Novel insulin-like growth factor-binding protein proteases : detection and characterization

<p>Members of the insulin-like growth factor (IGF) system are known to have an impact on cell differentiation, proliferation and survival, and to be important regulators for nutrient metabolism. The IGF-binding proteins (IGFBP-1 to -6) control the availability of IGFs (IGF-I and IGF-II) to their receptors. In the circulation IGFs and IGFBP-3 or IGFBP-5 can bind an acid-labile subunit (ALS) to form a high molecular mass complex, which constitutes a circulating reservoir of IGFs. Post-translational modification and binding to cells and extracellular matrix may affect the IGF-dependent effects of IGFBPs. For example highly phosphorylated IGFBP-1, which is increased in catabolic conditions, has a high affinity for, and inhibits the actions of IGF-I. IGFBPs also have IGF-independent effects, through the interaction with cell surface structure or extracellular molecules. For example, the RGD sequence in the C-terminus of IGFBP-1 can interact with a cell surface á5â1 integrin to regulate cell migration and apoptosis. Proteolysis is another important posttranslational modification that affects IGFBP function at the target tissue.</p><p>The aims of this thesis are to characterise two novel IGFBP protease activities: (i) an IGFBP-3 protease activity detected in the thyroid of male mice and (ii) an IGFBP-1-specific protease activity discovered in human urine. In normal mice there is gender dimorphism in the circulating IGF system, with higher fasting IGF-I and IGFBP-3 ternary complex in male animals. IGFBP-3 protease activity was found in trunk blood from male mice and not females. This protease activity was due to tissue contamination likely to derive from thyroid. IGFBP-3 protease activity is present in the thyroid extracts from male mice. This protease has the characteristics of a calcium-activated serine protease and resulted in an 8-fold reduction in the IGF-I affinity of IGFBP-3.</p><p>We discovered a novel and specific IGFBP-1 protease activity that we purified from the urine of a patient with multiple myeloma and an inflammatory skin disorder. The proteolytic fraction contained azurocidin, which is a protease homologue hitherto considered inactive. The activity was abolished by an azurocidin monoclonal antibody. This protease efficiently cleaves both phosphorylated and nonphosphorylated IGFBP-1 at Ile130-Ser131 to generate fragments that, on BIAcore analysis, have higher association and dissociation rates than the intact IGFBP-1. In addition to having reduced affinity for IGFs, after cleavage of IGFBP-1. there was also a higher capacity for IGF-I binding, suggesting that both N- and C-terminal fragments may interact with ligand independently. Cleavage of IGFBP-1 decreased the inhibitory effect of IGF-II effect on MCF-7 breast cancer cell growth and on glucose uptake into cultures of human skeletal myotubes. Alone, proteolysed IGFBP-1. stimulated glucose uptake in muscle. No IGFBP-1. protease activity was detected in scrum from the patient; however there was evidence of immunoreactive fragments in the circulation.</p><p>In conclusion, novel IGFBP proteases may have important tissue-specific roles in determining the IGF-dependent and independent actions of IGFBPs.</p><h3>List of scientific papers</h3><p>I. Lewitt MS, Brismar K, Wang J, Wivall-Helleryd IL, Sindelar P, Gonzalez FJ, Bergman T, Bobek GA (2001). Responses of insulin-like growth factor (IGF)-I and IGF-binding proteins to nutritional status in peroxisome proliferator-activated receptor-alpha knockout mice. Growth Horm IGF Res. 11(5): 303-13. <br><a href="https://doi.org/10.1054/ghir.2001.0247">https://doi.org/10.1054/ghir.2001.0247</a><br><br> </p><p>II. Wang J, Grunler J, Lewitt MS (2004). Gender-specific pattern of insulin-like growth factor-binding protein-3 protease activity in mouse thyroid. Endocrinology. 145(3): 1137-43. <br><a href="https://doi.org/10.1210/en.2003-1378">https://doi.org/10.1210/en.2003-1378</a><br><br> </p><p>III. Wang J, Shafqat J, Hall K, Stahlberg M, Wivall-Helleryd IL, Bouzakri K, Zierath JR, Brismar K, Jornvall H, Lewitt MS. (2006). Specific cleavage of insulin-like growth factor-binding protein-1 by a novel protease activity. Cell Mol Life Sci. [Accepted] <br><a href="https://doi.org/10.1007/s00018-006-6248-7">https://doi.org/10.1007/s00018-006-6248-7</a><br><br> </p><p>IV. Wang J, Brandt K, Wivall-Helleryd IL, Horn HV, Hall, K, Lewitt MS (2006). Detection of IGFBP-1 fragments in clinical samples. [Manuscript]</p>