Basic Science Tower, Stony Brook University, Stony Brook, NY 11794-8651 / 631-444-3219
Medical Scientist (M.D./Ph.D.) Training Program

Ian C. Brett
B.S. College of Mount St. Vincent, 1996
Ph.D. Biochemistry & Structurial Biology, 2012

4th Year Medical Student

Advisor: Steven O. Smith, Ph.D.

Department: Biochemistry & Cell Biology

Graduate Program: Biochemistry & Structural Biology

Title:
 The role of the transmembrane domain in the structure and function of the cytokine receptor family

Abstract:

         Activation of the erythropoietin receptor (EpoR) by the soluble cytokine erythropoietin (Epo) is essential for the differentiation of erythrocyte progenitors and their development into red blood cells. The single transmembrane (TM) helix of the EpoR mediates dimerization of the receptor in the inactive state and is responsible for coupling ligand binding to activation of an intracellular Janus kinase. Neither the structure of the inactive dimer nor the structural changes in the TM region that occur upon ligand binding are known. This work presents the solution NMR structures of peptides corresponding to the TM and juxtamembrane (JM) sequences that bridge the extracellular and intracellular domains. The N terminal end of the TM-JM peptides (LTASDLDPLILT) contains the transition point between the last b-strand of the extracellular D2 domain of the receptor and the TM a-helix. NMR measurements indicate that the TM helix extends to Leu226. The proline residue allows Asp224 to fold back and form side chain hydrogen bonds to the backbone NH of Leu226. Structural studies on the TM region of EpoR alone reveal intermolecular contacts between polar residues (Ser231, Ser238 and Thr242). At the intracellular TM-JM boundary, the defined a-helical secondary structure appears to break at the Arg250-Arg251 sequence. However, Leu253-Lys256 exhibit downfield carbonyl chemical shifts consistent with helical structure for the JM switch region. To stabilize the TM-JM peptides in an active conformation, two approaches were undertaken. The first approach was to substitute Leu223 with cysteine. The full length L223C mutant of EpoR was previously shown to have constitutive activity. The second approach was to characterize the complex between the TM-JM dimer and the TM domain of an EpoR-activating viral membrane protein, Gp55-P. In both cases, the largest chemical shift changes were at the intracellular TM-JM boundary, particularly His249. A mechanism of receptor activation that unites biophysical and biochemical data is presented.

Publications:
(pre-MSTP publications indicated with an *)

Itaya M, Brett IC, Smith SO. (2012). Synthesis, purification, and characterization of single helix membrane peptides and proteins for NMR spectroscopy. Methods Mol Biol. 831:333-57. PMID:22167682.

Staerk J, Defour JP, Pecquet C, Leroy E, Antoine-Poirel H, Brett I, Itaya M, Smith SO, Vainchenker W, Constantinescu SN. (2011). Orientation-specific signalling by thrombopoietin receptor dimers. EMBO J. 30(21):4398-413. doi: 10.1038/emboj.2011.315. PMID:21892137.

Johansson BE, Brett IC. (2009). Mouse models of influenza. Handbook on Immunosenescence: basic understanding and clinical applications. Fulop, T.; Franceschi, C.; Hirokawa, K.; Pawelec, G. (Eds.) Springer Science+Business Media BV.

Johansson BE, Brett IC. (2008). Recombinant influenza B virus HA and NA antigens administered in equivalent amounts are immunogenically equivalent and induce equivalent homotypic and broader heterovariant protection in mice than conventional and live influenza vaccines. Human Vaccine. 4(6):420-4.

Mohiyiddeen G, Brett I, Jude E. (2008). Infective endocarditis caused by taphylococcus aureus in a patient with atopic dermatitis: a case report. J Med Case Rep. 2:143. PMID:18454875.

Johansson BE, Brett IC. (2007). Changing perspective on immunization against influenza. Vaccine. 25(16):3062-5.

*Brett IC, Johansson BE. (2006). Variation of divalent cation requirements of influenza A virus N1 neuraminidases. J Biochem. 136:439-447.

*Johansson BE, Brett IC. (2006). An inactivated subvirion influenza A (H5N1) vaccine. NEJM. 354(25):2724-5. (Letter to the editor)

*Muddu BN, Umaar R, Kim WY, Zenios M, Brett I, Sharma Y. (2005). Whiplash injury of the shoulder: is it a distinct clinical entity? Acta Orthop Belg. 71(4):385-7. PMID:16184990.


*Brett IC, Johansson BE. (2005). Immunization against Influenza A virus: Comparison of conventional inactivated, live-attenuated, and recombinant baculovirus produced purified hemagglutinin and neuraminidase vaccines in a murine model system. Virology. 339(2):273-80.

*Noy A, Yahalom J, Zaretsky L, Brett I, Zelenetz AD. (2005). Gastric mucosa-associated lymphoid tissue detected by clonotypic polymerase chain reaction despite continuous pathologic remission induced by involved-field radiotherapy. J Clin Oncol. 23(16):3768-72.

*Kilbourne ED, Pokorny BA, Johansson B, Brett I, Milev Y, Matthews JT. (2004). Protection of mice with recombinant influenza virus neuraminidase. J Infect Dis. 189(3):459-61.

*Johansson, BE and Brett, IC. (2003). Variation in divalent cation requirements of influenza A virus N2 neuraminidases. J Biochem. 134(2):345-352.

*Brown ST, Brett I, Almenoff PL, Lesser M, Terrin M, Teirstein AS. (2003). Recovery of cell wall deficient organisms from blood does not distinguish between subjects with sarcoidosis and controls. Chest. 123(2):413-7.

*Kilbourne ED, Smith C, Brett I, Pokorny BA, Johansson B, Cox N. The total influenza vaccine failure of 1947 revisited: major intrasubtypic antigenic change can explain failure of vaccine in a post-World War II epidemic. Proc Natl Acad Sci U S A. 2002 Aug 6;99(16):10748-52. Epub 2002 Jul 22. Erratum in: Proc Natl Acad Sci U S A. 2003 Jan 21;100(2):764. PMID:12136133.

*Brett I, Werber J, Kilbourne ED. (2002). Rapid confirmation by RFLP of transfer to vaccine candidate reassortment viruses of the principal ‘high yield’ gene of influenza A viruses. J Virol Meth. 100:133-140.

*Kilbourne ED, Smith C, Brett I, Pokorny BA, Johansson B, Cox N. (2002). The total influenza vaccine failure of 1947 revisited: Major intrasubtypic antigenic change can explain failure of vaccine in a post-World War II epidemic. Proc Nat Acad Sci. 99(16):10748-10752.

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