R J Simpson

University of Melbourne, Melbourne, Victoria, Australia

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Publications (112)529.42 Total impact

  • Journal of Proteomics & Bioinformatics 07/2008; DOI:10.4172/jpb.s1000171
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    O. K. Bernhard, T. W. Barnes, R. J. Simpson
    Journal of Proteomics & Bioinformatics 07/2008; DOI:10.4172/jpb.s1000080
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    Journal of Proteomics & Bioinformatics 07/2008; DOI:10.4172/jpb.s1000090
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    Journal of Proteomics & Bioinformatics 07/2008; DOI:10.4172/jpb.s1000189
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    Journal of Proteomics & Bioinformatics 07/2008; DOI:10.4172/jpb.s1000143
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    Journal of Proteomics & Bioinformatics 07/2008; DOI:10.4172/jpb.s1000116
  • Molecular &amp Cellular Proteomics 01/2006; 5(10):S346-S346. · 7.25 Impact Factor
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    ABSTRACT: Phosphorylation is a key posttranslational modification for modulating biological interactions. Biosensor technology is ideally suited for examining in real time the role of phosphorylation on protein-protein interactions in signaling pathways. We have developed processes for on-chip phosphorylation of immobilized receptors on biosensor surfaces. These processes have been used to analyze E-cadherin/beta-catenin interactions. Phosphorylation of the intracellular domain (ICD) of E-cadherin modulates its affinity to beta-catenin and consequently the strength of cell-cell adhesion. We have phosphorylated immobilized E-cadherin ICD in situ using casein kinase 1 (CK1), casein kinase 2 (CK2), and src. On-chip phosphorylation of E-cadherin was confirmed using anti-phosphoserine and anti-phosphotyrosine antibodies. The binding of beta-catenin to E-cadherin was analyzed quantitatively. CK1 phosphorylation of E-cadherin increased the binding affinity to beta-catenin from approximately 230 to 4 nM. A similar increase in affinity, from 260 to 4 nM, was obtained with CK2 phosphorylation of E-cadherin. However, phosphorylation by src kinase decreased the affinity constant from approximately 260 nM to 4 microM. Interestingly, phosphorylation of E-cadherin by CK1 or CK2 prevented the inhibition of beta-catenin binding by src phosphorylation.
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    F Schütz, E A Kapp, R J Simpson, T P Speed
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    ABSTRACT: Improved search algorithms and scoring functions are required before the identification of peptide tandem MS data can be considered to be fully reliable and automatable. The development of models that can accurately predict product ion spectra from a peptide sequence would certainly help achieve this goal, but this firstly requires a better understanding of the process of fragmentation of peptides in the gas-phase. We summarize recent developments in this area and show that the prediction of product ion spectra is feasible and should improve the identification of peptide tandem MS data, especially for peptides that currently give low or insignificant scores with current search algorithms.
    Biochemical Society Transactions 01/2004; 31(Pt 6):1479-83. DOI:10.1042/BST0311479 · 3.24 Impact Factor
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    ABSTRACT: Dysregulated production of IL-6 and its receptor (IL-6R) are implicated in the pathogenesis of multiple myeloma, autoimmune diseases and prostate cancer. The IL-6R complex comprises two molecules each of IL-6, IL-6R, and the signaling molecule, gp130. Here, we report the x-ray structure (2.4 A) of the IL-6R ectodomains. The N-terminal strand of the Ig-like domain (D(1)) is disulfide-bonded to domain D(2), and domains D(2) and D(3), the cytokine-binding domain, are structurally similar to known cytokine-binding domains. The head-to-tail packing of two closely associated IL-6R molecules observed in the crystal may be representative of the configuration of the physiological dimer of IL-6R and provides new insight into the architecture of the IL-6R complex.
    Proceedings of the National Academy of Sciences 01/2003; 99(25):15959-64. DOI:10.1073/pnas.232432399 · 9.81 Impact Factor
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    ABSTRACT: A series of three aromatic to alanine mutants of recombinant murine interleukin-6 lacking the 22 N-terminal residues (DeltaN22mIL-6) were constructed to investigate the role of these residues in the structure and function of mIL-6. While Y78A and Y97A have activities similar to that of DeltaN22mIL-6, F173A lacks biological activity. F173A retains high levels of secondary structure, as determined by far-UV circular dichroism (CD), but has substantially reduced levels of tertiary structure, as determined by near-UV CD and (1)H NMR spectroscopy. F173A also binds the hydrophobic dye 1-anilino-8-naphthalenesulfonic acid (ANS) over a range of pH values and exhibits noncooperative equilibrium unfolding (as judged by the noncoincidence of monophasic unfolding transitions monitored by far-UV CD and lambda(max), with midpoints of unfolding at 2.6 +/- 0. 1 and 3.5 +/- 0.3 M urea, respectively, and the lack of an observable thermal unfolding transition). These are all properties of molten globule states, suggesting that the loss of activity of F173A results from the disruption of the fine structure of the protein, rather than from the loss of a side chain that is important for ligand-receptor interactions. Surprisingly, under some conditions, this loosened conformation is no more susceptible to proteolytic attack than the parent protein. By analogy with human IL-6, Phe173 in DeltaN22mIL-6 makes multiple interhelical interactions, the removal of which appear to be sufficient to induce a molten globule-like conformation.
    Biochemistry 03/2000; 39(8):1942-50. · 3.01 Impact Factor
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    ABSTRACT: Vascular endothelial growth factor-D (VEGF-D) binds and activates the endothelial cell tyrosine kinase receptors VEGF receptor-2 (VEGFR-2) and VEGF receptor-3 (VEGFR-3), is mitogenic for endothelial cells, and shares structural homology and receptor specificity with VEGF-C. The primary translation product of VEGF-D has long N- and C-terminal polypeptide extensions in addition to a central VEGF homology domain (VHD). The VHD of VEGF-D is sufficient to bind and activate VEGFR-2 and VEGFR-3. Here we report that VEGF-D is proteolytically processed to release the VHD. Studies in 293EBNA cells demonstrated that VEGF-D undergoes N- and C-terminal cleavage events to produce numerous secreted polypeptides including a fully processed form of M(r) approximately 21,000 consisting only of the VHD, which is predominantly a non-covalent dimer. Biosensor analysis demonstrated that the VHD has approximately 290- and approximately 40-fold greater affinity for VEGFR-2 and VEGFR-3, respectively, compared with unprocessed VEGF-D. In situ hybridization demonstrated that embryonic lung is a major site of expression of the VEGF-D gene. Processed forms of VEGF-D were detected in embryonic lung indicating that VEGF-D is proteolytically processed in vivo.
    Journal of Biological Chemistry 12/1999; 274(45):32127-36. DOI:10.1074/jbc.274.45.32127 · 4.57 Impact Factor
  • G.E. Reid, R.J. Simpson, R.A.J. O'Hair
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    ABSTRACT: An ion trap mass spectrometer equipped with electrospray ionization has been modified to study the structure of protonated polyglycyl peptides Gn (where n = 2–5 glycine residues) and their product ions formed by collision induced dissociation tandem mass spectrometry (CID MS/MS) via the novel application of gas phase ion–molecule hydrogen/deuterium (H/D) exchange reactions. In particular, the structures of the b2, b3, b4, and b5 ions formed via CID MS/MS from various protonated glycine oligomer precursors have been examined. The b2 ions, formed from the protonated G2 and G3 precursor ions, the b3 ion from the protonated G3 precursor, and the b4 ion from the protonated G5 ion all undergo CID and gas phase H/D exchange consistent with formation of protonated oxazolone structures previously proposed for bn-type ions. However, CID MS/MS, MS3, and H/D exchange of the putative b4 and b5 arising from the protonated G4 and G5 precursor ions, respectively, as well as experiments with various methylated derivatives of G4, suggest that the major portion of these ions are not bn ions, but are instead formed via backbone–backbone neighboring group participation reactions remote to the C-terminal amino acid. Efforts to elucidate the mechanisms behind this loss of H2O are described.
    International Journal of Mass Spectrometry 08/1999; 190(191). DOI:10.1016/S1387-3806(99)00023-8 · 2.23 Impact Factor
  • Proteome and Protein Analysis, First edited by RM Kamp, D Kyriakidis, T Choli-Papadopoulou, 05/1999: chapter Chapter 3: pages 29-52; Springer Verlag Berlin Heidelberg New York., ISBN: 3-540-65891-2
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    ABSTRACT: The suppressors of cytokine signaling (SOCS) family of proteins act as intracellular inhibitors of several cytokine signal transduction pathways. Their expression is induced by cytokine activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway and they act as a negative feedback loop by subsequently inhibiting the JAK/STAT pathway either by direct interaction with activated JAKs or with the receptors. These interactions are mediated at least in part by the SH2 domain of SOCS proteins but these proteins also contain a highly conserved C-terminal homology domain termed the SOCS box. Here we show that the SOCS box mediates interactions with elongins B and C, which in turn may couple SOCS proteins and their substrates to the proteasomal protein degradation pathway. Analogous to the family of F-box-containing proteins, it appears that the SOCS proteins may act as adaptor molecules that target activated cell signaling proteins to the protein degradation pathway.
    Proceedings of the National Academy of Sciences 04/1999; 96(5):2071-6. DOI:10.1073/pnas.96.5.2071 · 9.81 Impact Factor
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    ABSTRACT: The high affinity interleukin-6 (IL-6) receptor is a hexameric complex consisting of two molecules each of IL-6, IL-6 receptor (IL-6R), and the high affinity converter and signaling molecule, gp130. The extracellular "soluble" part of the IL-6R (sIL-6R) consists of three domains: an amino-terminal Ig-like domain and two fibronectin-type III (FN III) domains. The two FN III domains comprise the cytokine-binding domain defined by a set of 4 conserved cysteine residues and a WSXWS sequence motif. Here, we have determined the disulfide structure of the human sIL-6R by peptide mapping in the absence and presence of reducing agent. Mass spectrometric analysis of these peptides revealed four disulfide bonds and two free cysteines. The disulfides Cys102-Cys113 and Cys146-Cys157 are consistent with known cytokine-binding domain motifs, and Cys28-Cys77 with known Ig superfamily domains. An unusual cysteine connectivity between Cys6-Cys174, which links the Ig-like and NH2-terminal FN III domains causing them to fold back onto each other, has not previously been observed among cytokine receptors. The two free cysteines (Cys192 and Cys258) were detected as cysteinyl-cysteines, although a small proportion of Cys258 was reactive with the alkylating agent 4-vinylpyridine. Of the four potential N-glycosylation sites, carbohydrate moieties were identified on Asn36, Asn74, and Asn202, but not on Asn226.
    Journal of Biological Chemistry 04/1999; 274(11):7207-15. DOI:10.1074/jbc.274.11.7207 · 4.57 Impact Factor
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    ABSTRACT: Protozoan parasites of the genus Leishmania secrete a number of glycoproteins and mucin-like proteoglycans that appear to be important parasite virulence factors. We have previously proposed that the polypeptide backbones of these molecules are extensively modified with a complex array of phosphoglycan chains that are linked to Ser/Thr-rich domains via a common Manalpha1-PO4-Ser linkage (Ilg, T., Overath, P., Ferguson, M. A. J., Rutherford, T., Campbell, D. G., and McConville, M. J. (1994) J. Biol. Chem. 269, 24073-24081). In this study, we show that Leishmania mexicana promastigotes contain a peptide-specific mannose-1-phosphotransferase (pep-MPT) activity that adds Manalpha1-P to serine residues in a range of defined peptides. The presence and location of the Manalpha1-PO4-Ser linkage in these peptides were determined by electrospray ionization mass spectrometry and chemical and enzymatic treatments. The pep-MPT activity was solubilized in non-ionic detergents, was dependent on Mn2+, utilized GDP-Man as the mannose donor, and was expressed in all developmental stages of the parasite. The pep-MPT activity was maximal against peptides containing Ser/Thr-rich domains of the endogenous acceptors and, based on competition assays with oligosaccharide acceptors, was distinct from other leishmanial MPTs involved in the initiation and elongation of lipid-linked phosphoglycan chains. In subcellular fractionation experiments, pep-MPT was resolved from the endoplasmic reticulum marker BiP, but had an overlapping distribution with the cis-Golgi marker Rab1. Although Man-PO4 residues in the mature secreted glycoproteins are extensively modified with mannose oligosaccharides and phosphoglycan chains, similar modifications were not added to peptide-linked Man-PO4 residues in the in vitro assays. Similarly, Man-PO4 residues on endogenous polypeptide acceptors were also poorly extended, although the elongating enzymes were still active, suggesting that the pep-MPT activity and elongating enzymes may be present in separate subcellular compartments.
    Journal of Biological Chemistry 04/1999; 274(10):6678-88. DOI:10.1074/jbc.274.10.6678 · 4.57 Impact Factor
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    ABSTRACT: Lysosomes degrade a wide range of macromolecules to yield monomer products which are exported out of the lysosome by a series of transporters. In addition, lysosomes perform a range of other functions which are cell or tissue specific. In order to gain insight into the tissue specific role of lysosomes, carrier-ampholyte two-dimensional electrophoresis (2-DE) was used in combination with N-terminal sequencing to identify the major proteins present in both the membrane and luminal space of placental lysosomes. From the 45 N-terminal peptide sequences generated, 14 luminal and five membrane proteins were identified while three other sequences were novel. The sequenced proteins were a mixture of lysosomal and non-lysosomal proteins. The lysosomal proteins consisted of gamma-interferon-inducible protein (IP-30), Saposin D, cathepsins B and D, beta-hexosaminidase, palmitoyl protein thioesterase, alpha-glucosidase, and LAMP-1. The non-lysosomal proteins were serum albumin, serotransferrin, haemoglobin gamma G chain, alpha-1-antitrypsin, placental lactogen, endoplasmin, peptide binding protein 74, p60 lymphocyte protein, p450 side chain cleavage enzyme and placental alkaline phosphatase. The 2-DE maps obtained in this study are the first to identify the major proteins in both the lumen and membrane of placental lysosomes through sequence analysis, and thus provide the basis upon which to build a complete 2-DE database of the lysosome. Furthermore, the identities of the proteins sequenced from the placental lysosomes suggest a role for lysosomes in the transport of nutrients across the trophoblastic layer.
    Placenta 12/1998; 19(8):643-54. DOI:10.1016/S0143-4004(98)90026-1 · 3.29 Impact Factor
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    ABSTRACT: Mixed-lineage kinase 2 (MLK2) is a cytoplasmic protein kinase expressed at high levels in mammalian brain. The MLK2 structure is composed of a Src homology 3 (SH3) domain, two leucine zippers, a basic motif, a Cdc42/Rac interactive binding motif and a large C-terminal domain rich in proline, serine and threonine residues. To begin to define the role of MLK2 in mammalian brain, we used an MLK2-SH3 domain-glutathione S-transferase fusion protein (GST-MLK2-SH3) to isolate MLK2-binding proteins from rat brain extract. This analysis revealed that the major MLK2-SH3-domain-binding protein in rat brain is the GTPase dynamin. By using two different forms of the dynamin proline-rich domain as affinity ligands, the binding site for MLK2-SH3 was mapped to the C-terminal region of dynamin between residues 832 and 864. In GTPase assays, the addition of MLK2-SH3 stimulated the activity of purified dynamin I by 3-fold over the basal level, whereas the addition of a known dynamin activator, phosphatidylserine (PtdSer), stimulated a 6-fold increase. When MLK2-SH3 was added to the assay together with PtdSer, however, dynamin GTPase activity accelerated by more than 23-fold over basal level. An MLK2 mutant (MLK2-W59A-SH3), with alanine replacing a conserved tryptophan residue in the SH3 domain consensus motif, had no effect on dynamin activity, either alone or in the presence of PtdSer. In the same assay the SH3 domain from the regulatory subunit of phosphatidylinositol 3'-kinase stimulated a similar synergistic acceleration of dynamin GTPase activity in the presence of PtdSer. These results suggest that synergy between phospholipid and SH3 domain binding might be a general mechanism for the regulation of GTP hydrolysis by dynamin.
    Biochemical Journal 11/1998; 335 ( Pt 1)(1):119-24. · 4.78 Impact Factor
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    ABSTRACT: The transmembrane protein gp130 is a shared component of the receptor complexes for the interleukin-6 (IL-6)-type cytokines, which include IL-6, leukemia inhibitory factor (LIF) and oncostatin M (OSM). In addition to its role in the generation of high affinity receptors, gp130 is required for signal transduction by these cytokines. In the present study we have examined the role of the N-terminal located, extracellular immunoglobulin (Ig)-like module of gp130 in signal transduction by IL-6 and LIF. We have expressed wild-type human gp130 or three mutants in murine myeloid M1-UR21 cells that lack functional endogenous gp130 but express the IL-6 receptor (IL-6R) and the LIF receptor (LIFR). By measuring cellular responses, such as morphological changes upon differentiation, soft agar colony formation, and induction of tyrosine phosphorylation of the signal transducer and activator of transcription, STAT3, we show that signaling by IL-6, but not LIF, is significantly reduced by mutations in the Ig-like module of gp130. However, the binding of 125I-labeled IL-6 or LIF is not affected by these mutations. We also present evidence that the Ig-like module forms part of the epitope of an anti-gp130 monoclonal antibody that neutralizes the bioactivity of IL-6, but not of LIF or OSM. The data suggest that gp130-activation by IL-6 and LIF requires different regions of gp130, that the Ig-like module of gp130 may be required for IL-6-induced gp130 dimerization, and that the stoichiometry of the high affinity IL-6 receptor-complex differs from those of the receptor-complexes for LIF and OSM.
    Journal of Biological Chemistry 09/1998; 273(35):22701-7. DOI:10.1074/jbc.273.35.22701 · 4.57 Impact Factor

Publication Stats

6k Citations
529.42 Total Impact Points

Institutions

  • 2008
    • University of Melbourne
      • Department of Biochemistry and Molecular Biology
      Melbourne, Victoria, Australia
  • 1986–2008
    • Ludwig Institute for Cancer Research
      La Jolla, California, United States
  • 1985–2008
    • The Walter and Eliza Hall Institute of Medical Research
      • Division of Infection and Immunity
      Melbourne, Victoria, Australia
  • 1985–2000
    • Royal Melbourne Hospital
      Melbourne, Victoria, Australia
  • 1986–1997
    • Ludwig Institute for Cancer Research Australia
      Melbourne, Victoria, Australia
  • 1995
    • Peter MacCallum Cancer Centre
      Melbourne, Victoria, Australia
  • 1993
    • La Trobe University
      • Department of Biochemistry
      Melbourne, Victoria, Australia
    • Sir Charles Gairdner Hospital
      Perth City, Western Australia, Australia
  • 1992–1993
    • Heart Research Institute
      Newtown, New South Wales, Australia
    • The Princess Margaret Hospital
      Toronto, Ontario, Canada
  • 1991
    • University of Toronto
      • Hospital for Sick Children
      Toronto, Ontario, Canada
  • 1989
    • University of Western Australia
      Perth City, Western Australia, Australia
  • 1988
    • Ludwig von Mises Institute
      AUO, Alabama, United States
  • 1987
    • University of New South Wales
      Kensington, New South Wales, Australia
    • Catholic University of Louvain
      Walloon Region, Belgium