Role of the hydrophobic and charged residues in the 218–226 region of apoA-I in the biogenesis of HDL

We investigated the significance of hydrophobic and charged residues 218–226 on the structure and functions of apoA-I and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of apoA-I[L218A/L219A/V221A/L222A] in apoA-I^−/− mice decreased plasma cholesterol and apoA-I levels to 15% of wild-type (WT) control mice and generated pre-β- and α4-HDL particles. In apoA-I^−/− × apoE^−/− mice, the same mutant formed few discoidal and pre-β-HDL particles that could not be converted to mature α-HDL particles by excess LCAT. Expression of the apoA-I[E223A/K226A] mutant in apoA-I^−/− mice caused lesser but discrete alterations in the HDL phenotype. The apoA-I[218–222] and apoA-I[E223A/K226A] mutants had 20% and normal capacity, respectively, to promote ABCA1-mediated cholesterol efflux. Both mutants had ∼65% of normal capacity to activate LCAT in vitro. Biophysical analyses suggested that both mutants affected in a distinct manner the structural integrity and plasticity of apoA-I that is necessary for normal functions. We conclude that the alteration of the hydrophobic 218–222 residues of apoA-I disrupts apoA-I/ABCA1 interactions and promotes the generation of defective pre-β particles that fail to mature into α-HDL subpopulations, thus resulting in low plasma apoA-I and HDL. Alterations of the charged 223, 226 residues caused milder but discrete changes in HDL phenotype.     

Loop C and the mechanism of acetylcholine receptor–channel gating

Agonist molecules at the two neuromuscular acetylcholine (ACh) receptor (AChR) transmitter-binding sites increase the probability of channel opening. In one hypothesis for AChR activation (“priming”), the capping of loop C at each binding site transfers energy independently to the distant gate over a discrete structural pathway. We used single-channel analyses to examine the experimental support for this proposal with regard to brief unliganded openings, the effects of loop-C modifications, the effects of mutations to residues either on or off the putative pathway, and state models for describing currents at low [ACh]. The results show that (a) diliganded and brief unliganded openings are generated by the same essential, global transition; (b) the radical manipulation of loop C does not prevent channel opening but impairs agonist binding; (c) both on- and off-pathway mutations alter gating by changing the relative stability of the open-channel conformation by local interactions rather than by perturbing a specific site–gate communication link; and (d) it is possible to estimate directly the rate constants for agonist dissociation from and association to both the low and high affinity forms of the AChR-binding site by using a cyclic kinetic model. We conclude that the mechanism of energy transfer between the binding sites and the gate remains an open question.     

Beyond the Binding Site: The Role of the β2 – β3 Loop and Extra-Domain Structures in PDZ Domains

A general paradigm to understand protein function is to look at properties of isolated well conserved domains, such as SH3 or PDZ domains. While common features of domain families are well understood, the role of subtle differences among members of these families is less clear. Here, molecular dynamics simulations indicate that the binding mechanism in PSD95-PDZ3 is critically regulated via interactions outside the canonical binding site, involving both the poorly conserved loop and an extra-domain helix. Using the CRIPT peptide as a prototypical ligand, our simulations suggest that a network of salt-bridges between the ligand and this loop is necessary for binding. These contacts interconvert between each other on a time scale of a few tens of nanoseconds, making them elusive to X-ray crystallography. The loop is stabilized by an extra-domain helix. The latter influences the global dynamics of the domain, considerably increasing binding affinity. We found that two key contacts between the helix and the domain, one involving the loop, provide an atomistic interpretation of the increased affinity. Our analysis indicates that both extra-domain segments and loosely conserved regions play critical roles in PDZ binding affinity and specificity.     

Does Actin Polymerization Status Modulate CaStorage in Human Neutrophils? Release and Coalescence of CaStores by Cytochalasins

The aim of this paper was to establish whether actin polymerization modulated cytosolic Ca²⁺storage in human neutrophils. Over the concentration ranges which inhibit actin polymerization, cytochalasins A, B, and D liberated Ca²⁺from membrane-bound stores within neutrophils. Two Ca²⁺storage sites were identified in neutrophils by the accumulation of the Ca²⁺binding probe, chlortetracycline: one at the center of the cell and the other at the cell periphery. Confocal imaging demonstrated that cytochalasins released Ca²⁺from the neutrophil periphery, but not from the central Ca²⁺store. Ca²⁺store release was coupled to Ca²⁺influx, suggesting that the peripheral site may be a physiological store containing a Ca²⁺influx factor. 3,3′-Dihexyloxacarbocyanine iodide staining organelles, which correlate with Ca²⁺release sites, coalesced in neutrophils after treatment with cytochalasins. We propose that peripheral Ca²⁺storage sites are restricted from coalescence by cortical polymerized actin and that Ca²⁺store coalescence and Ca²⁺release are coupled events.     

Role of gelatinases in pathological and physiological processes involving the dystrophin–glycoprotein complex

Dystrophin is a cytosolic protein belonging to a membrane-spanning glycoprotein complex, called dystrophin–glycoprotein complex (DGC) that is expressed in many tissues, especially in skeletal muscle and in the nervous system. The DGC connects the cytoskeleton to the extracellular matrix and, although none of the proteins of the DGC displays kinase or phosphatase activity, it is involved in many signal transduction pathways. Mutations in some components of the DGC are linked to many forms of inherited muscular dystrophies. In particular, a mutation in the dystrophin gene, leading to a complete loss of the protein, provokes one of the most prominent muscular dystrophies, the Duchenne muscular dystrophy, which affects 1 out of 3500 newborn males. What is observed in these circumstances, is a dramatic alteration of the expression levels of a multitude of metalloproteinases (MMPs), a family of extracellular Zn²⁺-dependent endopeptidases, in particular of MMP-2 and MMP-9, also called gelatinases. Indeed, the enzymatic activity of MMP-2 and MMP-9 on dystroglycan, an important member of the DGC, plays a significant role also in physiological processes taking place in the central and peripheral nervous system. This mini-review discusses the role of MMP-2 and MMP-9, in physiological as well as pathological processes involving members of the DGC.     

Role of the autotaxin–lysophosphatidate axis in cancer resistance to chemotherapy and radiotherapy

High expression of autotaxin in cancers is often associated with increased tumor progression, angiogenesis and metastasis. This is explained mainly since autotaxin produces the lipid growth factor, lysophosphatidate (LPA), which stimulates cell division, survival and migration. It has recently become evident that these signaling effects of LPA also produce resistance to chemotherapy and radiation-induced cell death. This results especially from the stimulation of LPA₂ receptors, which depletes the cell of Siva-1, a pro-apoptotic signaling protein and stimulates prosurvival kinase pathways through a mechanism mediated via TRIP-6. LPA signaling also increases the formation of sphingosine 1-phosphate, a pro-survival lipid. At the same time, LPA decreases the accumulation of ceramides, which are used in radiation therapy and by many chemotherapeutic agents to stimulate apoptosis. The signaling actions of extracellular LPA are terminated by its dephosphorylation by a family of lipid phosphate phosphatases (LPP) that act as ecto-enzymes. In addition, lipid phosphate phoshatase-1 attenuates signaling downstream of the activation of both LPA receptors and receptor tyrosine kinases. This makes many cancer cells hypersensitive to the action of various growth factors since they often express low LPP1/3 activity. Increasing our understanding of the complicated signaling pathways that are used by LPA to stimulate cell survival should identify new therapeutic targets that can be exploited to increase the efficacy of chemo- and radio-therapy. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Expanding horizons in iron chelation and the treatment of cancer: Role of iron in the regulation of ER stress and the epithelial–mesenchymal transition

Cancer is a major public health issue and, despite recent advances, effective clinical management remains elusive due to intra-tumoural heterogeneity and therapeutic resistance. Iron is a trace element integral to a multitude of metabolic processes, including DNA synthesis and energy transduction. Due to their generally heightened proliferative potential, cancer cells have a greater metabolic demand for iron than normal cells. As such, iron metabolism represents an important “Achilles' heel” for cancer that can be targeted by ligands that bind and sequester intracellular iron. Indeed, novel thiosemicarbazone chelators that act by a “double punch” mechanism to both bind intracellular iron and promote redox cycling reactions demonstrate marked potency and selectivity in vitro and in vivo against a range of tumours. The general mechanisms by which iron chelators selectively target tumour cells through the sequestration of intracellular iron fall into the following categories: (1) inhibition of cellular iron uptake/promotion of iron mobilisation; (2) inhibition of ribonucleotide reductase, the rate-limiting, iron-containing enzyme for DNA synthesis; (3) induction of cell cycle arrest; (4) promotion of localised and cytotoxic reactive oxygen species production by copper and iron complexes of thiosemicarbazones (e.g., Triapine^® and Dp44mT); and (5) induction of metastasis and tumour suppressors (e.g., NDRG1 and p53, respectively). Emerging evidence indicates that chelators can further undermine the cancer phenotype via inhibiting the epithelial–mesenchymal transition that is critical for metastasis and by modulating ER stress. This review explores the “expanding horizons” for iron chelators in selectively targeting cancer cells.     

A Single Mutation in Human Mitochondrial DNA Polymerase Pol γA Affects Both Polymerization and Proofreading Activities of Only the Holoenzyme

Common causes of human mitochondrial diseases are mutations affecting DNA polymerase (Pol) γ, the sole polymerase responsible for DNA synthesis in mitochondria. Although the polymerase and exonuclease active sites are located on the catalytic subunit Pol γA, in holoenzyme both activities are regulated by the accessory subunit Pol γB. Several patients with severe neurological and muscular disorders were reported to carry the Pol γA substitutions R232G or R232H, which lie outside of either active site. We report that Arg²³² substitutions have no effect on independent Pol γA activities but show major defects in the Pol γA-Pol γB holoenzyme, including decreased polymerase and increased exonuclease activities, the latter with decreased selectivity for mismatches. We show that Pol γB facilitates distinguishing mismatched from base-paired primer termini and that Pol γA Arg²³² is essential for mediating this regulatory function of the accessory subunit. This study provides a molecular basis for the disease symptoms exhibited by patients carrying those substitutions.     

Role of the conserved Ser–Tyr–Lys triad of the SDR family in sepiapterin reductase

Sepiapterin reductase (EC 1.1.1.153; SPR) is an enzyme involved in the biosynthesis of tetrahydrobiopterin; and SPR has been identified as a member of the NADP(H)-preferring short-chain dehydrogenase/reductase (SDR) family based on its catalytic properties for exogenous carbonyl compounds and molecular structure. To examine possible differences in the catalytic sites of SPR for exogenous carbonyl compounds and the native pteridine substrates, we investigated by site-directed mutagenesis the role of the highly conserved Ser–Tyr–Lys triad (Ser and YXXXK motif) in SPR, which was shown to be the catalytic site of SDR-family enzymes. From the analysis of catalytic constants for single- and double-point mutants against the triad, Ser and YXXXK motif, in the SPR molecule, participate in the reduction of the carbonyl group of both pteridine and exogenous carbonyl compounds. The Ser and the Tyr of the triad may co-act in proton transfer and stabilization for the carbonyl group of substrates, as was demonstrated for those in the SDR family. But either the Tyr or the Ser of SPR can function alone for proton transfer to a certain extent and show low activity for both substrates.     

Role of the tof gene in the production and perpetuation of the λdv plasmid

Molecular Genetics and Genomics - A series of λ derivatives carrying tof mutations were tested for their ability to give rise to plasmid λ dv. Phages carrying tof mutations that distorted...     

The Role of the Transport System in the Control of the Source–Sink Relations in Pinus sylvestris

Morphophysiological correlations were studied in medium-aged (20- to 60-year-old) Scots pine trees under the northern taiga conditions. Under various ecological conditions, pine trees developed a well-balanced structure, with close linear relationships between needle and root weight and their cross-section areas in all components of the continuous transport network (the coefficient of determination was between 0.88 and 0.999). When the annual cycle of soluble and insoluble carbohydrate contents was followed in various pine tissues, the total concentrations of soluble and insoluble carbohydrates were maintained at constant and tissue-specific levels, except in the growth period. The maximum level of carbohydrates was observed in all tissues at the beginning of rapid growth, and the minimum, at growth cessation. The qualitative composition and amount of carbohydrates matched the phenological phases of development and were not affected by the ecological growth conditions pertinent to the particular environment. The authors conclude that assimilate synthesis and partitioning are related to structural development, and the state of sink centers determines the attracting capacity, whereas the transport network, from roots to needles, and its conducting capacity are essential for the realization of systemic relationships and the control over growth and development in Pinus sylvestris L.     

Role of the ubiquitin–proteasome system on macrophages in the tumor microenvironment

Tumor-associated macrophages (TAMs) are key components of the tumor microenvironment, and their different polarization states play multiple roles in tumors by secreting cytokines, chemokines, and so on, which are closely related to tumor development. In addition, the enrichment of TAMs is often associated with poor prognosis of tumors. Thus, targeting TAMs is a potential tumor treatment strategy, in which therapeutic approaches such as reducing TAMs numbers, remodeling TAMs phenotypes, and altering their functions are being extensively investigated. Meanwhile, the ubiquitin–proteasome system (UPS), an important mechanism of protein hydrolysis in eukaryotic cells, participates in cellular processes by regulating the activity and stability of key proteins. Interestingly, UPS plays a dual role in the process of tumor development, and its role in TAMs deserve to be investigated in depth. This review builds on this foundation to further explore the multiple roles of UPS on TAMs and identifies a promising approach to treat tumors by targeting TAMs with UPS.     

Bistratene A Causes Phosphorylation of Talin and Redistribution of Actin Microfilaments in Fibroblasts: Possible Role for PKC-δ

Bistratene A is a marine toxin which induces phosphorylation of cellular proteins. Our current evidence indicates that this occurs through activation of protein kinase C-δ. In fibroblasts bistratene A causes rounding up of the cells and a rapid disappearance of vinculin staining and actin stress fibers as detected by fluorescence immunohistochemistry. Phosphorylation of the focal adhesion protein, talin, is increased after bistratene A treatment and this is inhibited by calphostin C, a specific inhibitor of PKC. No changes in the phosphorylation status of vinculin, tubulin, or vimentin were observed in the presence of the toxin. Treatment with bistratene A caused a redistribution of PKC-δ from cytosolic and membrane compartments to the nuclear fraction. There was no effect on the subcellular distribution of any other PKC isoform. These results demonstrate that phosphorylation of talin is implicated in the disruption of actin microfilaments in fibroblasts by bistratene A and that this is most likely mediated by PKC-δ.     

Role of the autotaxin–lysophosphatidate axis in the development of resistance to cancer therapy

Autotaxin (ATX) is a secreted enzyme that hydrolyzes lysophosphatidylcholine to produce lysophosphatidate (LPA), which signals through six G-protein coupled receptors (GPCRs). Signaling through LPA is terminated by its degradation by a family of three lipid phosphate phosphatases (LPPs). LPP1 also attenuates signaling downstream of the activation of LPA receptors and some other GPCRs. The ATX-LPA axis mediates a plethora of activities such as cell proliferation, survival, migration, angiogenesis and inflammation, which perform an important role in facilitating wound healing. This wound healing response is hijacked by cancers where there is decreased expression of LPP1 and LPP3 and increased expression of ATX. This maladaptive regulation of LPA signaling also causes chronic inflammation, which has been recognized as one of the hallmarks in cancer. The increased LPA signaling promotes cell survival and migration and attenuates apoptosis, which stimulates tumor growth and metastasis. The wound healing functions of increased LPA signaling also protect cancer cells from effects of chemotherapy and radiotherapy. In this review, we will summarize knowledge of the ATX-LPA axis and its role in the development of resistance to chemotherapy and radiotherapy. We will also offer insights for developing strategies of targeting ATX-LPA axis as a novel part of cancer treatment. This article is part of a Special Issue entitled Lysophospholipids and their receptors: New data and new insights into their function edited by Susan Smyth, Viswanathan Natarajan and Colleen McMullen.     

Role of the pentose phosphate pathway and the Entner–Doudoroff pathway in glucose metabolism of Gluconobacter oxydans 621H

Glucose catabolism by the obligatory aerobic acetic acid bacterium Gluconobacter oxydans 621H proceeds in two phases comprising rapid periplasmic oxidation of glucose to gluconate (phase I) and oxidation of gluconate to 2-ketogluconate or 5-ketogluconate (phase II). Only a small amount of glucose and part of the gluconate is taken up into the cells. To determine the roles of the pentose phosphate pathway (PPP) and the Entner–Doudoroff pathway (EDP) for intracellular glucose and gluconate catabolism, mutants defective in either the PPP (Δgnd, Δgnd zwf*) or the EDP (Δedd–eda) were characterized under defined conditions of pH 6 and 15 % dissolved oxygen. In the presence of yeast extract, neither of the two pathways was essential for growth with glucose. However, the PPP mutants showed a reduced growth rate in phase I and completely lacked growth in phase II. In contrast, the EDP mutant showed the same growth behavior as the reference strain. These results demonstrate that the PPP is of major importance for cytoplasmic glucose and gluconate catabolism, whereas the EDP is dispensable. Reasons for this difference are discussed.     

Role of Müller glia in neuroprotection and regeneration in the retina

Glial cells are thought to protect neurons from various neurological insults. When there is injury to retina, Müller cells, which are the predominant glial element in the retina, undergo significant morphological, cellular and molecular changes. Some of these changes reflect Müller cell involvement in protecting the retina from further damage. Müller cells express growth factors, neurotransmitter transporters and antioxidant agents that could have an important role in preventing excitotoxic damage to retinal neurons. Moreover, Müller cells contact to endothelial cells to facilitate the neovascularization process during hypoxic conditions. Finally, recent studies have pointed to a role of Müller cells in retina regeneration after damage, dedifferentiating to progenitor cells and then giving rise to different neuronal cell types. In this article we will review the role of Müller glia in neuroprotection and regeneration after damage in the retina.