encoded by L. pneumophila. A proteomic analysis of fractionated membranes revealed that TMD-effectors are targeted to and inserted into the bacterial inner membranes of L. pneumophila independent of the presence of a functional T4bSS. While the T4bSS chaperones IcmS and IcmW were critical for secretion of all tested TMD-effectors, they did not influence inner membrane targeting of these proteins. As for soluble effector proteins, translocation of all investigated TMD-effectors depended on a C-terminal secretion signal. A deeper analysis of the TMD-effector SidF showed that this signal needed to be presented towards the cytoplasmic side of the inner membrane and that a small periplasmic loop was required for efficient translocation. We propose that strongly hydrophobic TMD-effectors are secreted in a two-step secretion process: Initially, an inner membrane intermediate is formed, that is extracted towards the cytoplasmic side, possibly by the help of the type IV coupling protein complex and subsequently secreted into eukaryotic host cells by the T4bSS core complex. Overall, our study highlights the amazing versatility of T4bSS to secrete soluble and TMD-effectors from different subcellular locations of the bacterial cell.

The vacuolar K+/H+ exchangers and calmodulin-like CML18 constitute a pH-sensing module that regulates K+ status in Arabidopsis.

Shifts in cytosolic pH have been recognized as key signaling events and mounting evidence supports the interdependence between H+ and Ca2+ signaling in eukaryotic cells. Among the cellular pH-stats, K+/H+ exchange at various membranes is paramount in plant cells. Vacuolar K+/H+ exchangers of the NHX (Na+,K+/H+ exchanger) family control luminal pH and, together with K+ and H+ transporters at the plasma membrane, have been suggested to also regulate cytoplasmic pH. We show the regulation of vacuolar K+/H+ exchange by cytoplasmic pH and the calmodulin-like protein CML18 in Arabidopsis. The crystal structure and physicochemical properties of CML18 indicate that this protein senses pH shifts. Interaction of CML18 with tonoplast exchangers NHX1 and NHX2 was favored at acidic pH, a physiological condition elicited by K+ starvation in Arabidopsis roots, whereas excess K+ produced cytoplasmic alkalinization and CML18 dissociation. These results imply that the pH-responsive NHX-CML18 module is an essential component of the cellular K+- and pH-stats.

Bacterial Organelles in Iron Physiology.

Bacteria were once thought to be simple organisms, lacking the membrane-bound organelles found in eukaryotic cells. However, recent advancements in microscopy have changed this view, revealing a diverse array of organelles within bacterial cells. These organelles, surrounded by lipid bilayers, protein-lipid monolayers, or proteinaceous shells, play crucial roles in facilitating biochemical reactions and protecting cells from harmful byproducts. Unlike eukaryotic organelles, which are universally present, bacterial organelles are species-specific and induced only under certain conditions. This review focuses on the bacterial organelles that contain iron, an essential micronutrient for all life forms but potentially toxic when present in excess. To date, three types of iron-related bacterial organelles have been identified: two membrane-bound organelles, magnetosomes and ferrosomes, and one protein-enclosed organelle, the encapsulated ferritin-like proteins. This article provides an updated overview of the genetics, biogenesis, and physiological functions of these organelles. Furthermore, we discuss how bacteria utilize these specialized structures to adapt, grow, and survive under various environmental conditions.

Ribozyme-activated mRNA trans-ligation enables large gene delivery to treat muscular dystrophies.

Ribozymes are small catalytic RNA sequences capable of nucleotide-specific self-cleavage found widespread in nature. Ribozyme cleavage generates distinct 2',3'-phosphate and 5'-hydroxyl termini that resemble substrates for recently characterized RNA repair pathways in cells. We report that ribozyme cleavage of two separate mRNAs activated their scarless trans-ligation and translation into full-length protein in eukaryotic cells, a process that we named StitchR (for Stitch RNA). Optimization of StitchR activity in mammalian cells resulted in a ~900-fold increase in protein expression that approached levels observed for genes expressed from single vectors. We demonstrate that StitchR can be harnessed for effective dual adeno-associated virus gene therapies to correct muscular dystrophies by restoring large functional muscle proteins to endogenous levels in vivo.

Structural response of microtubule and actin cytoskeletons to direct intracellular load.

Microtubule and actin are the two major cytoskeletal polymers that form organized functional structures in the interior of eukaryotic cells. Although the structural mechanics of the cytoskeleton has been extensively studied by direct manipulations in in vitro reconstitution systems, such unambiguous characterizations inside the living cell are sparse. Here, we report a comprehensive analysis of how the microtubule and actin cytoskeletons structurally respond to direct intracellular load. Ferrofluid-based intracellular magnetic tweezers reveal rheological properties of the microtubule complex primarily determined by filamentous actin. The strain fields of the microtubule complex and actin meshwork follow the same scaling, suggesting that the two cytoskeletal systems behave as an integrated elastic body. The structural responses of single microtubules to contact and remote forces further evidence that the individual microtubules are enclosed by the elastic medium of actin. These results, directly characterizing the microtubule and actin cytoskeletons as an interacting continuum throughout the cytoplasm, serve as a cornerstone for the physical understanding of intracellular organization.

SITP: A single cell bioinformatics analysis flow captures proteasome markers in the development of breast cancer.

Single cell sequencing and related databases have been widely used in the exploration of cancer occurrence and development, but there is still no in-depth explanation of specific and complicated cellular protein modification processes. Ubiquitin-Proteasome System (UPS), as a specific and precise protein modification and degradation process, plays an important role in the biological functions of cancer cell proliferation and apoptosis. Proteasomes, vital multi-catalytic proteinases in eukaryotic cells, play a crucial role in protein degradation and contribute to tumor regulation. The 26S proteasome, part of the ubiquitin-proteasome system. In this study, we have enrolled a common SITP process including analysis of single cell sequencing to elucidate a flow that can capture typical proteasome markers in the oncogenesis and progression of breast cancer. PSMD11, a key component of the 26S proteasome regulatory particle, has been identified as a critical survival factor in cancer cells. Results suggest that PSMD11's rapid degradation is linked to acute apoptosis in cancer cells, making it a potential target for cancer treatment. Our study explored the potential mechanisms of PSMD11 in breast cancer development. The findings revealed the feasibility of disclosing ubiquitinating biomarkers from public database, as well as presented new evidence supporting PSMD11 as a potential therapeutic biomarker for breast cancer.

Comprehensive identification of LEA protein family genes and functional analysis of MdLEA60 involved in abiotic stress responses in apple (Malus domestica).

Late embryogenesis abundant (LEA) proteins are important proteins that exists widely in many plants and contribute to physiological processes of plant stress resistance. Despite LEA proteins being identified in many plants, none have been reported in apple (Malus domestica) until this study. In this study, a total of 87 MdLEA proteins were identified in apple, and a comprehensive analysis was conducted to elucidate the functions of MdLEA proteins in response to abiotic stress. Results showed that they were classified into 7 groups and distributed on 16 chromosomes. Collineation analysis revealed that segmental duplication primarily drove the expansion of MdLEA genes. The MdLEA promoters were enriched with elements associated with various stress responses. Through transcriptome and qRT-PCR analysis, several MdLEA genes related to drought/salinity/cold were excavated, and MdLEA60 was selected for transgenic validation. The ectopic expression of MdLEA60 enhanced osmotic and extreme temperature tolerance in both prokaryotic and eukaryotic cells, providing stress resistance support via antioxidant protection. Overall, the comprehensive analyses and identification not only establish a basis for future investigation into the functional mechanism of MdLEA proteins but also provide potential candidate genes for apple resistance breeding optimization.

Advances in the structure and function of the nucleolar protein fibrillarin

Fibrillarin (FBL) is a highly conserved and well-researched nucleolar protein found in eukaryotes. Its presence was first identified in 1985 through protein immunoblotting analyses using antisera from patients with autoimmune scleroderma. Through immunoelectron microscopy, FBL was shown to be localized in the dense fibrillar component of the nucleolus, leading to the term “fibrillarin”. The FBL protein is composed of 321 amino acids and contains two significant functional domains: the GAR domain and the methyltransferase domain. It is expressed in the nucleolus of eukaryotes. This makes FBL one of the most studied nucleolar proteins. While methylation is not essential for cell survival, the FBL gene is crucial for eukaryotic cells, underscoring the importance of investigating additional functions that do not rely on FBL methylation. This review will primarily examine the protein structural domains of FBL and its classic methyltransferase activity. Additionally, our review will examine the importance of the eukaryote-specific GAR structural domain of FBL in regulating intracellular phase separation. Furthermore, this paper analyzes recent developments in the utilization of FBL in the study of pathogen infections and cancer research over the past decade.

Investigating the Structure of the Components of the PolyADP-Ribosylation System in Fusarium Fungi and Evaluating the Expression Dynamics of Its Key Genes

Poly(ADP-ribose) polymerase (PARP) is the key enzyme in polyADP-ribosylation, one of the main post-translational modifications. This enzyme is abundant in eukaryotic organisms. However, information on the PARP structure and its functions in members of the Fungi kingdom is very limited. In this study, we performed a bioinformatic search for homologs of PARP and its antagonist, PARG, in the genomes of four Fusarium strains using their whole-genome sequences annotated and deposited in databases. The F. graminearum PH-1, F. proliferatum ET-1, and F. oxysporum Fo47 strains were shown to possess a single homolog of both PARP and PARG. In addition, the F. oxysporum f. sp. lycopersici strain 4287 contained four additional proteins comprising PARP catalytic domains whose structure was different from that of the remaining identified homologs. Partial nucleotide sequences encoding the catalytic domains of the PARP and PARG homologs were determined in 11 strains of 9 Fusarium species deposited in all-Russian collections, and the phylogenetic properties of the analyzed genes were evaluated. In the toxigenic F. graminearum strain, we demonstrated up-regulation of the gene encoding the PARP homolog upon culturing under conditions stimulating the production of the DON mycotoxin, as well as up-regulation of the gene encoding PARG at later stages of growth. These findings indirectly indicate involvement of the polyADP-ribosylation system in the regulation of the genes responsible for DON biosynthesis.

Flow in filopodia: re-organization and the representation of biological entities as computational objects

AbstractActin is the most abundant protein in eukaryotic cells. They form filamentous polymers that are organized in different ways within the cell to perform various functions. For instance, prominent parallel bundles of F-actins mediate the formation and dynamics of filopodia that are long, finger-like protrusions of cell membrane occurring in certain cells, like growing neurons. Understanding actin organization dynamics and its regulation is a crucial problem for biologists that cannot be solved exclusively by biological methods, requiring the support of mathematical and computational modelling. In this work, grounded on a previous hypothesis of ours about the cytosol flow within filopodia, we address several modelling challenges posed by the growth of filopodia in neurons. We use alternative stochastic models and particle-centered numerical methods for transport and elongations, as well as an innovative object-oriented modelling-strategy to represent chemical transformations, polymerization, and their regulation. These modelling strategies allowed for simulating elongations 20 times longer than the typical ranges attained by commonly used filopodia diffusion models, and show that our hypothesis is feasible, acting as a proof-of-concept about the importance of considering organization as a key element in biological explanations.

A statistical mechanics investigation of unfolded protein response across organisms.

Living systems rely on coordinated molecular interactions, especially those related to gene expression and protein activity. The Unfolded Protein Response is a crucial mechanism in eukaryotic cells, activated when unfolded proteins exceed a critical threshold. It maintains cell homeostasis by enhancing protein folding, initiating quality control, and activating degradation pathways when damage is irreversible. This response functions as a dynamic signaling network, with proteins as nodes and their interactions as edges. We analyze these protein-protein networks across different organisms to understand their intricate intra-cellular interactions and behaviors. In this work, analyzing twelve organisms, we assess how fundamental measures in network theory can individuate seed proteins and specific pathways across organisms. We employ network robustness to evaluate and compare the strength of the investigated protein-protein interaction networks, and the structural controllability of complex networks to find and compare the sets of driver nodes necessary to control the overall networks. We find that network measures are related to phylogenetics, and advanced network methods can identify main pathways of significance in the complete Unfolded Protein Response mechanism.

Saccharomyces Genome Database: Advances in Genome Annotation, Expanded Biochemical Pathways, and Other Key Enhancements.

Budding yeast (Saccharomyces cerevisiae) is the most extensively characterized eukaryotic model organism and has long been used to gain insight into the fundamentals of genetics, cellular biology, and the functions of specific genes and proteins. The Saccharomyces Genome Database (SGD) is a scientific resource that provides information about the genome and biology of S. cerevisiae. For more than 30 years, SGD has maintained the genetic nomenclature, chromosome maps, and functional annotation for budding yeast along with search and analysis tools to explore these data. Here we describe recent updates at SGD, including the two most recent reference genome annotation updates, expanded biochemical pathways representation, changes to SGD search and data files, and other enhancements to the SGD website and user interface. These activities are part of our continuing effort to promote insights gained from yeast to enable the discovery of functional relationships between sequence and gene products in fungi and higher eukaryotes.

Physiological and pathological roles of the transcriptional kinases CDK12 and CDK13 in the central nervous system.

The cyclin-dependent kinases 12 (CDK12) and 13 (CDK13) govern several steps of gene expression, including transcription, RNA processing and translation. The main target of CDK12/13 is the serine 2 residue of the carboxy-terminal domain of RNA polymerase II (RNAPII), thus influencing the directionality, elongation rate and processivity of the enzyme. The CDK12/13-dependent regulation of RNAPII activity influences the expression of selected target genes with important functional roles in the proliferation and viability of all eukaryotic cells. Neuronal cells are particularly affected by the loss of CDK12/13, as result of the high dependency of neuronal genes on RNAPII processivity for their expression. Deregulation of CDK12/13 activity strongly affects brain physiology by influencingthe stemness potential and differentiation properties of neuronal precursor cells. Moreover, mounting evidence also suggest the involvement of CDK12/13 in brain tumours. Herein, we discuss the functional role(s) of CDK12 and CDK13 in gene expression regulation and highlight similarities and differences between these highly homologous kinases, with particular attention to their impact on brain physiology and pathology. Lastly, we provide an overview of CDK12/13 inhibitors and of their efficacy in brain tumours and other neoplastic diseases.

DNAR-02. M33 CONDENSES CHROMATIN THROUGH NUCLEAR BODY FORMATION AND METHYLATION OF BOTH HISTONE H3 LYSINE 9 AND LYSINE 27

Abstract Heterochromatin, a type of condensed DNA in eukaryotic cells, has two main categories: Constitutive heterochromatin, which contains H3K9 methylation, and facultative heterochromatin, which contains H3K27 methylation. Methylated H3K9 and H3K27 serve as docking sites for chromodomain-containing proteins that compact chromatin. M33 (also known as CBX2) is a chromodomain-containing protein that binds H3K27me3 and compacts chromatin in vitro. However, whether M33 mediates chromatin compaction in cellulo remains unknown. Here we show that M33 compacts chromatin into DAPI-intense heterochromatin domains in cells. The formation of these heterochromatin domains requires H3K27me3, which recruits M33 to form nuclear bodies. G9a and SUV39H1 are sequentially recruited into M33 nuclear bodies to create H3K9 methylated chromatin in a process that is independent of HP1α. Finally, M33 decreases progerin-induced nuclear envelope disruption caused by loss of heterochromatin. Our findings demonstrate that M33 mediates the formation of condensed chromatin by forming nuclear bodies containing both H3K27me3 and H3K9me3. Our model of M33-dependent chromatin condensation suggests H3K27 methylation corroborates with H3K9 methylation during the formation of facultative heterochromatin and provides the theoretical basis for developing novel therapies to treat heterochromatin-related diseases. Keywords: H3K27 methylation; H3K9 methylation; Heterochromatin; M33 (CBX2); Nuclear body (NB); Progerin.

Conservation of the Polyamines Pathway in Ustilaginomycetes A Genomic and Experimental Approach.

Polyamines are organic and aliphatic molecules essential for the growth, development, and survival of both eukaryotes and prokaryotes. In fungi, polyamines play a crucial role in cellular differentiation and pathogenesis. Since fungi and animals are closely related evolutionarily, and fungi can be easily genetically manipulated in the lab, they serve as excellent models for studying polyamine metabolism and the molecular mechanisms controlled by these biomolecules. Although the metabolism of polyamines has been extensively studied in model fungi such as Saccharomyces cerevisiae and Ustilago maydis, the conservation of the polyamine biosynthesis pathway in other Ustilaginomycetes, a class of fungi that includes phytopathogens, saprophytes, mutualists, and mycorrhizae, has not been thoroughly investigated. In this study, using a genomic and bioinformatics approach, we analyzed the conservation of the polyamine biosynthesis pathway in Ustilaginomycetes. Additionally, we confirmed the functional conservation of ornithine decarboxylase (Odc), which is involved in the synthesis of putrescine, one of the most important polyamines in fungi and complex multicellular eukaryotic organisms, using genetics and molecular biology tools. Moreover, we identified the differentially regulated genes by this polyamine in U. maydis. This research provides insights into the similarities and differences in the conservation of the polyamine biosynthesis pathway in fungi, and it expands our understanding of the role of polyamines and the mechanisms regulated by these molecules in eukaryotes.

Genetic potential for aerobic respiration and denitrification in globally distributed respiratory endosymbionts

The endosymbiont Candidatus Azoamicus ciliaticola was proposed to generate ATP for its eukaryotic host, an anaerobic ciliate of the Plagiopylea class, fulfilling a function analogous to mitochondria in other eukaryotic cells. The discovery of this respiratory endosymbiosis has major implications for both evolutionary history and ecology of microbial eukaryotes. However, with only a single species described, knowledge of its environmental distribution and diversity is limited. Here we report four complete, circular metagenome assembled genomes (cMAGs) representing respiratory endosymbionts inhabiting groundwater in California, Ohio, and Germany. These cMAGs form two lineages comprising a monophyletic clade within the uncharacterized gammaproteobacterial order UBA6186, enabling evolutionary analysis of their key protein complexes. Strikingly, all four cMAGs encode a cytochrome cbb3 oxidase, which indicates that these endosymbionts have the capacity for aerobic respiration. Accordingly, we detect these respiratory endosymbionts in diverse habitats worldwide, thus further expanding the ecological scope of this respiratory symbiosis.

The Warburg Effect is the result of faster ATP production by glycolysis than respiration

Many prokaryotic and eukaryotic cells metabolize glucose to organism-specific by-products instead of fully oxidizing it to carbon dioxide and water-a phenomenon referred to as the Warburg Effect. The benefit to a cell is not fully understood, given that partial metabolism of glucose yields an order of magnitude less adenosine triphosphate (ATP) per molecule of glucose than complete oxidation. Here, we test a previously formulated hypothesis that the benefit of the Warburg Effect is to increase ATP production rate by switching from high-yielding respiration to faster glycolysis when excess glucose is available and respiration rate becomes limited by proteome occupancy. We show that glycolysis produces ATP faster per gram of pathway protein than respiration in Escherichia coli, Saccharomyces cerevisiae, and mammalian cells. We then develop a simple mathematical model of energy metabolism that uses five experimentally estimated parameters and show that this model can accurately predict absolute rates of glycolysis and respiration in all three organisms under diverse conditions, providing strong support for the validity of the ATP production rate maximization hypothesis. In addition, our measurements show that mammalian respiration produces ATP up to 10-fold slower than respiration in E. coli or S. cerevisiae, suggesting that the ATP production rate per gram of pathway protein is a highly evolvable trait that is heavily optimized in microbes. We also find that E. coli respiration is faster than fermentation, explaining the observation that E. coli, unlike S. cerevisiae or mammalian cells, never switch to pure fermentation in the presence of oxygen.

Ascorbic acid regulates in vitro and in vivo toxicogenetic effects of hydroxyurea on eukaryotic cells

Hydroxyurea (HU) exerts unique and diverse biological effects as an anti-leukemic agent, irradiation sensitizer, and HbS inducer in patients with sickle cell anemia. Herein, we assessed the potential toxicogenic and/or oxidant effects of hydroxyurea associated with ascorbic acid by in vivo examinations in Allium cepa and human cancer cells and systemically on mice tissues. Growing A. cepa roots and HCT-116 colorectal tumor cells were examined after HU and HU plus ascorbic acid exposure. DNA damage and antioxidant enzymatic activity were quantified in peripheral blood mononuclear cells (PBMC), bone marrow leukocytes and livers of mice after 7 day-HU treatment (7.5, 15 and 30 mg/kg/day) and Vitamin C 2 μM. Hydroxyurea presented toxic effects on meristematic Allium cepa cells, causing chromosomal abnormalities and reduction of mitotic index, killed HCT-116 colorectal carcinoma cells and induced DNA injuries upon mice cells (hepatocytes, bone marrow leukocytes and PBMC). Simultaneously, hydroxyurea decreased levels of CAT and GSH activities and expand lipid peroxidation. All these biochemical and physiological changes were ameliorated when associated with ascorbic acid, indicating it restored antioxidant enzymes, decreased MDA levels, removed peroxides and, consequently, presented cytoprotection against HU-provoked cellular damage in normal cells. On the other hand, antioxidants compounds may interfere on effectiveness of HU during anticancer chemotherapies.