- Email: [email protected]
Growth factors as neuronal rescue agents
INSULIN-LIKE GROWTH FACTOR I (IGF-I), I G F - B I N D I N G PROTEINS AND IGF-RECEPTORS; PROGRESS REPORT OF A MOLECULAR STUDY OF INTERACTIONS AND STRUCTURES, 13. Nilsson, M. Jansson, M. Lake & J. K6rdel PHARMACIA AB, Biopharmaceuticals, 112 87 Stockholm, SWEDEN
GROWTH FACTORS AS NEURONAL RESCUE AGENTS P.D. Gluckrnan and C.E. Williams Research Centre for DevelopmentalMedicine and Biology, University of Auckland, Private Bag 92019, Auckland, New Zealand. Fax: 64-9-3737497
Insulin-like Growth Factor I (IGF-I) is a polypeptide growth factor which is thought to mediate most of the effects of Growth Hormone. IGF-I is produced mainly by the liver, and thereafter transported to the target tissues. IGF-I may also be produced locally and act through an autocrine and/or paracrine mechanism. The molecular endocrinological scheme of IGF-I-is rather complex, involving at least six different IGF-Binding Proteins (IGF-BP-1 through IGFBP-6) which act by carrying IGFI in circulation. These IGFBPs are known to modulate many of the properties of IGF-I, including half-life, activity, specificity as well as targeting. We are undertaking a structural biological, biochemical and biophysical approach to understand mechanisms and interactions which are involved in the molecular endocrinology of IGF-I. These studies include binding properties of mutant IGF-I molecules with single amino acid substitutions in amino acids Val 11, Asp12, Ash15 and Phe 16. Mutant proteins were produced in Escherichia coil and their respective binding properties t o IGFBP-1 and soluble recombinant IGF-I-receptor (IGF-IR), respectively, have been analyzed using BIAcore. The soluble IGF-I R molecule was expressed in mammalian cells. Further, disulfide exchange folding properties of IGF-I have been analyzed using an equilibrium glutathione buffer system showing that IGF-I is not able to form or maintain its native disulfides under in vivo conditions. Further, attempts to grow crystals of IGF-I in complex with IGFBP-1 and the IGF-I R, respectively, for x-ray diffraction studies have been initiated. Taken together, results of our biophysical studies are very useful to understand the molecular endocrinology of IGF-I. Such information can be useful in future design of more potent IGF-I analogous or antagonists.
It is now apparent that neuronal and glial death following asphyxial and other forms of injury occurs in two phases; a primary phase associated with the insult and a secondary phase occurring 12-72 hours later. This secondary loss is due to several mechanisms including apoptosis and macrophage activation. Therapies given after the injury which target these mechanisms and thus reduce the degree of delayed neuronal and glial cell death are termed neuronal rescue therapies. Using molecular biological approaches we have shown that specific growth factors are induced in areas of cell loss. In particular insulin-like growth factor 1 (IGF-1) and two of its specific binding proteins IGFBP-2 and IGFBP-3 are induced 3-5 days after injury whereas IGF-2 and IGFBP-5 are induced much later. The neurotrophins, NGFfl, BDNF and NT-3 are not induced in the areas of injury whereas the inhibitory cytokine transforming growth factor beta (TGF/3) is. IGF-1, but not IGF-2, when administered icy 2h after, but not before, injury in the rat and sheep markedly reduces neuronal loss in all regions, glial cell loss and improves behavioural outcome. Further studies show that the induction of IGFBPs in the region of injury is important to the action of IGF-1. IGF-1 acts both at the IGF type 1 receptor and by acting as a prohormone for the N terminal tripeptide glu-pro-gly which has independent neuroprotective mechanisms. TGF/~ also acts in vivo as a neuronal rescue agent perhaps by inhibiting macrophage activation. The degree of neuronal Protection achieved is marked compared to other stratagems and appears safe: the route of administration being the only limitation on early clinical introduction.
RECOMBINANT ANTIBODIES FOR THE STUDY OF NEUROTROPHINS AND NEUROPEPTIDES A. Cattaneo International School for Advanced Studies (SISSA), Biophysics Sector, Trieste (Italy)
PHARMACOLOGY OF G-PROTEIN-COUPLED : RECEPTORS IN ENGINEERED CELLS
M.J. Lohse, P. S6hlemann Institute of Pharmacology and Toxicology, University of Wfirzburg, 97078 Wfirzburg, Germany
We have developed a new e x p e r i m e n t a l strategy ( n e u r o a n t i b o d i e s ) , based on the local expression in the CNS of recombinant versions of monoclonal antibodies directed against target molecules of interest. By suitable engineering of the antibody genes the corresponding, antibody proteins can be targeted in a functional form to different s u b c e l l u l a r compartments, where they can interfere with the function of the corresponding antigen. Thus, according to the localization of the target antigen of 'interest, the ectopic expression of recombinant antibodies can be used for intracellular or intercellular immunization. The recombinant antibodies can be expressed in the CNS i) by t r a n s g e n i c mice technology, with the use of suitable promoters, or ii) by transplanting cells secreting the antibodies. Results will be presented on the application of these experimental strategies to the study of the Substance P neuropeptide and of the neurotrophic growth factor NGF. The results presented prospect the neuroantibody appproach as a complementary one to gene knock-out by homologous recombination.
m
Membrane-bound receptors are among the primary targets of drugs. Consequently, there is great interest in expression systems which would permit investigations of the signalling and ligand binding properties of such receptors. Today, molecular biology techniques allow the expression of a variety of human receptor genes in several expression systems, which, in addition to providing the quantities of receptor required for large scale screening, also obviate the problems of species differences, Using the human 132-adrenergic receptor as a prototype, we have examined and optimised various expression systems. These range from transient expression in eukaryotic cells such as HEK-293 and COS-7 ceils and stable expression in cell lines such as CHO to baculovirus-driven expression in insect cells, expression in E. coli, in halobacteria and in dictyostelium. In all cases, the receptors were found to retain their typical ligand binding properties. However, different systems appear to be particularly useful for specific questions: large-scale production is best achieved with the help of the baculovirus system, which allows expression of up to 50 pmol receptors per mg membrane protein. Purification of such membranes on sucrose gradients results in receptor densities of up to 250 pmol/mg protein. Because insect cell G proteins are also present in these membranes, they are not optimal for the study of receptor signalling. Such signalling can be studied in mammalian cell lines using their endogenous G proteins. As an alternative, the receptors can be expressed in the membranes of E .coli, which do not contain any G proteins, and then defined G proteins or their subunits can be added and coupling can be studied. Taken together, expression techniques for receptors allow a variety of studies with a specificity unattainable before.
17 m
GROWTH FACTORS AS NEURONAL RESCUE AGENTS P.D. Gluckrnan and C.E. Williams Research Centre for DevelopmentalMedicine and Biology, University of Auckland, Private Bag 92019, Auckland, New Zealand. Fax: 64-9-3737497
Insulin-like Growth Factor I (IGF-I) is a polypeptide growth factor which is thought to mediate most of the effects of Growth Hormone. IGF-I is produced mainly by the liver, and thereafter transported to the target tissues. IGF-I may also be produced locally and act through an autocrine and/or paracrine mechanism. The molecular endocrinological scheme of IGF-I-is rather complex, involving at least six different IGF-Binding Proteins (IGF-BP-1 through IGFBP-6) which act by carrying IGFI in circulation. These IGFBPs are known to modulate many of the properties of IGF-I, including half-life, activity, specificity as well as targeting. We are undertaking a structural biological, biochemical and biophysical approach to understand mechanisms and interactions which are involved in the molecular endocrinology of IGF-I. These studies include binding properties of mutant IGF-I molecules with single amino acid substitutions in amino acids Val 11, Asp12, Ash15 and Phe 16. Mutant proteins were produced in Escherichia coil and their respective binding properties t o IGFBP-1 and soluble recombinant IGF-I-receptor (IGF-IR), respectively, have been analyzed using BIAcore. The soluble IGF-I R molecule was expressed in mammalian cells. Further, disulfide exchange folding properties of IGF-I have been analyzed using an equilibrium glutathione buffer system showing that IGF-I is not able to form or maintain its native disulfides under in vivo conditions. Further, attempts to grow crystals of IGF-I in complex with IGFBP-1 and the IGF-I R, respectively, for x-ray diffraction studies have been initiated. Taken together, results of our biophysical studies are very useful to understand the molecular endocrinology of IGF-I. Such information can be useful in future design of more potent IGF-I analogous or antagonists.
It is now apparent that neuronal and glial death following asphyxial and other forms of injury occurs in two phases; a primary phase associated with the insult and a secondary phase occurring 12-72 hours later. This secondary loss is due to several mechanisms including apoptosis and macrophage activation. Therapies given after the injury which target these mechanisms and thus reduce the degree of delayed neuronal and glial cell death are termed neuronal rescue therapies. Using molecular biological approaches we have shown that specific growth factors are induced in areas of cell loss. In particular insulin-like growth factor 1 (IGF-1) and two of its specific binding proteins IGFBP-2 and IGFBP-3 are induced 3-5 days after injury whereas IGF-2 and IGFBP-5 are induced much later. The neurotrophins, NGFfl, BDNF and NT-3 are not induced in the areas of injury whereas the inhibitory cytokine transforming growth factor beta (TGF/3) is. IGF-1, but not IGF-2, when administered icy 2h after, but not before, injury in the rat and sheep markedly reduces neuronal loss in all regions, glial cell loss and improves behavioural outcome. Further studies show that the induction of IGFBPs in the region of injury is important to the action of IGF-1. IGF-1 acts both at the IGF type 1 receptor and by acting as a prohormone for the N terminal tripeptide glu-pro-gly which has independent neuroprotective mechanisms. TGF/~ also acts in vivo as a neuronal rescue agent perhaps by inhibiting macrophage activation. The degree of neuronal Protection achieved is marked compared to other stratagems and appears safe: the route of administration being the only limitation on early clinical introduction.
RECOMBINANT ANTIBODIES FOR THE STUDY OF NEUROTROPHINS AND NEUROPEPTIDES A. Cattaneo International School for Advanced Studies (SISSA), Biophysics Sector, Trieste (Italy)
PHARMACOLOGY OF G-PROTEIN-COUPLED : RECEPTORS IN ENGINEERED CELLS
M.J. Lohse, P. S6hlemann Institute of Pharmacology and Toxicology, University of Wfirzburg, 97078 Wfirzburg, Germany
We have developed a new e x p e r i m e n t a l strategy ( n e u r o a n t i b o d i e s ) , based on the local expression in the CNS of recombinant versions of monoclonal antibodies directed against target molecules of interest. By suitable engineering of the antibody genes the corresponding, antibody proteins can be targeted in a functional form to different s u b c e l l u l a r compartments, where they can interfere with the function of the corresponding antigen. Thus, according to the localization of the target antigen of 'interest, the ectopic expression of recombinant antibodies can be used for intracellular or intercellular immunization. The recombinant antibodies can be expressed in the CNS i) by t r a n s g e n i c mice technology, with the use of suitable promoters, or ii) by transplanting cells secreting the antibodies. Results will be presented on the application of these experimental strategies to the study of the Substance P neuropeptide and of the neurotrophic growth factor NGF. The results presented prospect the neuroantibody appproach as a complementary one to gene knock-out by homologous recombination.
m
Membrane-bound receptors are among the primary targets of drugs. Consequently, there is great interest in expression systems which would permit investigations of the signalling and ligand binding properties of such receptors. Today, molecular biology techniques allow the expression of a variety of human receptor genes in several expression systems, which, in addition to providing the quantities of receptor required for large scale screening, also obviate the problems of species differences, Using the human 132-adrenergic receptor as a prototype, we have examined and optimised various expression systems. These range from transient expression in eukaryotic cells such as HEK-293 and COS-7 ceils and stable expression in cell lines such as CHO to baculovirus-driven expression in insect cells, expression in E. coli, in halobacteria and in dictyostelium. In all cases, the receptors were found to retain their typical ligand binding properties. However, different systems appear to be particularly useful for specific questions: large-scale production is best achieved with the help of the baculovirus system, which allows expression of up to 50 pmol receptors per mg membrane protein. Purification of such membranes on sucrose gradients results in receptor densities of up to 250 pmol/mg protein. Because insect cell G proteins are also present in these membranes, they are not optimal for the study of receptor signalling. Such signalling can be studied in mammalian cell lines using their endogenous G proteins. As an alternative, the receptors can be expressed in the membranes of E .coli, which do not contain any G proteins, and then defined G proteins or their subunits can be added and coupling can be studied. Taken together, expression techniques for receptors allow a variety of studies with a specificity unattainable before.
17 m