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biopharmaceuticals biochemistry and biotecnology - Walsh G.

Walsh G. biopharmaceuticals biochemistry and biotecnology - John Wiley & Sons, 2003. - 572 p.
ISBN 0-470-84327-6
Download (direct link): biochemistryandbiotechnology2003.pdf
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Each b-subunit is composed of three regions; the extracellular domain, the transmembrane domain and a large cytoplasmic domain that displays tyrosine kinase activity. In the absence of insulin, tyrosine kinase activity is very weak. Proteolytic digestion of the a-subunit results in activation of this kinase activity. It is believed that the intact a-subunit exerts a negative influence on the endogenous kinase of the b-subunit and that binding of insulin, by causing a conformational shift in a-subunits, relieves this negative influence.
The cytoplasmic domain of the b-subunit displays three distinct sub-domains: (a) the ‘juxtamembrane domain’, implicated in recognition/binding of intracellular substrate molecules;
(b) the tyrosine kinase domain, which (upon receptor activation) displays tyrosine kinase activity; and (c) the C-terminal domain, whose exact function is less clear although site-directed mutagenesis studies implicate it promoting insulin’s mitogenic effects.
The molecular mechanisms central to insulin signal transduction are complex and have yet to be fully elucidated. However, considerable progress in this regard has been made over the last decade. Binding of insulin to its receptor promotes the autophosphorylation of three specific tyrosine residues in the tyrosine kinase domain (Figure 8.2(b)). This in turn promotes an alteration in the conformational state of the entire b-subunit, unmasking ATP (the phosphate donor) binding sites as well as substrate docking sites and activating its tyrosine kinase activity. Depending upon which specific intracellular substrates are then phosphorylated, at least two different signal transduction pathways are initiated (Figure 8.2(c)). Activation of the ‘MAP kinase’ pathway is ultimately responsible for triggering insulin’s mitogenic effects, whereas activation of the PI-3 kinase pathway apparently mediates the majority of insulin’s metabolic effects. Many of these effects, particularly the mitogenic effects, are promoted via transcriptional regulation of insulin-sensitive genes, of which there are probably in excess of 100 (Table 8.2).
Insulin production
Insulin preparations used initially were little more than crude pancreatic extracts. The therapeutic value of such products was marginal, as severe adverse reactions were commonplace (due to the presence of impurities). This was made worse by the frequency of injections required. The introduction of an acid-alcohol precipitation step yielded insulin preparations of moderate purity, thus partially overcoming the range and severity of side effects noted.
Although insulin was first crystallized in 1926, the factors promoting crystal growth were poorly understood and yielded inconsistent results. It was almost 10 years later when researchers discovered that the addition of zinc to a crude extract promoted reproducible crystallization (zinc addition yields a characteristic rhombohedral crystal, the basic crystal unit being the insulin hexamer, stabilized by the two zinc atoms).
308 BIOPHARMACEUTICALS
Figure 8.2. Structure of the insulin receptor (a). Binding of insulin promotes autophosphorylation of the b-subunits, where each b-subunit phosphorylates the other b-subunit. Phosphate groups are attached to three specific tyrosine residues (1158, 1162 and 1163), as indicated in (b). Activation of the b-subunit’s tyrosine kinase activity in turn results in the phosphorylation of various intracellular (protein) substrates which trigger the MAP kinase and/or the IP-3 kinase pathway, responsible for inducing insulin’s mitogenic and metabolic effects. The underlying molecular events occurring in these pathways are complex (see e.g. Combettes-Souverain and Issad, 1998)
HORMONES OF THERAPEUTIC INTEREST 309
Table 8.2. Selected genes whose rate of transcription is altered by binding of insulin to its receptor. In virtually all instances, the ultimate effect is to promote anabolic events characteristic of insulin action. Two-dimensional gel electrophoresis has also pinpointed dozens of proteins of unknown function whose cellular level is altered by insulin
Protein class Gene product Insulin effect (" or #
in transcription rate)
Integral membrane proteins Insulin receptor
Growth hormone receptor
Glucose transporters
Enzymes Fatty acid synthetase
Glutamine synthetase
Pyruvate kinase
Fructose 1,6 bis-phosphatase
Phosphoenolpyruvate carboxykinase
Glucokinase
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