Intrinsically Disordered Proteins. Alex Cioffi June 22 nd 2013

Transcription

1 Intrinsically Disordered Proteins Alex Cioffi June 22 nd 2013

2 Implications in Human Disease Adenovirus Early Region 1A (E1A) and Cancer α-synuclein and Parkinson s Disease Amyloid β and Alzheimer s Disease Prion Protein and Creutzfeldt-Jakob Disease

3 General Characteristics Low Sequence Complexity Low content of bulky hydrophobic amino acids (Val, Leu, Ile, Met, Phe, Trp, Tyr) High proportion of polar and charged amino acids (Gln, Ser, Pro, Glu, Lys). small/uncharged/hydrophilic residues and Pro Hydrophobic residues acidic residues basic residues Romero, P. et al. Proteins 2001, 42, Vucetic, S., Brown, C.J., Dunker, A.K. & Obradovic, Z. Proteins 2003, 52, About 20-30% of eukaryotic proteins are mostly disordered. More than 50% of eukaryotic proteins have long regions of disorder. More than 70% of signaling proteins and oncoproteins have long disordered regions.

4 The Continuum of Protein Structure Dyson, J.H. & Wright, P.E. Nat. Rev. Mol. Cell Biol. 2005, 6,

5 The Energy Landscape Uversky, V.N., Oldfield, C.J. & Dunker, A.K. Annu. Rev. Biophys. 2008, 37,

6 Role in Transcription Domain Structure of Human CBP Transcription factor cyclic-ampresponse-element-binding protein (CREB). CREB-binding protein (CBP) and its paralogue p300 are transcriptional co-activators. Co-activators modify chromatin and transcription factors through acetyltransferase activity. Also function as scaffolds for recruitment and assembly of transcriptional machinery. More than 50% of human CBP is intrinsically disordered. = structured domains NBCD = unstructured nuclear-receptor coactivator-binding domain NRID = disordered nuclear-receptor-interaction domain Giordano, A. & Avantaggiati, M.L. J. Cell Phsiol. 1999, 181, Goodman, R.H. & Smolik, S. Genes Dev. 2000, 14, HAT = histone acetyltransferase domain KIX = KID binding domain PHD = plant homeodomain TAZ1/2 = transcriptional-adapter zinc-finger ZZ = zinc-binding domain

7 Role of TAZ1 in Hypoxic Response Hypoxia TAZ1-domain-HIF1α complex Hypoxiainducible factor-1α (HIF1α) Hypoxic Conditions Asn hydroxlayase FIH-HIF1α complex Arany, Z. et al. Proc. Natl. Acad. Sci. USA 1996, 93, Freedman, S.J. et al. Proc. Natl. Acad. Sci. USA 2002, 99,

8 Lill, N.L. et al. Nature 1997, 387, Gu, W., Shi, X.L., & Roeder, R.G. Nature 1997, 387, Ayed, A. et al. Nature Stuct. Biol. 2001, 8, Role of p53 in Cancer p53 the guardian of the genome p53 pathways CBP-bromodomain-p53 complex

9 Role of E1A/TAZ2 in cancer retinoblastoma protein (prb) adenovirus early region 1A (E1A) oncoprotein Ferreon, A.C.M., Ferreon, J.C., Wright, P.E. & Deniz, A.A. Nature 2013, 498,

10 Probing prb-e1a-taz2 complex E1A Constructs E1A-TAZ2-pRb Ternary Complex Alexa Fluor 488 Maleimide Dye Ferreon, A.C.M., Ferreon, J.C., Wright, P.E. & Deniz, A.A. Nature 2013, 498,

11 Single-Molecule Fluorescence Resonance Energy Transfer Ferreon, A.C.M., Moran, C.R., Gambin, Y. & Deniz, A.A. Nature 2013, 498,

12 Allosteric Interactions of prb-e1a- TAZ2 complex Ferreon, A.C.M., Ferreon, J.C., Wright, P.E. & Deniz, A.A. Nature 2013, 498,

13 Modulation of Allostery Ferreon, A.C.M., Ferreon, J.C., Wright, P.E. & Deniz, A.A. Nature 2013, 498,

14 Functional Complexity through Binding Promiscuity Ferreon, A.C.M., Ferreon, J.C., Wright, P.E. & Deniz, A.A. Nature 2013, 498,

15 THANKS!

16 Transcription Activation

17 Hypoxia Mechanism The alpha subunits of HIF are hydroxylated at conserved proline residues by HIF prolylhydroxylases, allowing their recognition and ubiquitination by the VHL E3 ubiquitin ligase, which labels them for rapid degradation by the proteasome. [11] This occurs only in normoxic conditions. In hypoxic conditions, HIF prolyl-hydroxylase is inhibited, since it utilizes oxygen as a cosubstrate. [12] Inhibition of electron transfer in the succinate dehydrogenase complex due to mutations in the SDHB or SDHD genes can cause a build-up of succinate that inhibits HIF prolyl-hydroxylase, stabilizing HIF-1α. This is termed pseudohypoxia. HIF-1, when stabilized by hypoxic conditions, upregulates several genes to promote survival in low-oxygen conditions. These include glycolysis enzymes, which allow ATP synthesis in an oxygen-independent manner, and vascular endothelial growth factor (VEGF), which promotes angiogenesis. HIF-1 acts by binding to HIF-responsive elements (HREs) in promoters that contain the sequence NCGTG. It has been shown that muscle A kinase anchoring protein (makap) organized E3 ubiquitin ligases, affecting stability and positioning of HIF-1 inside its action site in the nucleus. Depletion of makap or disruption of its targeting to the perinuclear (in cardiomyocytes) region altered the stability of HIF-1 and transcriptional activation of genes associated with hypoxia. Thus, "compartmentalization" of oxygen-sensitive signaling components may influence the hypoxic response. [13]