BBA - General Subjects (v.1860, #2)

Preferential nitrite inhibition of the mitochondrial F1FO-ATPase activities when activated by Ca2 + in replacement of the natural cofactor Mg2 + by Salvatore Nesci; Vittoria Ventrella; Fabiana Trombetti; Maurizio Pirini; Alessandra Pagliarani (345-353).
The mitochondrial F1FO-ATP synthase has not only the known life function in building most cellular ATP, but also, as recently hinted, an amazing involvement in cell death. Accordingly, the two-faced enzyme complex, which catalyzes both ATP synthesis and ATP hydrolysis, has been involved in the mitochondrial permeability transition, the master player in apoptosis and necrosis. Nitrite, a cellular nitric oxide reservoir, has a recognized role in cardiovascular protection, through still unclear mechanisms.In swine heart mitochondria the effect of nitrite on the F1FO-ATPase activity activated by Ca2 +, henceforth defined as Ca-ATPase(s), or by the natural cofactor Mg2 +, was investigated by evaluating ATP hydrolysis under different assay conditions.Ca2 + is far less efficient than the natural cofactor Mg2+ in the ATPase activation. However, when activated by Ca2+ the ATPase activity is especially responsive to nitrite, which acts as uncompetitive inhibitor and up to 2 mM inhibits the Ca2+-activated-ATPase(s), probably by promoting dytirosine formation on the enzyme proteins, leaving the Mg-ATPase(s) unaffected. Most likely these ATPases refer to the same F1FO complex, even if coexistent ATPases may overlap.The preferential inhibition by nitrite of the Ca-ATPase(s), due to post-translational tyrosine modifications, may prevent the calcium-dependent functionality of the mitochondrial F1FO complex and related events.In mitochondria the preferential inhibition of the Ca-ATPase activity/ies by nitrite concentrations which do not affect the coexistent Mg-ATPase(s) may quench the negative events linked to the calcium-dependent functioning mode of the F1FO complex under pathological conditions.Display Omitted
Keywords: Mitochondria; F1FO-ATPase; Nitrite; Post-translational modifications; Calcium; Dityrosine;

Influence of a family 29 carbohydrate binding module on the activity of galactose oxidase from Fusarium graminearum by Filip Mollerup; Kirsti Parikka; Thu V. Vuong; Maija Tenkanen; Emma Master (354-362).
Galactose oxidase (GaO) selectively oxidizes the primary hydroxyl of galactose to a carbonyl, facilitating targeted chemical derivatization of galactose-containing polysaccharides, leading to renewable polymers with tailored physical and chemical properties. Here we investigate the impact of a family 29 glucomannan binding module on the activity and binding of GaO towards various polysaccharides. Specifically, CBM29-1-2 from Piromyces equi was separately linked to the N- and C-termini of GaO.Both GaO–CBM29 and CBM29–GaO were successfully expressed in Pichia pastoris, and demonstrated enhanced binding to galactomannan, galactoglucomannan and galactoxyloglucan. The position of the CBM29 fusion affected the enzyme function. Particularly, C-terminal fusion led to greatest increases in galactomannan binding and catalytic efficiency, where relative to wild-type GaO, k cat /K m values increased by 7.5 and 19.8 times on guar galactomannan and locust bean galactomannan, respectively. The fusion of CBM29 also induced oligomerization of GaO–CBM29.Similar to impacts of cellulose-binding modules associated with cellulolytic enzymes, increased substrate binding impeded the action of GaO fusions on more concentrated preparations of galactomannan, galactoglucomannan and galactoxyloglucan; this was especially true for GaO–CBM29. Given the N-terminal positioning of the native galactose-binding CBM32 in GaO, the varying impacts of N-terminal versus C-terminal fusion of CBM29-1-2 may reflect competing action of neighboring CBMs.This study thoroughly examines and discusses the effects of CBM fusion to non-lignocellulytic enzymes on soluble polysaccharides. Herein kinetics of GaO on galactose containing polysaccharides is presented for the first time.
Keywords: Galactose oxidase; Carbohydrate binding module; Galactomannan; Galactoglucomannan; Galactoxyloglucan; Fusion proteins;

Iron-associated biology of Trypanosoma brucei by Somsuvro Basu; Eva Horáková; Julius Lukeš (363-370).
Every eukaryote requires iron, which is also true for the parasitic protist Trypanosoma brucei, the causative agent of sleeping sickness in humans and nagana in cattle. T. brucei undergoes a complex life cycle during which its single mitochondrion is subject to major metabolic and morphological changes.This review covers what is known about processes associated with iron–sulfur clusters and heme metabolism in T. brucei. We discuss strategies by which iron and heme are acquired and utilized by this model parasite, emphasizing the differences between its two life cycle stages residing in the bloodstream of the mammalian host and gut of the insect vector. Finally, the role of iron in the host–parasite interactions is discussed along with their possible exploitation in fighting these deadly parasites.The processes associated with acquisition and utilization of iron, distinct in the two life stages of T. brucei, are fine tuned for the dramatically different host environment occupied by them. Although the composition and compartmentalization of the iron–sulfur cluster assembly seem to be conserved, some unique features of the iron acquisition strategies may be exploited for medical interventions against these parasites.As early-branching protists, trypanosomes and related flagellates are known to harbor an array of unique features, with the acquisition of iron being another peculiarity. Thanks to intense research within the last decade, understanding of iron–sulfur cluster assembly and iron metabolism in T. brucei is among the most advanced of all eukaryotes.
Keywords: Iron; Fe/S cluster; Heme; Trypanosoma; TAO;

C-termini are essential and distinct for nucleic acid binding of human NABP1 and NABP2 by Venkatasubramanian Vidhyasagar; Yujiong He; Manhong Guo; Hao Ding; Tanu Talwar; Vi Nguyen; Jessica Nwosu; George Katselis; Yuliang Wu (371-383).
Human Nucleic Acid Binding Protein 1 and 2 (hNABP1 and 2; also known as hSSB2 and 1, respectively) are two newly identified single-stranded (ss) DNA binding proteins (SSB). Both NABP1 and NABP2 have a conserved oligonucleotide/oligosaccharide-binding (OB)-fold domain and a divergent carboxy-terminal domain, the functional importance of which is unknown.Recombinant hNABP1/2 proteins were purified using affinity and size exclusion chromatography and their identities confirmed by mass spectrometry. Oligomerization state was checked by sucrose gradient centrifugation. Secondary structure was determined by circular dichroism spectroscopy. Nucleic acid binding ability was examined by EMSA and ITC.Both hNABP1 and hNABP2 exist as monomers in solution; however, hNABP2 exhibits anomalous behavior. CD spectroscopy revealed that the C-terminus of hNABP2 is highly disordered. Deletion of the C-terminal tail diminishes the DNA binding ability and protein stability of hNABP2. Although both hNABP1 and hNABP2 prefer to bind ssDNA than double-stranded (ds) DNA, hNABP1 has a higher affinity for ssDNA than hNABP2. Unlike hNABP2, hNABP1 protein binds and multimerizes on ssDNA with the C-terminal tail responsible for its multimerization. Both hNABP1 and hNABP2 are able to bind single-stranded RNA, with hNABP2 having a higher affinity than hNABP1.Biochemical evidence suggests that the C-terminal region of NABP1 and NABP2 is essential for their functionality and may lead to different roles in DNA and RNA metabolism.This is the first report demonstrating the regulation and functional properties of the C-terminal domain of hNABP1/2, which might be a general characteristic of OB-fold proteins.
Keywords: NABP1; NABP2; OB fold; DNA binding; RNA binding; C-terminus;

Raf kinase inhibitor protein mediated signaling inhibits invasion and metastasis of hepatocellular carcinoma by Xiaohui Wu; Yongjiang Yang; Zhuo Xu; Jiankun Li; Baoming Yang; Ningning Feng; Yueshan Zhang; Shunxiang Wang (384-391).
Hepatocellular carcinoma (HCC) is the most common type of liver cancer with high mortality and poor prognosis. Mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways have been implicated in promoting tumor cell proliferation and invasion of HCC cells.As a potential inhibitor of tumor metastasis, the role of Raf kinase inhibitor protein (RKIP) in HCC development and the functional relevance with MAPK and NF-κB signaling pathways were investigated. The levels of RKIP expression were examined in human HCC tissues and correlated with tumor stages and metastatic status. Function of RKIP in cellular proliferation, migration, invasion and apoptosis was investigated in HCC cell lines by either overexpressing or knocking down RKIP expression. Mouse xenograft model was established to assess the effect of RKIP expression on tumor growth.Our results demonstrated decreased RKIP expression in HCC tissues and a strong correlation with tumor grade and distant metastasis. Manipulation of RKIP expression in HCCLM3 and HepG2 cells indicated that RKIP functioned to inhibit HCC cell motility and invasiveness, and contributed to tumor growth inhibition in vivo. Mechanistic studies showed that the function of RKIP was mediated through MAPK and NF-κB signaling pathways. However, cell type-dependent RKIP regulation on these two pathways was also suggested, indicating the complex nature of signaling network.Our study provides a better understanding on the molecular mechanisms of HCC metastasis and sets the foundation for the development of targeted therapeutics for HCC.
Keywords: Hepatocellular carcinoma; RKIP; Signaling transduction; Invasion; Metastasis;

Uptake of Marasmius oreades agglutinin disrupts integrin-dependent cell adhesion by Samuel Juillot; Catherine Cott; Josef Madl; Julie Claudinon; Niels Sebastiaan Johannes van der Velden; Markus Künzler; Roland Thuenauer; Winfried Römer (392-401).
Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a lectin from the fairy ring mushroom with specificity for Galα1-3Gal containing carbohydrates. This lectin is composed of an N-terminal carbohydrate-binding domain and a C-terminal dimerization domain. The dimerization domain of MOA shows in addition calcium-dependent cysteine protease activity, similar to the calpain family.Cell detachment assay, cell viability assay, immunofluorescence, live cell imaging and Western blot using MDCKII cell line.In this study, we demonstrate in MDCKII cells that after internalization, MOA protease activity induces profound physiological cellular responses, like cytoskeleton rearrangement, cell detachment and cell death. These changes are preceded by a decrease in FAK phosphorylation and an internalization and degradation of β1-integrin, consistent with a disruption of integrin-dependent cell adhesion signaling. Once internalized, MOA accumulates in late endosomal compartments.Our results suggest a possible toxic mechanism of MOA, which consists of disturbing the cell adhesion and the cell viability.After being ingested by a predator, MOA might exert a protective role by diminishing host cell integrity.
Keywords: Lectin; Cysteine protease; Cell adhesion; Focal adhesion kinase; Integrin;

Oxytocin opposes effects of bacterial endotoxin on ER-stress signaling in Caco2BB gut cells by Benjamin Y. Klein; Hadassah Tamir; David L. Hirschberg; Robert J. Ludwig; Sara B. Glickstein; Michael M. Myers; Martha G. Welch (402-411).
The neuropeptide neuromodulator and hormone oxytocin (OT) activates signaling pathways involved in mRNA translation in response to endoplasmic reticulum stress and reduces inflammation associated with experimental colitis in rats. The anti-inflammatory effects of OT may serve a vital role in the development, survival and function of newborn-type enterocytes during microbial gut colonization, which coincides with the milk suckling period when OT receptor expression peaks in the gut. Furthermore, mice deficient in the OT receptor have abnormal gut structure and function, underscoring OT's developmental importance.We tested the effect of OT upon lipopolysaccharide (LPS)-induced markers of the inflammatory response in Caco2BB gut cells in vitro using automated immunocapillary electrophoresis.We demonstrate that OT suppresses NF-κB signaling and presumably inflammatory transcriptional programs, which are unleashed by LPS through the modulation of IκB. We show that OT counteracts LPS-elicited silencing of the unfolded protein response, a pathway limiting endoplasmic reticulum stress by suppressing protein translation. OT selectively activates dsRNA-activated kinase (PKR), X-box binding protein 1 (XBP1), immunoglobulin binding protein (BiP), A20 (TNFα-induced protein 3) and inositol requiring enzyme 1a (IRE1a). OT inactivates eukaryotic translation initiation factor 2a (eIF2a) without significant activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK).Mild, preemptive stimulation of endoplasmic reticulum stress sensors by OT may precondition newborn enterocytes to resist apoptosis associated with inflammation and may support their differentiation and development by modulating cellular metabolism.OT may protect enterocytes and other cell types, such as neurons, from stress-related complications during postnatal development.
Keywords: Enterocytes; Unfolded protein response; Inositol requiring enzyme 1a; X-box binding protein 1; Eukaryotic requiring enzyme 1a, A20;

Study of the phase transition in lysozyme crystals by Raman spectroscopy by Anna V. Frontzek (neé Svanidze); Laurent Paccou; Yannick Guinet; Alain Hédoux (412-423).
Recently, it has been revealed that tetragonal lysozyme crystals show a phase transition at 307 K upon heating. The underlying mechanisms of the phase transition are still not fully understood. Here we focus on the study of high-frequency vibrational modes arising from the protein and their temperature evolution in the vicinity of T ph as well as on the detailed study of crystalline water dynamics near T ph .Raman experiments have been performed at temperatures 295–323 K including T ph . The low-frequency modes and the modes of fingerprint region, CH- and OH-stretching regions have been analyzed.In spite of the absence of noticeable rearrangements in protein structure, the high-frequency vibrational modes of lysozyme located in the fingerprint region have been found to exhibit the features of critical dynamics near T ph . Pronounced changes in the dynamics of α-helixes and Tyr residues exposed on the protein surface point to the important role of H-bond rearrangements at the phase transition. Additionally the study of temperature evolution of OH-stretching modes has shown an increase in distortions of tertahedral H-bond network of crystalline water above T ph . These changes in water dynamics could play a crucial role in the mechanisms of the phase transition.The present results shed light on the mechanisms of the phase transition in lysozyme crystals.
Keywords: Phase transition; Tetragonal lysozyme crystal; Raman spectroscopy;

Imipramine administration induces changes in the phosphorylation of FAK and PYK2 and modulates signaling pathways related to their activity by Teresa Zalewska; Adam Bielawski; Luiza Stanaszek; Krzysztof Wieczerzak; Małgorzata Ziemka-Nałęcz; Irena Nalepa (424-433).
Antidepressants can modify neuronal functioning by affecting many levels of signal transduction pathways that are involved in neuroplasticity. We investigated whether the phosphorylation status of focal adhesion kinase (FAK/PTK2) and its homolog, PYK2/PTK2B, and their complex with the downstream effectors (Src kinase, p130Cas, and paxillin) are affected by administration of the antidepressant drug, imipramine. The treatment influence on the levels of ERK1/2 kinases and their phosphorylated forms (pERK1/2) or the Gαq, Gα11 and Gα12 proteins were also assessed.Rats were injected with imipramine (10 mg/kg, twice daily) for 21 days. The levels of proteins investigated in their prefrontal cortices were measured by Western blotting.Imipramine induced contrasting changes in the phosphorylation of FAK and PYK2 at Tyr397 and Tyr402, respectively. The decreased FAK phosphorylation and increased PYK2 phosphorylation were reflected by changes in the levels of their complex with Src and p130Cas, which was observed predominantly after chronic imipramine treatment. Similarly only chronic imipramine decreased the Gαq expression while Gα11 and Gα12 proteins were untouched. Acute and chronic treatment with imipramine elevated ERK1 and ERK2 total protein levels, whereas only the pERK1 was significantly affected by the drug.The enhanced activation of PYK2 observed here could function as compensation for FAK inhibition.These data demonstrate that treatment with imipramine, which is a routine in counteracting depressive disorders, enhances the phosphorylation of PYK2, a non-receptor kinase instrumental in promoting synaptic plasticity. This effect documents as yet not considered target in the mechanism of imipramine action.
Keywords: Antidepressant drug; Focal adhesion kinases; ERK1/2; G proteins; Rat prefrontal cortex;

Protein conformational perturbations in hereditary amyloidosis: Differential impact of single point mutations in ApoAI amyloidogenic variants by Rita Del Giudice; Angela Arciello; Francesco Itri; Antonello Merlino; Maria Monti; Martina Buonanno; Amanda Penco; Diana Canetti; Ganna Petruk; Simona Maria Monti; Annalisa Relini; Piero Pucci; Renata Piccoli; Daria Maria Monti (434-444).
Amyloidoses are devastating diseases characterized by accumulation of misfolded proteins which aggregate in fibrils. Specific gene mutations in Apolipoprotein A I (ApoAI) are associated with systemic amyloidoses. Little is known on the effect of mutations on ApoAI structure and amyloid properties. Here we performed a physico-chemical characterization of L75P- and L174S-amyloidogenic ApoAI (AApoAI) variants to shed light on the effects of two single point mutations on protein stability, proteolytic susceptibility and aggregation propensity. Both variants are destabilized in their N-terminal region and generate fibrils with different morphological features. L75P-AApoAI is significantly altered in its conformation and compactness, whereas a more flexible and pronounced aggregation-competent state is associated to L174S-AApoAI. These observations point out how single point mutations in ApoAI gene evocate differences in the physico-chemical and conformational behavior of the corresponding protein variants, with the common feature of diverting ApoAI from its natural role towards a pathogenic pathway.
Keywords: Apolipoprotein A I; Protein stability; Amyloidosis; Conformational diseases;

The structure of Resuscitation promoting factor B from M. tuberculosis reveals unexpected ubiquitin-like domains by Alessia Ruggiero; Flavia Squeglia; Maria Romano; Luigi Vitagliano; Alfonso De Simone; Rita Berisio (445-451).
RpfB is a key factor in resuscitation from dormancy of Mycobacterium tuberculosis. This protein is a cell-wall glycosidase, which cleaves cell-wall peptidoglycan. RpfB is structurally complex and is composed of three types of domains, including a catalytic, a G5 and three DUF348 domains. Structural information is currently limited to a portion of the protein including only the catalytic and G5 domains. To gain insights into the structure and function of all domains we have undertaken structural investigations on a large protein fragment containing all three types of domains that constitute RpfB (RpfB3D).The structural features of RpfB3D have been investigated combining x-ray crystallography and biophysical studies.The crystal structure of RpfB3D provides the first structural characterization of a DUF348 domain and revealed an unexpected structural relationship with ubiquitin. The crystal structure also provides specific structural features of these domains explaining their frequent association with G5 domains.Results provided novel insights into the mechanism of peptidoglycan degradation necessary to the resuscitation of M. tuberculosis. Features of the DUF348 domain add structural data to a large set of proteins embedding this domain. Based on its structural similarity to ubiquitin and frequent association to the G5 domain, we propose to name this domain as G5-linked-Ubiquitin-like domain, UBLG5.
Keywords: Cell wall; Crystal structure; Peptidoglycan; Tuberculosis;

Protective role against hydrogen peroxide and fibroblast stimulation via Ce-doped TiO2 nanostructured materials by Noel Gravina; Karim Maghni; Mélanie Welman; L'Hocine Yahia; Doris A. Mbeh; Paula V. Messina (452-464).
Cerium oxide (CeO2) and Ce-doped nanostructured materials (NMs) are being seen as innovative therapeutic tools due to their exceptional antioxidant effects; nevertheless their bio-applications are still in their infancy.TiO2, Ce–TiO2 and CeO2–TiO2 NMs were synthesized by a bottom-up microemulsion-mediated strategy and calcined during 7 h at 650 °C under air flux. The samples were compared to elucidate the physicochemical characteristics that determine cellular uptake, toxicity and the influence of redox balance between the Ce3 +/Ce4 + on the cytoprotective role against an exogenous ROS source: H2O2. Fibroblasts were selected as a cell model because of their participation in wound healing and fibrotic diseases.Ce–TiO2 NM obtained via sol–gel reaction chemistry of metallic organic precursors exerts a real cytoprotective effect against H2O2 over fibroblast proliferation, while CeO2 pre-formed nanoparticles incorporated to TiO2 crystalline matrix lead to a harmful CeO2–TiO2 material. TiO2 was processed by the same pathways as Ce–TiO2 and CeO2–TiO2 NM but did not elicit any adverse or protective influence compared to controls.It was found that the Ce atoms source and its concentration have a clear effect on material's physicochemical properties and its subsequent influence in the cellular response. It can induce a range of biological reactions that vary from cytotoxic to cytoprotective.Even though there are still some unresolved issues and challenges, the unique physical and chemical properties of Ce-based NMs are fascinating and versatile resources for different biomedical applications.Display Omitted
Keywords: Reactive oxygen species; Fibroblast; Nanoparticles; Ce-doped TiO2; Anatase; Nano-ceria;