Diverse Cellular Compartments Research Articles

Oxidative Stress in the Healthy and Wounded Hepatocyte: A Cellular Organelles Perspective.

Accurate control of the cell redox state is mandatory for maintaining the structural integrity and physiological functions. This control is achieved both by a fine-tuned balance between prooxidant and anti-oxidant molecules and by spatial and temporal confinement of the oxidative species. The diverse cellular compartments each, although structurally and functionally related, actively maintain their own redox balance, which is necessary to fulfill specialized tasks. Many fundamental cellular processes such as insulin signaling, cell proliferation and differentiation and cell migration and adhesion, rely on localized changes in the redox state of signal transducers, which is mainly mediated by hydrogen peroxide (H2O2). Therefore, oxidative stress can also occur long before direct structural damage to cellular components, by disruption of the redox circuits that regulate the cellular organelles homeostasis. The hepatocyte is a systemic hub integrating the whole body metabolic demand, iron homeostasis and detoxification processes, all of which are redox-regulated processes. Imbalance of the hepatocyte's organelles redox homeostasis underlies virtually any liver disease and is a field of intense research activity. This review recapitulates the evolving concept of oxidative stress in the diverse cellular compartments, highlighting the principle mechanisms of oxidative stress occurring in the healthy and wounded hepatocyte.

Editorial: Mechanisms of neuroinflammation and inflammatory neurodegeneration in acute brain injury.

The current research topic and eBook “Mechanisms of neuroinflammation and inflammatory neurodegeneration in acute brain injury” was initiated as a reaction to the rapidly expanding literature on inflammatory mechanisms in the pathophysiology of acute brain injuries. The scope of this compilation of reviews, opinion, and original research articles was to give a broad overview of the diverse cellular compartments and mechanisms involved in the inflammatory response to brain tissue injury. Although a specific aspect of the pathophysiology of acute brain injuries, the immune system interacts in highly complex as well as diverse mechanisms with the damaged brain. On one side acute brain lesions, such as brain ischemia, hemorrhage or traumatic injury, induce a local neuroinflammatory reaction, wherein microglial cells represent the local immune cells (Benakis et al., 2015; Lourbopoulos et al., 2015). This local inflammatory response has a major impact on outcome with differential effects during the phases of post-stroke lesion evolution and recovery (Shichita et al., 2014). Intriguingly, besides abundant evidence on post-stroke neuroinflammation immunological mechanisms similar mechanisms are also observed in traumatic brain injuries (Schwarzmaier and Plesnila, 2014), intracerebral hemorrhage (Mracsko and Veltkamp, 2014) and even ethanol-induced neurotoxicity (Alfonso-Loeches et al., 2014; Sokolowski et al., 2014) or direct application of exogenous pathogens (Gullo et al., 2014) with functional consequences for neuronal outcome. Moreover, Gauberti et al. (2014) present an overview on state-of-the-art molecular magnetic resonance imaging of neuroinflammatory markers. In recent years also the molecular pathways and effector molecules of inflammation-induced neurotoxicity after acute injuries have been investigated in great detail: Murray et al. (2015) describe in their review the prominent role of the pro-inflammatory cytokine IL-1, Orsini et al. (2014) give an overview on the complement system in neuroinflammation, while Albert-Weissenberger et al. (2014a) focus on the contribution of the kallikrein-kinin system in traumatic brain injury and Zhao et al. (2014) review the current knowledge on programed death-1/programed death ligand signaling. In addition to the activation of local inflammatory pathways in the injured brain, invasion of peripheral immune cells to the brain is a critical step in secondary neuroinflammation. Gelderblom et al. (2014) review the role of gdT cells as a pro-inflammatory invariant T cell subpopulation recruited to the injured brain. In contrast, Urra et al. (2014) discuss potential mechanisms of antigen-specific autoimmunity after acute brain injury. In addition, the original research article by Kim et al. (2014) underlines that the cellular immune response to ischemic brain injury might differ substantially between commonly used mouse strains. In addition to an overview and discussion of basic mechanisms and involved pathways in secondary neuroinflammation after acute brain injury, our research topic also contains several reviews and original articles on novel therapeutic approaches to modulate the immune response. Rissiek et al. (2014) introduce nanobodies as a novel tool for targeting neuroinflammation. Brunkhorst et al. (2014) provide an overview on the promising approach of blocking cellular neuroinflammation with Fingolimod. Bodhankar et al. (2014) review the current literature on targeting the PD-L1 and PD-L2 pathways. The original article by Mouihate (2014) reports a novel role for hormonal replacement therapy in neuroinflammation and the original article by Albert-Weissenberger et al. (2014b) the use of C1-inhibitors in a cortical cryolesion model. Dotson et al. (2014) have tested the use of recombinant TCR ligand with differential effects in young and old mice (see also commentary by Pennypacker, 2014). In summary this research topic gathered contributions from the leading laboratories working in the field of secondary neuroinflammation after brain injury with nearly 100 authors from 4 continents. We are confident that this compilation covers most established and emerging research questions in this specific research field and presents an up-to-date overview on inflammatory mechanisms and drug targets in acute brain injuries.

Sirtuins Function as the Modulators in Aging-related Diseases in Common or Respectively.

Sirtuins Function as the Modulators in Aging-related Diseases in Common or Respectively.

Analysis of signaling networks distributed over intracellular compartments based on protein-protein interactions.

BackgroundBiological processes are usually distributed over various intracellular compartments. Proteins from diverse cellular compartments are often involved in similar signaling networks. However, the difference in the reaction rates between similar proteins among different compartments is usually quite high. We suggest that the estimation of frequency of intracompartmental as well as intercompartmental protein-protein interactions is an appropriate approach to predict the efficiency of a pathway.ResultsUsing data from the databases STRING, ANDSystem, IntAct and UniProt, a PPI frequency matrix of intra/inter-compartmental interactions efficiencies was constructed. This matrix included 15 human-specific cellular compartments. An approach for estimating pathway efficiency using the matrix of intra/inter-compartmental PPI frequency, based on analysis of reactions efficiencies distribution was suggested. An investigation of KEGG pathway efficiencies was conducted using the developed method. The clusterization and the ranking of KEGG pathways based on their efficiency were performed. "Amino acid metabolism" and "Genetic information processing" revealed the highest efficiencies among other functional classes of KEGG pathways. "Nervous system" and "Signaling molecules interaction" contained the most inefficient pathways. Statistically significant differences were found between efficiencies of KEGG and randomly-generated pathways. Based on these observations, the validity of this approach was discussed.ConclusionThe estimation of efficiency of signaling networks is a complicated task because of the need for the data on the kinetic reactions. However, the proposed method does not require such data and can be used for preliminary analysis of different protein networks.

Is SIRT2 required for necroptosis?

Sirtuins can promote deacetylation of a wide range of substrates in diverse cellular compartments and regulate many cellular processes¹,². Recently Narayan et al., reported that SIRT2 was required for necroptosis based on their findings that SIRT2 inhibition, knock-down or knock-out prevented necroptosis. We sought to confirm and explore the role of SIRT2 in necroptosis and tested four different sources of the SIRT2 inhibitor AGK2, three independent siRNAs against SIRT2, and cells from two independently generated Sirt2−/− mouse strains, however we were unable to show that inhibiting or depleting SIRT2 protected cells from necroptosis. Furthermore, Sirt2−/− mice succumbed to TNF induced Systemic Inflammatory Response Syndrome (SIRS) more rapidly than wild type mice while Ripk3−/− mice were resistant. Our results therefore question the importance of SIRT2 in the necroptosis cell death pathway.

Specificity and Commonality of the Phosphoinositide-Binding Proteome Analyzed by Quantitative Mass Spectrometry

Phosphoinositides (PIPs) play key roles in signaling and disease. Using high-resolution quantitative mass spectrometry, we identified PIP-interacting proteins and profiled their binding specificities toward all seven PIP variants. This analysis revealed 405 PIP-binding proteins, which is greater than the total number of phospho- or ubiquitin-binding domains. Translocation and inhibitor assays of identified PIP-binding proteins confirmed that our methodology targets direct interactors. The PIP interactome encompasses proteins from diverse cellular compartments, prominently including the nucleus. Our data set revealed a consensus motif for PI(3,4,5)P3-interacting pleckstrin homology (PH) domains, which enabled in silico identification of phosphoinositide interactors. Members of the dedicator of cytokinesis family C exhibited specificity toward both PI(3,4,5)P3 and PI(4,5)P2. Structurally, this dual specificity is explained by a decreased number of positively charged residues in the L1 subdomain compared with DOCK1. The presented PIP-binding proteome and its specificity toward individual PIPs should be a valuable resource for the community.

The Kvβ2 subunit of voltage-gated potassium channels is interacting with ProSAP2/Shank3 in the PSD

The postsynaptic density is an electron dense meshwork composed of a variety of molecules facilitating neuronal signal transmission. ProSAP2/Shank3 represents a crucial player at postsynaptic sites, assembling large multimeric platforms and anchoring numerous other molecules, thereby linking the functional synapse with the cytoskeleton. ProSAP2/Shank3 is also implicated in the pathogenesis of numerous diseases, including autism spectrum disorders. KvBeta2 (Kvβ2) on the other hand serves as a regulatory subunit of voltage-gated potassium channels. Kvβ2 is located at various sites in the neuron including the axon (binding to Kv1.2), the dendrites (binding to Kv4.2) and the synapse. Binding of Kvβ2 to either Kv1.2 or Kv4 modulates not only the channel conformation but directs targeting of the channel protein complex to distinct loci within the cell. Thus an interaction between ProSAP2 and Kvβ2 could have important roles at diverse cellular compartments and moreover during maturation stages. We report here on the direct protein–protein interaction of the postsynaptic density anchoring molecule ProSAP2 and the potassium channel subunit Kvβ2, initially identified in a yeast-two-hybrid-screen. Furthermore, we characterize this interaction at synapses using primary hippocampal neurons in vitro.

Tumors as complex organs: are cancers manageable through the modification of their microenvironment?

Cancers behave as organoids, composed not only by genetically altered tumor cells, but also by a variety of non-tumoral cells, such as fibroblasts, macrophages and endothelial cells among others. Cellular interactions within this organoid dictate the rate of tumor progression, and therefore the accumulation of heterogeneous genotypes which characterize the tumor mass at the moment of diagnosis. Evidence for both homotypic and heterotypic cooperation between cells within the tumor microenvironment exist. We will show evidence for fluctuations in the expression of a specific tumor marker, namely galectin-3. Galectin-3 is an animal lectin that is found in diverse cellular compartments, depending on the cellular context and functional state of cells. Normal cells tend to accumulate galectin-3 in the nucleus. Upon malignant transformation, galectin-3 may concentrate in the cytoplasm and can be secreted to the extracellular matrix, where it acts as a matricellular protein, interfering with integrin function among other cell surface glycoproteins. We had shown a focal accumulation of galectin-3 in glioblastomas. Galectin-3 expression was associated to a very specific tumor microenvironment, the pseudopalisades- areas of viable tumor cells surrounding necrotic areas that are commonly found in advanced stages of glioblastomas. Galectin-3 expression in hypoxic and nutrient deprived cells is associated the activation of HIF-1a and it was inhibitable by NF-kB antagonists. In glioblastoma cells, galectin-3 expression was associated with its translocation to mitochondria, suggesting its involvement in mitochondrial homeostasis. Inhibition of galectin-3 expression led to increased cell death, indicating that galectin-3 acts as a prosurvival factor in very specific tumor microenvironments associated with glioblastoma progression. The protumoral role of galectin-3 was also observed in murine melanomas, engrafted in both wild type and galectin-3-deficient mice. Absence of galectin-3 from both tumoral and stromal compartments was associated with attenuated tumor growth, as compared to an experimental condition where both tumor and stromal cells expressed galectin-3. Intriguingly, de novo expression of galectin-3 was frequently observed in tumors derived from cells that did not express galectin-3 when these cells were engrafted in galectin-3 deficient mice. This observation prompted us to investigate whether galectin-3 expressing cells would favor tumor growth when admixed with galectin-3 negative cells. Indeed, when cells are admixed, tumors grew much more efficiently than those engraftments of either cell alone, indicating cooperation between galectin-3 expressing cells and galectin-3 negative cells. Further experiments indicated that galectin-3 expression is related with the induction of a proangiogenic tumor environment. Thus, targeting galectin-3 expression in selected tumors may prove to be an effective strategy to control tumor growth. Angiogenesis and vasculature function are critically altered in cancers, which are usually ill-perfused. Evidence for activation of both angiotensin and bradykinin-dependent pathways within the tumor microenvironment of melanomas will be discussed. Blockage of vasoactive peptide-dependent pathways may control tumor angiogenesis and therefore may control tumor growth. Indeed, epidemiological studies are now indicating that individuals receiving specific blockers of angiotensin receptors may be relatively protected from the development of tumors. In a clinical setting, however, antiangiogenic drugs would be preferably used in combination with chemotherapeutic drugs. Vasculature function, however is also critical for the proper delivery of chemotherapeutic drugs to tumors, specific therapeutic windows for the application of antiangiogenic drugs need to be identified. This notion will be further discussed. Altogether, the examples above call for a necessary understanding of tumor physiology, which seems to be under appreciated in the design of experimental/clinical trials.

Pathways linking Aβ and tau pathologies

Abeta (amyloid beta-peptide) and tau are the main proteins that misfold and accumulate in amyloid plaques and NFTs (neurofibrillary tangles) of Alzheimer's disease and other neurological disorders. Historically, because plaques and NFTs accumulate in diverse cellular compartments, i.e. mainly extracellularly for plaques and intracellularly for NFTs, it was long presumed that the constituent proteins formed these lesions via unrelated pathways. Animal and cell studies over the last decade, however, have provided convincing evidence to show that Abeta can facilitate the development of tau pathology by altering several cell-dependent and -independent mechanisms. In the present article, results are reviewed from several laboratories that show that modulating Abeta pathology can directly affect the development of tau pathology, which has significant implications for the treatment of Alzheimer's disease.

HIV reservoirs in vivo and new strategies for possible eradication of HIV from the reservoir sites.

Even though the treatment of human immunodeficiency virus (HIV)-infected individuals with highly active antiretroviral therapy (HAART) provides a complete control of plasma viremia to below detectable levels (<40 copies/mL plasma), there is an unequal distribution of all antiretroviral drugs across diverse cellular and anatomic compartments in vivo. The main consequence of this is the acquisition of resistance by HIV to all known classes of currently prescribed antiretroviral drugs and the establishment of HIV reservoirs in vivo. HIV has a distinct advantage of surviving in the host via both pre-and postintegration latency. The postintegration latency is caused by inert and metabolically inactive provirus, which cannot be accessed either by the immune system or the therapeutics. This integrated provirus provides HIV with a safe haven in the host where it is incessantly challenged by its immune selection pressure and also by HAART. Thus, the provirus is one of the strategies for viral concealment in the host and the provirus can be rekindled, through unknown stimuli, to create progeny for productive infection of the host. Thus, the reservoir establishment remains the biggest impediment to HIV eradication from the host. This review provides an overview of HIV reservoir sites and discusses both the virtues and problems associated with therapies/strategies targeting these reservoir sites in vivo.

Insight into substrate recognition and catalysis by the human neuraminidase 3 (NEU3) through molecular modeling and site-directed mutagenesis

The mammalian neuraminidase (NEU) enzymes are found in diverse cellular compartments. Members of the family, such as NEU2 and NEU1, are cytosolic or lysosomal, while NEU3 and NEU4 are membrane-associated. NEU enzymes that act on substrates in the plasma membrane could modulate cellular signaling, cell surface glycoforms and the composition of plasma membrane glycolipids. Therefore, their substrates and mechanism of action are of interest for discerning their physiological roles. We have studied the structure of the human NEU3 using molecular modeling to predict residues involved in the recognition and hydrolysis of glycolipid substrates. To test the model, we have used site-directed mutagenesis of the recombinant protein. Enzymatic studies of the relative activity of these mutants, as well as their pH profiles and inhibition by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, are reported. Using nuclear magnetic resonance spectroscopy, we confirmed that the enzyme is a retaining exo-sialidase, and we propose that the key catalytic residues of the enzyme consist of the general acid-base D50 and the nucleophilic Y370-E225 pair. Mutations of residues expected to interact directly with the sialic acid N5-acetyl (A160, M87, I105) and C7-C9 glycerol side-chain (E113, Y179, Y181) reduced enzymatic activity. We identified several active mutants of the enzyme which contain modifications at the periphery of the active site. Truncations at the N- or C-terminus of more than 10 residues abolished enzyme activity. We propose a catalytic mechanism consistent with the data and identify residues that contribute to glycolipid recognition.

Multiple Active Site Conformers in the Carbon Monoxide Complexes of Trematode Hemoglobins

Sequence alignment of hemoglobins of the trematodes Paramphistomum epiclitum and Gastrothylax crumenifer with myoglobin suggests the presence of an unusual active site structure in which two tyrosine residues occupy the E7 and B10 helical positions. In the crystal structure of P. epiclitum hemoglobin, such an E7-B10 tyrosine pair at the putative helical positions has been observed, although the E7 Tyr is displaced toward CD region of the polypeptide. Resonance Raman data on both P. epiclitum and G. crumenifer hemoglobins show that interactions of heme-bound ligands with neighboring amino acid residues are unusual. Multiple conformers in the CO complex, termed the C, O, and N conformers, are observed. The conformers are separated by a large difference (approximately 60 cm(-1)) in the frequencies of their Fe-CO stretching modes. In the C conformer the Fe-CO stretching frequency is very high, 539 and 535 cm(-1), for the P. epiclitum and G. crumenifer hemoglobins, respectively. The Fe-CO stretching of the N conformer appears at an unusually low frequency, 479 and 476 cm(-1), respectively, for the two globins. A population of an O conformer is seen in both hemoglobins, at 496 and 492 cm(-1), respectively. The C conformer is stabilized by a strong polar interaction of the CO with the distal B10 tyrosine residue. The O conformer is similar to the ones typically seen in mutant myoglobins in which there are no strong interactions between the CO and residues in the distal pocket. The N conformer possesses an unusual configuration in which a negatively charged group, assigned as the oxygen atom of the B10 Tyr side chain, interacts with the CO. In this conformer, the B10 Tyr assumes an alternative conformation consistent with one of the conformers seen the crystal structure. Implications of the multiple configurations on the ligand kinetics are discussed.

Scaffolding proteins organize multimolecular protein complexes for sensory signal transduction.

Scaffolding proteins composed of protein-protein interaction domains have emerged as organizers of multiprotein complexes in diverse cellular compartments, including neuronal synapses, cell-cell junctions of epithelial cells, and the stimulus perceiving structures of sensory neurons. This review focuses on the INAD-assembled signalling complex of Drosophila photoreceptors, which organizes key components of the phototransduction cascade into a multiprotein signal transduction unit. The structure, the physiological consequences, and the assembly and targeting of the members of the INAD signalling complex will be described. In addition, the existence of signalling complexes in vertebrate photoreceptors, olfactory neurons and mechanosensitive hair cells will be discussed.

Retargeting of the mitochondrial protein p32/gC1Qr to a cytoplasmic compartment and the cell surface.

p32/gC1qR is a small acidic protein that has been reported to have a broad range of distinct functions and to associate with a wide array of cellular, viral and bacterial proteins. It has been found in each of the main cellular compartments including mitochondria, nucleus and cytoplasm and is also thought to be located at the plasma membrane and secreted into the extracellular matrix. The true physiological role(s) of p32 remains controversial because it has been difficult to reconcile all of the findings on protein interactions and the seemingly disparate observations on compartmentalisation. However, it has been proposed that p32 is somehow involved in transport processes connecting diverse cellular compartments and the cell surface. Here we show that native p32 appears to be localised mainly in the mitochondria and is not detectable on the cell surface. However, addition of a short tag to the N-terminus of p32 appears to block its mitochondrial targeting, resulting in redirection into a cytoplasmic vesicular pattern, overlapping with the endoplasmic reticulum. The redirection of p32 results in an alteration in and co-localisation with ER markers including calreticulin, a lumenal ER chaperone. Furthermore, we show both by immunofluorescence and cross-linking studies that this also results in cell-surface expression of p32. These results indicate that, at least under certain circumstances, p32 can be retargeted and may help to provide an explanation for the diverse observations on its localization.

Protein kinases

Protein kinases catalyze the transfer of the γ-phosphate of a nucleoside triphosphate to form the phosphomonoesters of amino acid residues of a substrate polypeptide. Protein kinases are probably present in all eukaryotic cells, although most is known for the enzymes of vertebrates. In the cells of higher species, protein kinases are to be found in diverse cellular compartments. The isozymes of protein kinases can also exist and it is exemplified by the two well-characterized enzymes: cAMP-dependent protein kinase and phosphorylase kinase. Specificity for protein substrates is central to the function of protein kinases. The assessment of the selectivity of a protein kinase, however, must be made at both chemical and biological levels. Another aspect of protein kinase specificity is the phenomenon of autophosphorylation. Most protein kinases studied can catalyze the phosphorylation of one or more constituent subunits. Autophosphorylation is detected using purified enzymes at relatively high concentrations under conditions that are not physiological. In some cases, autophosphorylation can affect protein kinase activity.