Structural caracterization of Stanniocalcin-1 by advanced structural proteomics

Abstract

The Stanniocalcin-1 protein (STC1) is an endocrine glycoprotein hormone of approximately 27 kDa and composed of 247 amino acids. Discovered firstly in glans of bone fishes and late identified in humans by differential expression of mRNA related to cellular immortalization, being also involved in many physiological, pathological and development process, including carcinogenesis, pregnancy, lactation, angiogenesis, organogenesis, cerebral ischemia, and others. Recently (2011) research in patients with Acute Lymphoblastic Leukemia (ALL) has shown large increase in the expression of the STC1 gene in leukemic cells, reinforcing its potential role in cancer. The amount of papers and information available in the literature with respect to STC1 is very limited. Despite this protein being associated to the appearance and evolution of ALL and already have important interaction partners identified, as SUMO1, acting as SUMO E3 ligase, the way how it happens requires deeper investigations with respect to a more detailed structural model and its involved sites. Both the analysis by SAXS and circular dichroism show that STC1 is a structured protein and, curiously, the sequence of amino acids associated does not present similarities with any other protein in databank and, therefore, the comparative strategy cannot be used. Thus, perhaps we could be facing a new protein folding which endorse the need for further structural studies. The comprehension of the structural bases that determine the specificity of certain ligand to a protein is one of the challenges of structural biology. The Protein Crystallography (PC) and Nuclear Magnetic Resonance (NMR) are methods well established to the study of tertiary structure of proteins and their interactions. These techniques allow to obtain structures of proteins and proteins complexes in atomic level and are reference techniques to structural determination of proteins. Despite the high structural detailing given by these techniques, both show a limited applicability to proteins in general due to some experimental restrictions, among which stands out the need of a large amount of sample (the order of several milligrams) with high purity grade level. In the case of NMR, there is also the need of the sample being stable in some buffer that not interferes in the analysis for a long range of time (days to weeks) in room temperature besides the fact that the current instruments restrict the maximum sample size approximately 50 kDa, what a lot of times represent the mass of a single component of a protein complex. The PC on the other hand requires the sample being in the monocrystal form. This limitation is still more restrictive when one intends to characterize protein complexes due to the greater difficult to obtain pure complexes in large quantities, to a large size of the system and the large difficulty in to obtain monocrystal of those species. In this sense, there is interest in the development and application of methods of structural determination of proteins with an integrative approach. The utilization of mass spectrometry to the characterization of proteins is extremely interesting once it combines the intrinsic advantages of the technique, as high sensibility, velocity, versatility, ease of operation and high reliability of the results. These features promote new developments in mass spectrometry for the analysis of tertiary and quaternary structure of proteins, in which exchange hydrogen/deuterium and the use of cross linking agents in combination to molecular modeling stand out as indispensable tools in the resolution of these problems. (AU)