Biochemistry and Pharmacology - Research Publications

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    Opposing Actions of Extracellular Signal-regulated Kinase (ERK) and Signal Transducer and Activator of Transcription 3 (STAT3) in Regulating Microtubule Stabilization during Cardiac Hypertrophy
    Ng, DCH ; Ng, IHW ; Yeap, YYC ; Badrian, B ; Tsoutsman, T ; McMullen, JR ; Semsarian, C ; Bogoyevitch, MA (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011-01-14)
    Excessive proliferation and stabilization of the microtubule (MT) array in cardiac myocytes can accompany pathological cardiac hypertrophy, but the molecular control of these changes remains poorly characterized. In this study, we examined MT stabilization in two independent murine models of heart failure and revealed increases in the levels of post-translationally modified stable MTs, which were closely associated with STAT3 activation. To explore the molecular signaling events contributing to control of the cardiac MT network, we stimulated cardiac myocytes with an α-adrenergic agonist phenylephrine (PE), and observed increased tubulin content without changes in detyrosinated (glu-tubulin) stable MTs. In contrast, the hypertrophic interleukin-6 (IL6) family cytokines increased both the glu-tubulin content and glu-MT density. When we examined a role for ERK in regulating cardiac MTs, we showed that the MEK/ERK-inhibitor U0126 increased glu-MT density in either control cardiac myocytes or following exposure to hypertrophic agents. Conversely, expression of an activated MEK1 mutant reduced glu-tubulin levels. Thus, ERK signaling antagonizes stabilization of the cardiac MT array. In contrast, inhibiting either JAK2 with AG490, or STAT3 signaling with Stattic or siRNA knockdown, blocked cytokine-stimulated increases in glu-MT density. Furthermore, the expression of a constitutively active STAT3 mutant triggered increased glu-MT density in the absence of hypertrophic stimulation. Thus, STAT3 activation contributes substantially to cytokine-stimulated glu-MT changes. Taken together, our results highlight the opposing actions of STAT3 and ERK pathways in the regulation of MT changes associated with cardiac myocyte hypertrophy.
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    The Minimal Active Structure of Human Relaxin-2
    Hossain, MA ; Rosengren, KJ ; Samuel, CS ; Shabanpoor, F ; Chan, LJ ; Bathgate, RAD ; Wade, JD (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2011-10-28)
    H2 relaxin is a peptide hormone associated with a number of therapeutically relevant physiological effects, including regulation of collagen metabolism and multiple vascular control pathways. It is currently in phase III clinical trials for the treatment of acute heart failure due to its ability to induce vasodilation and influence renal function. It comprises 53 amino acids and is characterized by two separate polypeptide chains (A-B) that are cross-linked by three disulfide bonds. This size and complex structure represents a considerable challenge for the chemical synthesis of H2 relaxin, a major limiting factor for the exploration of modifications and derivatizations of this peptide, to optimize effect and drug-like characteristics. To address this issue, we describe the solid phase peptide synthesis and structural and functional evaluation of 24 analogues of H2 relaxin with truncations at the termini of its peptide chains. We show that it is possible to significantly truncate both the N and C termini of the B-chain while still retaining potent biological activity. This suggests that these regions are not critical for interactions with the H2 relaxin receptor, RXFP1. In contrast, truncations do reduce the activity of H2 relaxin for the related receptor RXFP2 by improving RXFP1 selectivity. In addition to new mechanistic insights into the function of H2 relaxin, this study identifies a critical active core with 38 amino acids. This minimized core shows similar antifibrotic activity as native H2 relaxin when tested in human BJ3 cells and thus represents an attractive receptor-selective lead for the development of novel relaxin therapeutics.
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    c-Jun N-terminal Kinase Phosphorylation of Stathmin Confers Protection against Cellular Stress
    Ng, DCH ; Zhao, TT ; Yeap, YYC ; Ngoei, KR ; Bogoyevitch, MA (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010-09-10)
    The cell stress response encompasses the range of intracellular events required for adaptation to stimuli detrimental to cell survival. Although the c-Jun N-terminal kinase (JNK) is a stress-activated kinase that can promote either cell survival or death in response to detrimental stimuli, the JNK-regulated mechanisms involved in survival are not fully characterized. Here we show that in response to hyperosmotic stress, JNK phosphorylates a key cytoplasmic microtubule regulatory protein, stathmin (STMN), on conserved Ser-25 and Ser-38 residues. In in vitro biochemical studies, we identified STMN Ser-38 as the critical residue required for efficient phosphorylation by JNK and identified a novel kinase interaction domain in STMN required for recognition by JNK. We revealed that JNK was required for microtubule stabilization in response to hyperosmotic stress. Importantly, we also demonstrated a novel cytoprotective function for STMN, as the knockdown of STMN levels by siRNA was sufficient to augment viability in response to hyperosmotic stress. Our findings show that JNK targeting of STMN represents a novel stress-activated cytoprotective mechanism involving microtubule network changes.
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    Conservation of a Glycine-rich Region in the Prion Protein Is Required for Uptake of Prion Infectivity
    Harrison, CF ; Lawson, VA ; Coleman, BM ; Kim, Y-S ; Masters, CL ; Cappai, R ; Barnham, KJ ; Hill, AF (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010-06-25)
    Prion diseases are associated with the misfolding of the endogenously expressed prion protein (designated PrP(C)) into an abnormal isoform (PrP(Sc)) that has infectious properties. The hydrophobic domain of PrP(C) is highly conserved and contains a series of glycine residues that show perfect conservation among all species, strongly suggesting it has functional and evolutionary significance. These glycine residues appear to form repeats of the GXXXG protein-protein interaction motif (two glycines separated by any three residues); the retention of these residues is significant and presumably relates to the functionality of PrP(C). Mutagenesis studies demonstrate that minor alterations to this highly conserved region of PrP(C) drastically affect the ability of cells to uptake and replicate prion infection in both cell and animal bioassay. The localization and processing of mutant PrP(C) are not affected, although in vitro and in vivo studies demonstrate that this region is not essential for interaction with PrP(Sc), suggesting these residues provide conformational flexibility. These data suggest that this region of PrP(C) is critical in the misfolding process and could serve as a novel, species-independent target for prion disease therapeutics.
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    Anionic Phospholipid Interactions of the Prion Protein N Terminus Are Minimally Perturbing and Not Driven Solely by the Octapeptide Repeat Domain
    Boland, MP ; Hatty, CR ; Separovic, F ; Hill, AF ; Tew, DJ ; Barnham, KJ ; Haigh, CL ; James, M ; Masters, CL ; Collins, SJ (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2010-10-15)
    Although the N terminus of the prion protein (PrP(C)) has been shown to directly associate with lipid membranes, the precise determinants, biophysical basis, and functional implications of such binding, particularly in relation to endogenously occurring fragments, are unresolved. To better understand these issues, we studied a range of synthetic peptides: specifically those equating to the N1 (residues 23-110) and N2 (23-89) fragments derived from constitutive processing of PrP(C) and including those representing arbitrarily defined component domains of the N terminus of mouse prion protein. Utilizing more physiologically relevant large unilamellar vesicles, fluorescence studies at synaptosomal pH (7.4) showed absent binding of all peptides to lipids containing the zwitterionic headgroup phosphatidylcholine and mixtures containing the anionic headgroups phosphatidylglycerol or phosphatidylserine. At pH 5, typical of early endosomes, quartz crystal microbalance with dissipation showed the highest affinity binding occurred with N1 and N2, selective for anionic lipid species. Of particular note, the absence of binding by individual peptides representing component domains underscored the importance of the combination of the octapeptide repeat and the N-terminal polybasic regions for effective membrane interaction. In addition, using quartz crystal microbalance with dissipation and solid-state NMR, we characterized for the first time that both N1 and N2 deeply insert into the lipid bilayer with minimal disruption. Potential functional implications related to cellular stress responses are discussed.
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    A systems biology approach sheds new light on Escherichia coli acid resistance
    Stincone, A ; Daudi, N ; Rahman, AS ; Antczak, P ; Henderson, I ; Cole, J ; Johnson, MD ; Lund, P ; Falciani, F (OXFORD UNIV PRESS, 2011-09)
    In order to develop an infection, diarrhogenic Escherichia coli has to pass through the stomach, where the pH can be as low as 1. Mechanisms that enable E. coli to survive in low pH are thus potentially relevant for pathogenicity. Four acid response systems involved in reducing the concentration of intracellular protons have been identified so far. However, it is still unclear to what extent the regulation of other important cellular functions may be required for survival in acid conditions. Here, we have combined molecular and phenotypic analysis of wild-type and mutant strains with computational network inference to identify molecular pathways underlying E. coli response to mild and strong acid conditions. The interpretative model we have developed led to the hypothesis that a complex transcriptional programme, dependent on the two-component system regulator OmpR and involving a switch between aerobic and anaerobic metabolism, may be key for survival. Experimental validation has shown that the OmpR is responsible for controlling a sizeable component of the transcriptional programme to acid exposure. Moreover, we found that a ΔompR strain was unable to mount any transcriptional response to acid exposure and had one of the strongest acid sensitive phenotype observed.
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    Loss of the SPHF Homologue Slr1768 Leads to a Catastrophic Failure in the Maintenance of Thylakoid Membranes in Synechocystis sp PCC 6803
    Bryan, SJ ; Burroughs, NJ ; Evered, C ; Sacharz, J ; Nenninger, A ; Mullineaux, CW ; Spence, EM ; Stal, LJ (PUBLIC LIBRARY SCIENCE, 2011-05-23)
    BACKGROUND: In cyanobacteria the photosystems are localised to, and maintained in, specialist membranes called the thylakoids. The mechanism driving the biogenesis of the thylakoid membranes is still an open question, with only two potential biogenesis factors, Vipp1 and Alb3 currently identified. METHODOLOGY/PRINCIPAL FINDINGS: We generated a slr1768 knockout using the pGEM T-easy vector and REDIRECT. By comparing growth and pigment content (chlorophyll a fluoresence) of the Δslr1768 mutant with the wild-type, we found that Δslr1768 has a conditional phenotype; specifically under high light conditions (130 µmol m(-2) s(-1)) thylakoid biogenesis is disrupted leading to cell death on a scale of days. The thylakoids show considerable disruption, with loss of both structure and density, while chlorophyll a density decreases with the loss of thylakoids, although photosynthetic efficiency is unaffected. Under low light (30 µmol m(-2) s(-1)) the phenotype is significantly reduced, with a growth rate similar to the wild-type and only a low frequency of cells with evident thylakoid disruption. CONCLUSIONS/SIGNIFICANCE: This is the first example of a gene that affects the maintenance of the thylakoid membranes specifically under high light, and which displays a phenotype dependent on light intensity. Our results demonstrate that Slr1768 has a leading role in acclimatisation, linking light damage with maintenance of the thylakoids.
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    Application of autologous bone marrow derived mesenchymal stem cells to an ovine model of growth plate cartilage injury.
    McCarty, RC ; Xian, CJ ; Gronthos, S ; Zannettino, ACW ; Foster, BK (Bentham Science Publishers Ltd., 2010-06-23)
    Injury to growth plate cartilage in children can lead to bone bridge formation and result in bone growth deformities, a significant clinical problem currently lacking biological treatment. Mesenchymal stem/stromal cells (MSC) offer a promising therapeutic option for regeneration of damaged cartilage, due to their self renewing and multi-lineage differentiation attributes. Although some small animal model studies highlight the therapeutic potential of MSC for growth plate repair, translational research in large animal models, which more closely resemble the human condition, are lacking. Our laboratory has recently characterised MSCs derived from ovine bone marrow, and demonstrated these cells form cartilage-like tissue when transplanted within the gelatin sponge, Gelfoam, in vivo. In the current study, autologous bone marrow MSC were seeded into Gelfoam scaffold containing TGF-beta1, and transplanted into a surgically created defect of the proximal ovine tibial growth plate. Examination of implants at 5 week post-operatively revealed transplanted autologous MSC failed to form new cartilage structure at the defect site, but contributed to an increase in formation of a dense fibrous tissue. Importantly, the extent of osteogenesis was diminished, and bone bridge formation was not accelerated due to transplantation of MSCs or the gelatin scaffold. The current study represents the first work that has utilised this ovine large animal model to investigate whether autologous bone marrow derived MSC can be used to initiate regeneration at the injured growth plate.
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    Assembly of the Mitochondrial Protein Import Channel Role of Tom5 in Two-Stage Interaction of Tom40 with the SAM Complex
    Becker, T ; Guiard, B ; Thornton, N ; Zufall, N ; Stroud, DA ; Wiedemann, N ; Pfanner, N ; Newmeyer, D (AMER SOC CELL BIOLOGY, 2010-09-15)
    The preprotein translocase of the outer mitochondrial membrane (TOM) consists of a central β-barrel channel, Tom40, and six proteins with α-helical transmembrane segments. The precursor of Tom40 is imported from the cytosol by a pre-existing TOM complex and inserted into the outer membrane by the sorting and assembly machinery (SAM). Tom40 then assembles with α-helical Tom proteins to the mature TOM complex. The outer membrane protein Mim1 promotes membrane insertion of several α-helical Tom proteins but also affects the biogenesis of Tom40 by an unknown mechanism. We have identified a novel intermediate in the assembly pathway of Tom40, revealing a two-stage interaction of the precursor with the SAM complex. The second SAM stage represents assembly of Tom5 with the precursor of Tom40. Mim1-deficient mitochondria accumulate Tom40 at the first SAM stage like Tom5-deficient mitochondria. Tom5 promotes formation of the second SAM stage and thus suppresses the Tom40 assembly defect of mim1Δ mitochondria. We conclude that the assembly of newly imported Tom40 is directly initiated at the SAM complex by its association with Tom5. The involvement of Mim1 in Tom40 biogenesis can be largely attributed to its role in import of Tom5.
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    Sexual dimorphism in lung function responses to acute influenza A infection
    Larcombe, AN ; Foong, RE ; Bozanich, EM ; Berry, LJ ; Garratt, LW ; Gualano, RC ; Jones, JE ; Dousha, LF ; Zosky, GR ; Sly, PD (WILEY, 2011-09)
    BACKGROUND: Males are generally more susceptible to respiratory infections; however, there are few data on the physiological responses to such infections in males and females. OBJECTIVES: To determine whether sexual dimorphism exists in the physiological/inflammatory responses of weanling and adult BALB/c mice to influenza. METHODS: Weanling and adult mice of both sexes were inoculated with influenza A or appropriate control solution. Respiratory mechanics, responsiveness to methacholine (MCh), viral titre and bronchoalveolar lavage (BAL) cellular inflammation/cytokines were measured 4 (acute) and 21 (resolution) days post-inoculation. RESULTS: Acute infection impaired lung function and induced hyperresponsiveness and cellular inflammation in both sexes at both ages. Males and females responded differently with female mice developing greater abnormalities in tissue damping and elastance and greater MCh responsiveness at both ages. BAL inflammation, cytokines and lung viral titres were similar between the sexes. At resolution, all parameters had returned to baseline levels in adults and weanling males; however, female weanlings had persisting hyperresponsiveness. CONCLUSIONS: We identified significant differences in the physiological responses of male and female mice to infection with influenza A, which occurred in the absence of variation in viral titre and cellular inflammation.