Structural Determinants within the Adenovirus Early Region 1A Protein Spacer Region Necessary for Tumorigenesis.

It has long been established that group A human adenoviruses (HAdV-A12, -A18, and -A31) can cause tumors in newborn rodents, with tumorigenicity related to the presence of a unique spacer region located between conserved regions 2 and 3 within the HAdV-A12 early region 1A (E1A) protein. Group B adenoviruses are weakly oncogenic, whereas most of the remaining human adenoviruses are nononcogenic. In an attempt to understand better the relationship between the structure of the AdE1A spacer region and oncogenicity of HAdVs, the structures of synthetic peptides identical or very similar to the adenovirus 12 E1A spacer region were determined and found to be α-helical using nuclear magnetic resonance (NMR) spectroscopy. This contrasts significantly with some previous suggestions that this region is unstructured. Using available predictive algorithms, the structures of spacer regions from other E1As were also examined, and the extent of the predicted α-helix was found to correlate reasonably well with the tumorigenicity of the respective virus. We suggest that this may represent an as-yet-unknown binding site for a partner protein required for tumorigenesis.IMPORTANCE This research analyzed small peptides equivalent to a region within the human adenovirus early region 1A protein that confers, in part, tumor-inducing properties to various degrees on several viral strains in rats and mice. The oncogenic spacer region is α-helical, which contrasts with previous suggestions that this region is unstructured. The helix is characterized by a stretch of amino acids rich in alanine residues that are organized into a hydrophobic, or "water-hating," surface that is considered to form a major site of interaction with cellular protein targets that mediate tumor formation. The extent of α-helix in E1A from other adenovirus species can be correlated to a limited degree to the tumorigenicity of that virus. Some serotypes show significant differences in predicted structural propensity, suggesting that the amino acid type and physicochemical properties are also of importance.

The Nucleolus: A Multiphase Condensate Balancing Ribosome Synthesis and Translational Capacity in Health, Aging and Ribosomopathies.

The nucleolus is the largest membrane-less structure in the eukaryotic nucleus. It is involved in the biogenesis of ribosomes, essential macromolecular machines responsible for synthesizing all proteins required by the cell. The assembly of ribosomes is evolutionarily conserved and is the most energy-consuming cellular process needed for cell growth, proliferation, and homeostasis. Despite the significance of this process, the intricate pathophysiological relationship between the nucleolus and protein synthesis has only recently begun to emerge. Here, we provide perspective on new principles governing nucleolar formation and the resulting multiphase organization driven by liquid-liquid phase separation. With recent advances in the structural analysis of ribosome formation, we highlight the current understanding of the step-wise assembly of pre-ribosomal subunits and the quality control required for proper function. Finally, we address how aging affects ribosome genesis and how genetic defects in ribosome formation cause ribosomopathies, complex diseases with a predisposition to cancer.

Inorganic polyphosphates and heavy metal resistance in microorganisms.

The mechanisms of heavy metal resistance in microbial cells involve multiple pathways. They include the formation of complexes with specific proteins and other compounds, the excretion from the cells via plasma membrane transporters in case of procaryotes, and the compartmentalization of toxic ions in vacuoles, cell wall and other organelles in case of eukaryotes. The relationship between heavy metal tolerance and inorganic polyphosphate metabolism was demonstrated both in prokaryotic and eukaryotic microorganisms. Polyphosphates, being polyanions, are involved in detoxification of heavy metals through complex formation and compartmentalization. The bacteria and fungi cultivated in the presence of some heavy metal cations contain the enhanced levels of polyphosphate. In bacteria, polyphosphate sequesters heavy metals; some of metal cations stimulate an exopolyphosphatase activity, which releases phosphate from polyphosphates, and MeHPO4- ions are then transported out of the cells. In fungi, the overcoming of heavy metal stresses is associated with the accumulation of polyphosphates in cytoplasmic inclusions, vacuoles and cell wall and the formation of cation/polyphosphate complexes. The effects of knockout mutations and overexpression of the genes encoding polyphosphate-metabolizing enzymes on heavy metal resistance are discussed.

CLIQ-BID: A method to quantify bacteria-induced damage to eukaryotic cells by automated live-imaging of bright nuclei.

Pathogenic bacteria induce eukaryotic cell damage which range from discrete modifications of signalling pathways, to morphological alterations and even to cell death. Accurate quantitative detection of these events is necessary for studying host-pathogen interactions and for developing strategies to protect host organisms from bacterial infections. Investigation of morphological changes is cumbersome and not adapted to high-throughput and kinetics measurements. Here, we describe a simple and cost-effective method based on automated analysis of live cells with stained nuclei, which allows real-time quantification of bacteria-induced eukaryotic cell damage at single-cell resolution. We demonstrate that this automated high-throughput microscopy approach permits screening of libraries composed of interference-RNA, bacterial strains, antibodies and chemical compounds in ex vivo infection settings. The use of fluorescently-labelled bacteria enables the concomitant detection of changes in bacterial growth. Using this method named CLIQ-BID (Cell Live Imaging Quantification of Bacteria Induced Damage), we were able to distinguish the virulence profiles of different pathogenic bacterial species and clinical strains.

Copper transporters are responsible for copper isotopic fractionation in eukaryotic cells.

Copper isotopic composition is altered in cancerous compared to healthy tissues. However, the rationale for this difference is yet unknown. As a model of Cu isotopic fractionation, we monitored Cu uptake in Saccharomyces cerevisiae, whose Cu import is similar to human. Wild type cells are enriched in 63Cu relative to 65Cu. Likewise, 63Cu isotope enrichment in cells without high-affinity Cu transporters is of slightly lower magnitude. In cells with compromised Cu reductase activity, however, no isotope fractionation is observed and when Cu is provided solely in reduced form for this strain, copper is enriched in 63Cu like in the case of the wild type. Our results demonstrate that Cu isotope fractionation is generated by membrane importers and that its amplitude is modulated by Cu reduction. Based on ab initio calculations, we propose that the fractionation may be due to Cu binding with sulfur-rich amino acids: methionine and cysteine. In hepatocellular carcinoma (HCC), lower expression of the STEAP3 copper reductase and heavy Cu isotope enrichment have been reported for the tumor mass, relative to the surrounding tissue. Our study suggests that copper isotope fractionation observed in HCC could be due to lower reductase activity in the tumor.

Mechanistic insights into selective autophagy pathways: lessons from yeast.

Autophagy has burgeoned rapidly as a field of study because of its evolutionary conservation, the diversity of intracellular cargoes degraded and recycled by this machinery, the mechanisms involved, as well as its physiological relevance to human health and disease. This self-eating process was initially viewed as a non-selective mechanism used by eukaryotic cells to degrade and recycle macromolecules in response to stress; we now know that various cellular constituents, as well as pathogens, can also undergo selective autophagy. In contrast to non-selective autophagy, selective autophagy pathways rely on a plethora of selective autophagy receptors (SARs) that recognize and direct intracellular protein aggregates, organelles and pathogens for specific degradation. Although SARs themselves are not highly conserved, their modes of action and the signalling cascades that activate and regulate them are. Recent yeast studies have provided novel mechanistic insights into selective autophagy pathways, revealing principles of how various cargoes can be marked and targeted for selective degradation.

[The role of mitochondrial dynamics in cell death].

Mitochondria are dynamic organelles whose homeostasis is defined by two opposite processes: fission (or fragmentation), or fusion. Fission of mitochondria results in generation of smaller organelles and fusion is when they produce tubular or net-like structures. Although a number of proteins are already known to control the process of fission/fusion additional regulators controlling these processes are being found. The Bcl-2 family members take part in the regulation of apoptosis and according to the current view are involved in the mitochondrial net-like structure maintenance. In this review we will discuss mechanisms of mitochondrial fission/fusion regulation and summarize the available information on the role of Bcl-2 family members in the regulation of mitochondrial fission/fusion dynamics.

FREE RADICALS, REACTIVE OXYGEN SPECIES, OXIDATIVE STRESSES AND THEIR CLASSIFICATIONS.

The phrases "free radicals" and "reactive oxygen species" (ROS) are frequently used interchangeably although this is not always correct. This article gives a brief description of two mentioned oxygen forms. During the first two-three decades after ROS discovery in biological systems (1950-1970 years) they were considered only as damaging agents, but later their involvement in organism protection and regulation of the expression of certain genes was found. The physiological state of increased steady-state ROS level along with certain physiological effects has been called oxidative stress. This paper describes ROS homeostasis and provides several classifications of oxidative stresses. The latter are based on time-course and intensity principles. Therefore distinguishing between acute and chronic stresses on the basis of the dynamics, and the basal oxidative stress, low intensity oxidative stress, strong oxidative stress, and finally a very strong oxidative stress based on the intensity of the action of the inductor of the stress are described. Potential areas of research include the development of this field with complex classification of oxidative stresses, an accurate identification of cellular targets of ROS action, determination of intracellular spatial and temporal distribution of ROS and their effects, deciphering the molecular mechanisms responsible for cell response to ROS attacks, and their participation in the normal cellular functions, i.e. cellular homeostasis and its regulation.

[Reparative autophagy and autophagy death of cells. Functional and regulatory aspects].

Molecular regulation of reparative/homeostatic autophagy induced by failure of vital resources and by cellular stress is considered. Extensive autophagy regulatory apparatus responds to starvation, insufficiency of growth factors and energy supply, accumulation of unfolded proteins (ER stress), reactive oxygen species, microbial invasion. Central sensor of the regulation is kinase mTOR. Part of the mTOR pool presented in lysosomes responds to the local level of amino acids and is able to induce autophagy under low rates of intralysosomal proteolysis. Autophagy is a self-regulated cell process, the peak of autophagy is followed by its regulatory weakening by amino acids formed in autophagolysosomes. Protective effect of autophagy is associated mainly with removal of permeabilized mitochondria generating ROS, and elimination of abnormally folded proteins. Autophagy has optimum of its activity: its deficiency leads to accelerated cellular aging, and the excessive autophagy brings to the deficiency of cellular survival resources and cell death. Autophagy cell death seems like a hyper-stimulated self-eating of the cell, but more probably that excessive autophagy disturbs cellular energy supply and switches on some specific cell death signalization (possibly associated with kinases c-Jun, DRP-1, PI3K class I etc.). Some approaches to use reparative autophagy for prevention of cell degeneration are considered.

Membranous replication factories induced by plus-strand RNA viruses.

In this review, we summarize the current knowledge about the membranous replication factories of members of plus-strand (+) RNA viruses. We discuss primarily the architecture of these complex membrane rearrangements, because this topic emerged in the last few years as electron tomography has become more widely available. A general denominator is that two "morphotypes" of membrane alterations can be found that are exemplified by flaviviruses and hepaciviruses: membrane invaginations towards the lumen of the endoplasmatic reticulum (ER) and double membrane vesicles, representing extrusions also originating from the ER, respectively. We hypothesize that either morphotype might reflect common pathways and principles that are used by these viruses to form their membranous replication compartments.

Antiviral atropisomers: conformational energy surfaces by NMR for host-directed myxovirus blockers.

Biologically active organic molecules characterized by a high single bond torsional barrier generate isolable isomers (atropisomers) and offer a unique stereochemical component to the design of selective therapeutic agents. The present work presents a nanomolar active inhibitor of myxoviruses, which most likely acts by blocking one or more cellular host proteins but also, serendipitously, exhibits axial chirality with an energy barrier of ΔG((++)) ≥30 kcal/mol. The latter has been probed by variable temperature NMR and microwave irradiation and by high level DFT transition state analysis and force field calculations. Full conformational profiles of the corresponding (aR,S) and (aS,S) atropisomers at ambient temperature were derived by conformer deconvolution with NAMFIS (NMR Analysis by Molecular Flexibility In Solution) methodology to generate seven and eight individual conformations, each assigned a % population. An accurate evaluation of a key torsion angle at the center of the molecules associated with a (3)JC-S-C-H coupling constant was obtained by mapping the S-C bond rotation with the MPW1PW91/6-31G-d,p DFT method followed by fitting the resulting dihedral angles and J-values to a Karplus expression. Accordingly, we have developed a complete conformational profile of diastereomeric atropisomers consistent with both high and low rotational barriers. We expect this assessment to assist the rationalization of the selectivity of the two (aR,S) and (aS,S) forms against host proteins, while offering insights into their divergent toxicity behavior.

Differential effects of CSF-1R D802V and KIT D816V homologous mutations on receptor tertiary structure and allosteric communication.

The colony stimulating factor-1 receptor (CSF-1R) and the stem cell factor receptor KIT, type III receptor tyrosine kinases (RTKs), are important mediators of signal transduction. The normal functions of these receptors can be compromised by gain-of-function mutations associated with different physiopatological impacts. Whereas KIT D816V/H mutation is a well-characterized oncogenic event and principal cause of systemic mastocytosis, the homologous CSF-1R D802V has not been identified in human cancers. The KIT D816V oncogenic mutation triggers resistance to the RTK inhibitor Imatinib used as first line treatment against chronic myeloid leukemia and gastrointestinal tumors. CSF-1R is also sensitive to Imatinib and this sensitivity is altered by mutation D802V. Previous in silico characterization of the D816V mutation in KIT evidenced that the mutation caused a structure reorganization of the juxtamembrane region (JMR) and facilitated its departure from the kinase domain (KD). In this study, we showed that the equivalent CSF-1R D802V mutation does not promote such structural effects on the JMR despite of a reduction on some key H-bonds interactions controlling the JMR binding to the KD. In addition, this mutation disrupts the allosteric communication between two essential regulatory fragments of the receptors, the JMR and the A-loop. Nevertheless, the mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. The distinct impact of equivalent mutation in two homologous RTKs could be associated with the sequence difference between both receptors in the native form, particularly in the JMR region. A local mutation-induced perturbation on the A-loop structure observed in both receptors indicates the stabilization of an inactive non-inhibited form, which Imatinib cannot bind.

"Three sources and three component parts" of free oligosaccharides.

Metabolism of glycoproteins and glycolipids is accompanied by the appearance of unbound structural analogues of the carbohydrate portion of glycoconjugates or so called free oligosaccharides. There are their several sources inside the cell: 1) multistep pathways of N-glycosylation, 2) the cell quality control and ER-associated degradation of misglycosylated and/or misfolded glycoproteins, 3) lysosomal degradation of mature glycoconjugates. In this review the information about the ways of free oligosaccharides appearance in different cell compartments and details of their structures depending on the source is summarized. In addition, extracellular free oligosaccharides, their structures and changes under normal and pathological conditions are discussed.

[Autophagy--molecular mechanism, apoptosis and cancer]. / Autofagia--mechanizm molekularny, apoptoza i nowotwory.

Autophagy is a catabolic process involving the degradation of long-lived proteins and organelles through the lysosomal machinery. In eukaryotic cells, among the three types of autophagy the most extensively studied is macroautophagy. Macroautophagy (hereafter referred to as autophagy) is characterized by sequestration of bulk cytoplasm in double-membrane vesicles, called autophagosomes, which ultimately fuse with lysosomes, resulting in degradation of their contents. Autophagy is responsible for the maintenance of intracellular homeostasis and enables cell survival under stress conditions. However, this process is also involved in the pathogenesis of diverse diseases, including cancers. In the cancer cell, autophagy plays a dual role, as a mechanism responsible for protecting or killing the cell. In most cases chemotherapy-induced autophagy in tumor cells is a prosurvival response which potentially leads to development of drug resistance. However, autophagy can also lead to cell death, thus enhancing treatment efficacy. It is important for the anticancer therapy to find the type of cancer cells which are susceptible to autophagy and to determine whether the autophagy induced by the applied therapy leads to cells' death or their survival and subsequently to therapy resistance. In this review, the molecular mechanism of macroautophagy and the most important signaling transduction pathways involved in regulation of this process in cancer cells are presented. The dual function of autophagy in tumorigenesis and the implications of autophagy modulation for cancer therapy are also discussed.

Immortalization and malignant transformation of eukaryotic cells.

The process of cellular transformation has been amply studied in vitro using immortalized cell lines. Immortalized cells never have the normal diploid karyotype, nevertheless, they cannot grow over one another in cell culture (contact inhibition), do not form colonies in soft agar (anchorage-dependent growth) and do not form tumors when injected into immunodeficient rodents. All these characteristics can be obtained with additional chromosome changes. Multiple genetic rearrangements, including whole chromosome and gene copy number gains and losses, chromosome translocations, gene mutations are necessary for establishing the malignant cell phenotype. Most of the experiments detecting transforming ability of genes overexpressed and/or mutated in tumors (oncogenes) were performed using mouse embryonic fibroblasts (MEFs), NIH3T3 mouse fibroblast cell line, human embryonic kidney 293 cell line (HEK293), and human mammary epithelial cell lines (mainly HMECs and MC-F10A). These cell lines have abnormal karyotypes and are prone to progress to malignantly transformed cells. This review is aimed at understanding the mechanisms of cell immortalization by different "immortalizing agents", oncogene-induced cell transformation of immortalized cells and moderate response of the advanced tumors to anticancer therapy in the light of tumor "oncogene and chromosome addiction", intra-/intertumor heterogeneity, and chromosome instability.

Phosphoinositides and cellular pathogens.

Phosphoinositides are considered as highly dynamic players in the spatiotemporal organization of key signaling pathways, actin cytoskeleton rearrangements, establishment of cell polarity and intracellular vesicle trafficking. Their metabolism is accurately controlled and mutations in several phosphoinositide metabolizing enzymes take part in the development of human pathologies. Interestingly, evidence is accumulating that modulation of the phosphoinositide metabolism is critical for pathogenicity and virulence of many human pathogens. Given the importance of phosphoinositides, which link membrane and cytoskeleton dynamics to cell responses, it is not surprising that many invasive pathogens hijack their metabolism as part of their strategies to establish infection. In fact, according to their lifestyle, cellular pathogens use the phosphoinositide metabolism in order to trigger their uptake in nonphagocytic cells and/or modulate the maturation of the pathogen-containing vacuole to establish their replicative niche or escape in the cytosol and promote host cell survival. The last two decades have been marked by the discovery of different tactics used by cellular pathogens to modulate the phosphoinositide metabolism as part of their strategies to survive, proliferate and disseminate in a hostile environment.

PI3Ks-drug targets in inflammation and cancer.

Phosphoinositide 3-kinases (PI3Ks) control cell growth, proliferation, cell survival, metabolic activity, vesicular trafficking, degranulation, and migration. Through these processes, PI3Ks modulate vital physiology. When over-activated in disease, PI3K promotes tumor growth, angiogenesis, metastasis or excessive immune cell activation in inflammation, allergy and autoimmunity. This chapter will introduce molecular activation and signaling of PI3Ks, and connections to target of rapamycin (TOR) and PI3K-related protein kinases (PIKKs). The focus will be on class I PI3Ks, and extend into current developments to exploit mechanistic knowledge for therapy.

Tumor stress inside out: cell-extrinsic effects of the unfolded protein response in tumor cells modulate the immunological landscape of the tumor microenvironment.

The unfolded protein response (UPR) is a eukaryotic cellular adaptive mechanism that functions to cope with stress of the endoplasmic reticulum (ER). Accumulating evidence demonstrates that the tumor microenvironment contains stressors that elicit a UPR, which has been demonstrated to be a cell-intrinsic mechanism crucial for tumorigenesis. In addition, the UPR is a source of proinflammatory signaling whose downstream mediators may hamper antitumor immunity. We discuss how the UPR may impair Ag presentation, which could result in defective T cell priming, also leading to tumor escape and growth. Further, we discuss the recent finding that ER stress and attendant proinflammation can be transmitted from ER-stressed tumor cells to myeloid cells. The ideas presented suggest that, in addition to being a cell-intrinsic mechanism of tumor survival, the tumor UPR can serve as a cell-extrinsic regulator of tumorigenesis by remodeling the immune response in the tumor microenvironment.

A model for sealing plasmalemmal damage in neurons and other eukaryotic cells.

Plasmalemmal repair is necessary for survival of damaged eukaryotic cells. Ca(2+) influx through plasmalemmal disruptions activates calpain, vesicle accumulation at lesion sites, and membrane fusion proteins; Ca(2+) influx also initiates competing apoptotic pathways. Using the formation of a dye barrier (seal) to assess plasmalemmal repair, we now report that B104 hippocampal cells with neurites transected nearer (<50 µm) to the soma seal at a lower frequency and slower rate compared to cells with neurites transected farther (>50 µm) from the soma. Analogs of cAMP, including protein kinase A (PKA)-specific and Epac-specific cAMP, each increase the frequency and rate of sealing and can even initiate sealing in the absence of Ca(2+) influx at both transection distances. Furthermore, Epac activates a cAMP-dependent, PKA-independent, pathway involved in plasmalemmal sealing. The frequency and rate of plasmalemmal sealing are decreased by a small molecule inhibitor of PKA targeted to its catalytic subunit (KT5720), a peptide inhibitor targeted to its regulatory subunits (PKI), an inhibitor of a novel PKC (an nPKCη pseudosubstrate fragment), and an antioxidant (melatonin). Given these and other data, we propose a model for redundant parallel pathways of Ca(2+)-dependent plasmalemmal sealing of injured neurons mediated in part by nPKCs, cytosolic oxidation, and cAMP activation of PKA and Epac. We also propose that the evolutionary origin of these pathways and substances was to repair plasmalemmal damage in eukaryotic cells. Greater understanding of vesicle interactions, proteins, and pathways involved in plasmalemmal sealing should suggest novel neuroprotective treatments for traumatic nerve injuries and neurodegenerative disorders.

Regulation of mammalian autophagy in physiology and pathophysiology.

(Macro)autophagy is a bulk degradation process that mediates the clearance of long-lived proteins and organelles. Autophagy is initiated by double-membraned structures, which engulf portions of cytoplasm. The resulting autophagosomes ultimately fuse with lysosomes, where their contents are degraded. Although the term autophagy was first used in 1963, the field has witnessed dramatic growth in the last 5 years, partly as a consequence of the discovery of key components of its cellular machinery. In this review we focus on mammalian autophagy, and we give an overview of the understanding of its machinery and the signaling cascades that regulate it. As recent studies have also shown that autophagy is critical in a range of normal human physiological processes, and defective autophagy is associated with diverse diseases, including neurodegeneration, lysosomal storage diseases, cancers, and Crohn's disease, we discuss the roles of autophagy in health and disease, while trying to critically evaluate if the coincidence between autophagy and these conditions is causal or an epiphenomenon. Finally, we consider the possibility of autophagy upregulation as a therapeutic approach for various conditions.