1. 1. Posttranslational modification in cell adhesion and migration with emphasis on acetylation Dipl.-Ing. Birgit Kastberger Department of Cellular Physiology and Metabolism, University Medical Center, University of Geneva Oral exam: November 7, 2014
2. 2. Cell Extracellular matrix (ECM) Integrins are involved in cell adhesion and migration provide a link from the ECM to the actin cytoskeleton a b1A Harvard University, BioVisions-The Inner Life Of A Cell, 2011 Integrins Actin
3. 3. adaptor and b1A integrin PTMs are involved in cell adhesion & migration Filopodial contacts first contact with ECM spreading Lamellipodia adhesion, maturation, signalling Focal adhesions ECM remodelling Fibrillar adhesions W. Hu et al, Plos One, 2014, Geiger, et al., Nature Reviews Molecular Cell Biology, 2001 Fibrillar adhesions: phospho-Tyrosine, Tensin Tensin Focal adhesions: phospho-Tyrosine pTyr integrins weblike/remodeling actin adaptor proteins bundled/contractile actin stress fibres
4. 4. b1A integrin tail is phosphorylated and acetylated Extracellular domain: 21-728 aa Transmembrane domain: 729-751 aa Cytoplasmic tail: 752-798 aa 1A LLMIIHDRREFAKFEKEKMNAKWDTGENPIYKSAVTTVVNPKYEGK What role do PTMs play? PP P PPP Ac
5. 5. Over 200 distinct covalent PTMs diversify the proteome KL Vermillion et al, JCB, 2014; Prabakaran et al, Wiley P., 2012, Thermo Scientific: post-translational-modification Most frequent PTMs experimentally observed Khoury et al., Sci. Rep. 1, 2014 (2011) P h o s p h o ry la tio n A c e ty la tio n M e th y la tio n O th e rs P P P P Ac Ac Me AcAc
6. 6. 3 examples of more widely studied reversible PTMs : phosphorylation t1/2=20min Donor: ATP Kinase Phosphatase x Okadaic acid, H2O2 x Imatinib, Dasatinib, Staurosporin EGF, PDGF experimental phosphoryl. frequencynorm to aa frequ. pY/pT/pS: 1/1/4 +PO4 -3 provoke charge modification Kinases > high Kd for ATP > makes them operate close to independently from ATP concentrations Arena et al, CMLS, 2005; S. Prabakaran et al., Wiley Periodicals, 2012 phosphorylation modulates activity conformation docking sites
7. 7. Phosphorylation in cell adhesion and migration: Cofilin K. Rottner et al 2011 Curr. Opin. Cell Biol; Y. Zhang, Genes Dev, 2012, Mizuno, Cellular Signalling, 2013; Moriyama, Genes Cells, 1996; Nagai et al, Genes & Cancer, 2012; Harvard University, BioVisions-The Inner Life Of A Cell, 2011 Cofilin pSer3 inactive Cofilin does not bind actin (phospho mimic S3D mutation) 1 integrin triggers downstream phosphorylation of pSer3 in Cofilin pSer3 active Cofilin binds to actin found at the leading edge of mobile cells is required for directional migration severs actin filaments Cofilin active Cofilin Ser-3 P inactive Ser-3 dephospho mimic S3A mutant > cell migration and invasiveness invading astrocytoma brain cancer cell number through the matrigel-precoated membrane activity regulated by phosphorylation > local K/P concentration
8. 8. 3 examples of widely studied reversible PTMs: methylation S. Prabakaran et al., Wiley Periodicals, 2012; S. Lanouette et al, Mol. Syst. B., 2014, Vermillion et al, JCB, 2014 Methyl Donor: SAM Lysine Methyl-Transferase (HMT) t1/2= very stable GSK J4 HCl x only small mass change protein stability modulates interactions between protein/DNA, protein/protein: docking site no change of positive charge crosstalks with PTMs x Demethylase (HDM) Mono- Di- Tri-methylation hydrogen bond formation not erasable by hydrolysis but via oxidative conversion > into hydrolytically labile imine methylation Competitive inhibitors for SAM: Trifluoroacetate salt, UNC0631 each methyl group > hydrophobicity of hydrophilic Lys
9. 9. Lysine-Methylation in cell adhesion and migration: elongation factor 1-a1 (EF1a1) methylation is required for neural crest migration K.L. Vermillion et al, JCB, 2014 IF Control chicken embryo no change in neural crest migration (at somite 9 stage) EF1a1 binds to the actin cytoskeleton at the leading edge of migratory neural crest cells Sox10: Protein specifically present in migrating neural crest cells GFP GFP GFP GFP 6x di-/tri-methylated K (found by MS) to methylation resistant A EF1a1 prevents or promotes actin polymerization (conc. dep.) methylation resistant EF1a1 expression > cell migration distance
10. 10. 3 examples of more widely studied reversible PTMs: acetylation t1/2= 1-60min Acetyl transferase (HAT) or equilibrium reaction Deacetylase (HDAC) Glucose, EtOH x SBHA, TSA, SAHA Anacardic acid Resveratrol, SRT-501 (are well tolerated in humans) Lundby et al, Cell Reports, 2012; Friis et al, Nucleic Acids Research 2009; Walsh et al, Angew. Chem. Int. Ed., 2005; Okanishi et al, J. proteome research, 2013; S. Prabakaran et al., Wiley Periodicals, 2012 Lys-e amine acetylation neutralizes its positive charge alters hydrogen bonds alters stability docking site hampers electrostatic interaction increases hydrophobicity of hydrophilic Lys tissue-type acetylation pattern cell compartment specific Lys acetylation x Donor: AcCoA
11. 11. Lysine is the most heavily modified amino acid Lys acetylation blocks other PTMs lengthens proteins lifetime (e.g. p53) XJ Yang, Mol. Cell., 2008
12. 12. 18 human HDAC enzymes classified into 4 groups based on yeast homology Kelly et al, Ashley Publications, 2002; Kim et al, Am J Transl Res, 2011; Sadoul et al, J. Biomed. and Biotech., 2011; Grant, Academic Press, p89, 2012; Michishita et al, MBC, 2005 HDACs class III SIRT 1/2cytop/3/4/5/6/7 NAD+ ubiquitously expressed high expression in brain, testis HDACs class I 1/2/3cytop/8cytop Zn2+ ubiquitously expressed TSA HDACs class II IIa: 4cytop/5cytop/7cytop/9cytop IIb: 6cytop/10cytop Zn2+ predominant in heart, smooth muscle, brain, kidney TSA HDACs class IV 11cytop Zn2+ predominant in heart, smooth muscle, brain, kidney TSA
13. 13. Acetylation can mingle with other PTMs to form complex regulatory programs XJ Yang, Mol. Cell., 2008; Rao et al, BMC Bioinformatics, 2013; Z. Lu et al, Plos One, 2011; Latham et al, Nat Struct Mol Biol, 2007 46.0% of studied Ac-Lys are predicted to have an effect on phosphorylation sites: creation/destruction of a phosphorylation site alterations in kinase binding Z. Lu et al, Plos One, 2011 #ofalteredphosphorylationsites Distance from K-Ac Distance distribution of altered phosphorylation site to K-Ac (Q) in silico perturbation of the microenvironment through substitution of Lys (K) (+) with Glu (Q) (neutral), mimicking a neutral Ac-Lys
14. 14. autophagy amino acid degradation Acetyl-CoA: the crossroads of fat, sugar and protein catabolism J. Patel et al., Nutrition & Metabolism, 2011; Choudhary et al, Nature reviews, 2014; Wellen et al, Nature reviews, 2012; Mario et al, cell press, 2014 acetate can be produced by deacetylation reactions ethanol metabolism in the liver by bacteria in the colon Citrate-Shuttle nutrient starvation > depletes AcCoA provokes autophagy caloric intake elevates Acetyl-CoA concentrations acetyl-CoA concentration influences HAT activity overall acetylation level
15. 15. 2 examples of protein acetylation in cell adhesion and migration: a-tubulin acetylation reduces cell motility XJ Yang et al, cell press, 2008; Joo et al, nature communications, 2014; Glozak, science direct, 2005; Harvard University, BioVisions-The Inner Life Of A Cell, 2011 welkescience Microtubles - and -tubulin dimers form long, hollow cylinders component of cytoskeleton platforms for intracellular transport (secretory vesicles, organelles) Microtubules Deacetylation: HDAC6, SIRT2 microtubule acetylation reduces cell migration increased stability microtubule acetylation found at leading edge prone to depolymerization HDAC6 >microtubule acetylation > cell migration speed
16. 16. 2 examples of protein acetylation in cell adhesion and migration: Cortactin acetylation reduces cell motility X. Zhang et al, Cell Press, 2007; Y. Zhang et al, Oncogene, 2009; XJ Yang et al, cell press, 2008; Kirkbridge et al, Cell Adhesion and Migration, 2011;p ; Harvard University, BioVisions-The Inner Life Of A Cell, 2011 Cortactin Cortactin cytoplasmic, monomeric regulates branched actin assembly & stabilization at leading edge abnormally expressed in many human tumours overexpression > invasiveness and migration depletion > impairs cell migration Acetylation: p300 Deacetylation: HDAC6 and Sirt1 deacetylated cortactin cells migrate faster translocates to cell periphery actin binding high levels of SIRT1 observed in tumours hyperacetylated cortactin (up to 10 residues) translocation to the cell periphery association with actin cell motility
17. 17. Drug effects on cell acetylation state and disease: HDAC inhibitors Singh et al, ERAT, 2010; Choudhary et al, Science, 2009; Wikipedia: SAHA, 2014; stocan.weebly.com, 2014; penn-medicine-lung-transplant.blogspot.ch, 2014 SAHA (Vorinostat) FDA approved treatment for cutaneous T-cell lymphoma (CTCL) anti-cancer drug with low toxicity competes with Zn2+ > blocks active site of HDACs affects growth/survival of tumor cells overall protein acetylation provokes tumour selective pro-apoptotic gene pro-survival gene HDAC-1 gastric cancer lung cancer HDAC-5/10 HDAC inhibitors are anticancer drugs cell cycle arrest/apoptosis/autophagy metastasis/angiogenesis acetylation mediated p53 activation > p53s half life SAHA HDACs are often deregulated in cancer
18. 18. Drug effects on cell acetylation state and disease: Glucose Miller et al, MBoC, 2014; Choudhary et al; Nature reviews, 2014 SIRT1 consequences of high glucose conditions for adhesion/matrix increase integrin cell adhesion stimulate FN polymerization diabetics > higher plasma glucose concentrations > may entail greater overall protein acetylation E.g. diabetic nephropathy comes with ECM expansion glucose restriction provokes glucose production from fatty acids > maintains blood glucose levels SIRT1 > acetylation of proteins? high concentrations of glucose acetyl-CoA levels overall protein acetylation fatty acid production
19. 19. Drug effects on cell acetylation state and disease: EtOH Blythe et al, alcoholism: Clinical and Experimental research, 2010; Kannarkat, Journal of Hepatology, 2006; Lieberman et al, Wolters Kluwer, 2012 3 male rats fed 5 weeks with 36% ETOH containing diet Arrows: 2 fold or greater increase of acetylation Acetyl-CoA livers EtOH metabolism EtOH transformed into acetate (liver) > acetate metabolized to AcCoA Anti-acetyl Lysine antibody of rat livers highly reactive with Lys not toxic treatment chronic liver disease > overall acetylation HDAC activator Resveratrol attenuates fatty liver in alcohol-exposed mice chronic EtOH consumption provokes liver protein hyperacetylation (3x ): tubulin, actin, cortactin, p53 key factor in liver injury mitochondrial dysfunction, altered protein trafficking after EtOH withdrawal > acetylation remains 0.04 endogenous EtOH in human blood bacterial fermentation caloric intake > 0.06 EtOH exposure to cells > protein acetylations, phosphorylations, methylations
20. 20. Conclusion: cell adhesion and migration are crucial for organisms structural organization embryogenesis body growth body function immune responses repair after injury constant dynamics tissue remodelling tissue renewal (adults cell age: 7-10 years) maintain body structure connective tissue epithelial tissue allow cell movement matrix modelling adhesion making/breaking Spalding et al, Cell, 2005; Alberts, 2008 Cell-Matrix adhesions: via integrins Cell migration: via cytoskeleton PTMs drive integrin function PTMs drive the function of cofilin, a-tubulin, cortactin, EF1a1 Cell adhesion and migration are essential for the structure and maintenance of multicellular organisms & PTMs are involved in their regulation
21. 21. Thank you for your attention! Now its time for questions..
22. 22. PTMs crosstalking evidence: the p53 example DNA damage> p53 activation via methylation-acetylation-phosphorylation cascade Lys372 methylation required for p53 acetylation Lys370 and Lys372 acetylation on p53 change DNA binding specificity forms docking sites for transcriptional co-activators enhances nuclear localization impairs methylation, ubiquitination > p53 half-life impacts on phosphorylation of Ser371 contributes to p53 stabilization cell cycle arrest through targeted protein expression Acetyltransferases (HAT): p300 Deacetylases (HDACs) : HDAC1, HDAC3, SIRT1, SIRT7 p53-transcription factor, tumour suppressor very unstable may be ac, p, m, ub,.. gene mutated in 50% of human cancers (eg Lys 120) in nucleus/cytoplasm at low levels (unstressed cells) activated p53 protein induces cell-cycle arrest/apoptosis mice with 7xKR mutations at the C-term including Lys370/372: viable and phenotypically normal Patel et al, Nutrition & Metabolism, 2011; XJ Yang et al, Cell, 2008; Walsh et al, Angew. Chem., 2005; Ashcroft, et al Mol. Cell. Biol., 1999