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Study on the mutant IL7RA mechanisms of dimerization and signaling

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Livia Weijenborg Campos
Total Authors: 1
Document type: Doctoral Thesis
Press: Campinas, SP.
Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
Defense date:
Examining board members:
José Andrés Yunes; Martin Herman Bonamino; Kenneth John Gollob; Jörg Kobarg; Carlos Henrique Inacio Ramos
Advisor: José Andrés Yunes

IL-7 is an essential cytokine for the development, survival, and proliferation of normal preterm thymocytes in the thymus and mature T lymphocytes in the peripheral lymphoid organs. The IL-7 receptor alpha chain (IL7Ralpha) is a transmembrane protein and has no kinase activity on its own. Signal transduction by IL7Ralpha is dependent on the associated tyrosine kinase protein JAK1 (Janus kinase 1), anchored to the intracellular domain of the receptor. To perform its physiological function IL7Ralpha forms a heterodimer with the common gammac transmembrane receptor (gammac), which brings JAK3 anchored. Signaling occurs only in the presence of IL-7, when JAK1 and JAK3 mutually transphosphorylate, leading to STAT activation. However, the IL7R signaling mechanism, i.e., the alignment and movement of the IL-7Ra and gammac interaction chains is still largely unknown. Unlike the wild-type receptor, IL7Ralpha mutants associated with leukemia, described by our group and others, form homodimers and signal constitutively in the absence of IL-7, gammac or JAK3 or form heterodimers more sensitive to IL-7. These mutant versions of the IL-7 receptor may help to understand the structural and dynamic characteristics that govern wild type IL7R signaling. In addition, a better understanding of the functioning of the mutant IL7R may aid in the development of therapeutic strategies for patients with leukemia. In this work we investigate the dimerization and signaling mechanisms of IL7R mutants with insertion of cysteine as well as IL7R mutants with addition of positively charged residues. The results showed that simple insertion of a cysteine into the extracellular juxtamembrane region (EJM) is not sufficient for signaling. The cysteine insertion should be finely positioned so that the interactive alpha-helix chains are aligned correctly. Amino acids introduced together with cysteine, such as proline, play an important role in the functioning of the mutant receptor. Removal of proline leads to reduction of signaling, proliferation and homodimerization capacity. Another factor to be considered is transmembrane (TM) serine residues that can stabilize the mutant IL7R homodimers, although they are not determinant in the constitutive signaling of cysteine mutants. In addition to mutants with cysteine insertion, there are mutations in the EJM region that are also associated with leukemia, but have positively charged residue insertion (example IL7R-RRI). In this work, we verified that IL7R-RRI forms heterodimers with the gammac chain, conferring greater sensitivity to IL-7 compared to IL7R-WT, thus being dependent on IL-7, gammac and JAK3. Apparently the positively charged amino acids RR in the IL7R-RRI are complemented with the negative residue E261 in the gammac by the difference in charges. In conclusion, this work reveals that simple insertion of cysteine and formation of a homodimer is not sufficient for constitutive IL7Ralpha-IL7Ralphareceptor signaling. The functionality of the homodimer depends on which face of the interacting alpha-hélices that are positioned face to face. In addition, insertion of proline cysteine as well as a Serine-X-Serine-X-Serine domain has been found to favor the functionality of the homodimer. Finally, our cysteine-free mutant data revealed that the greater approximation of the extracellular juxtamembrane portions of IL7Ralpha and gammac increases the affinity for IL-7 (AU)

FAPESP's process: 12/07992-6 - Study of dimerization and signaling mechanisms of the mutant IL7Ra
Grantee:Lívia Weijenborg Campos
Support Opportunities: Scholarships in Brazil - Doctorate