Structural and functional studies of human protein stanniocalcina-1, a new microenvironmental marker of leukemia

Author(s)

Daniel Maragno Trindade

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2009-07-17

Examining board members

Jörg Kobarg; Ana Carolina de Mattos Zeri; Marcelo Damario Gomes; Vanessa Schein; Celso Eduardo Benedetti

• Advisor: Jörg Kobarg; José Andrés Yunes

Abstract

Staniocalcins (STCs) represent a small family of glycoprotein hormones found in all vertebrates composed by STC1 and STC2, which have been initially implicated in calcium homeostasis and also recently implicated in several other processes. Stanniocalcin-1 (STC1), the first member found, was originally discovered in bony fishes and later identified in humans were it seems to have a role, although still unclear, in carcinogenesis and angiogenesis. In humans STC1 can be found in two forms: a dimer or a group of higher molecular weigh variants collectively called bigSTC. Since both leukemic cells and solid tumors depend on vascularization, angiogenesis is a fundamental step in tumor-host interaction and essential to the cancer progression. Previous microarray analysis resulted in the identification of several activated genes in bone marrow (BM) endothelial cells (BMEC) modulated by presence of leukemic cells. Here we present STC1 as a BM microenvironment marker during acute lymphoblastic leukemia (ALL) by validating previous microarray data by quantitative RealTime-PCR. In view of the lack of functional and structural information on STC1 we performed (1) a yeast two hybrid screen in order to find some of the human STC1 interacting proteins and (2) a initial structural lowresolution characterization of this protein. We were able to construct a protein-protein interaction map, derived from the 22 interactants found in our yeast two-hybrid screen. The proteins found are located in several cellular compartments in which STC1 has already been shown to be present, and the new information might help to clarify how and which role it performs at these sites. As a means to provide structural information about STC1, we performed biochemical and structural analyses of recombinant STC1 6xHis tagged protein produced in insect cells by using the baculovirus system. Circular dichroism analysis confirmed the in silico predicted high alpha-helical content. By mass spectroscopy analysis we provided experimental data that confirmed the conserved disulfide pattern previously described for fish STC1. Finally Small Angle X-ray Scattering data demonstrated that STC1 adopts a dimeric, slightly elongated structure in solution, providing there by the first low resolution structural data of this family of proteins. Additionally, we could obtain enough protein of high purity to perform crystallization trials that resulted in crystals diffracting at good resolution. (AU)