Cell biology of AB toxins

Transcription

1 Cell biology of AB toxins Gisou van der Goot, EPFL Lausanne EMBO/FEBS Advanced course at Spetses, Greece, September 1-9, 2010

2 Bacterial protein toxins Definition: Proteins released by bacteria into the extracellular medium that affect cellular functions of target cells

3 Classifications Where they act: Plasma membrane Cytoplasm Lumen of organelles What they act on: Membrane Cytoskeleton Protein synthesis - Activity: no enzymatic activity pore-forming toxins Enzymatic activity Lipase Protease ADP ribosyltransferase Glucosidase Adenylate cyclase. --> cytoplasmic targets

4 How do they reach their target?

5 Difference between toxins and type III or IV effectors: Mode of delivery A subunit: Enzymatic activity B-subunit Toxin: Multi subunit protein Or Independent polypeptide chains Spanò S, Galán JE. Curr Opin Microbiol. 2008

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7 They must therefore cross a membrane elsewhere in the cell To reach the cytosol, toxins must cross 1 membrane Figure 12-1 Molecular Biology of the Cell ( Garland Science 2008)

8 Membrane bound organelles probably originate from invaginations of the plasma membrane Space continuous with the outside of the cell Figure 12-4a Molecular Biology of the Cell ( Garland Science 2008) The lumen of all organelles are topologically equivalent to each other and to the extracellular space

9 Entry portals into the cell Marsh and Helenius, Cell 2006

10 Questions How do toxins get in? To what compartment do they go? How do they cross the membrane? What do they do once they get to the other side?.

11 Two examples Cholera toxin A subunit: Enzymatic activity B-subunit X X Anthrax toxin X 2 A subunits B-subunit

12 Is there a place in the cell where proteins get transported across membrane? 12

13 Figure Molecular Biology of the Cell ( Garland Science 2008)

14 Proteins are folded and assembled in the ER with the help of chaperones and folding enzymes (Anelli and Sitia, EMBO J. 2008)

15 In case of failure, misfolded proteins are retro translocated into the cytosol, polyubiquitinated and degraded by the proteasome The ERAD pathway Protein folding is not that efficient, for some proteins efficiency is 20% (Cystic fribosis CFTR)

16 ER resident proteins are either retained in the ER or retrieved from the Golgi through the KDEL receptor Golgi Endoplasmic reticulum

17 Cholera toxin: AB 5 toxin Petameric B subunit ADP-ribosyltransferase that modifies the Gα subunit of the host adenylate cyclase Receptor: Ganglioside GM1

18 Enzymatic activity of cholera ADP-ribosyltransferase Target is Gi of Adenylate cyclase: leads to a constitutively active form Increase in camp Opening of chloride channels Exit of Na + Exit of H 2 0

19 Cholera toxin can enter cells via multiple pathways Endocytosis is driven by the interaction with GM1 and depends on the clustering of the GM1 by the B pentamer and on the length of the GM1 acyl chains Marsh and Helenius, Cell 2006

20 Cholera toxin triggered signaling to the endocytic machineries requires transbilayer coupling

21 Transbilayer coupling Helenius group, ETHZ

22 Under control of the B-subunit Cholera toxin Early Endosomes Under control of the A-subunit Golgi Endoplasmic Reticulum Late Endosomes Lysosomes

23 How Cholera toxin hijacks the early secretory pathway Golgi A subunit has a C- terminal KDEL sequence -> signal for retrieval to the ER ER The A subunit is recognized as unfolded and undergoes retrotranslocation to the cytoplasm Due to the low lysine content of the A subunit the protein is not ubiquitinated and degraded

24 Proteins are folded and assembled in the ER with the help of chaperones and folding enzymes (Anelli and Sitia, EMBO J. 2008)

25 In case of failure, misfolded proteins are retro translocated into the cytosol, polyubiquitinated and degraded by the proteasome The ERAD pathway Protein folding is not that efficient, for some proteins efficiency is 20% (Cystic fribosis CFTR)

26 Very interesting novel variant of this class of toxin: SubAB produced by certain pathogenic E. coli strains SubAB: A-B5 like cholera toxin of Shiga toxin A sub-unit looks like subtilisin B subunit binds GM2 Never reaches the cytoplasm: 1st toxin that acts in the lumen of a comparment of the endomembrane system SubAB cleaves Bip/GRP78: ER lumen Hsp70 familly member

27 Bip: a master regulators of the ER Maintains the permeability barrier by blocking the non engaged translocons Acts as a chaprone for emerging nascent chains Bring misfolded proteins to the retrotranslocon a master regulator of the unfolded protein response SubAB cleaves between the ATPase domain and the petide binding domains and kills cells very rapidly

28 Anthrax toxin is tripartite PA: Protective antigen, bares similarities to pore-forming toxins EF: Edema factor, calmodulin dependent adenylate cyclase LF: Lethal factor, metalloprotease shown to cleave MAPKKs

29 Mode of action of PA Acidic ph (Modified from Prince, J Clin Invest. 2003)

30 Acidic ph (Prince, J Clin Invest. 2003)

31 Anthrax toxin receptor (ATRs) Von Willebrand A domain Ig-like domain vwa TM CT

32 Protective antigen Von Willebrand Factor A domain of the CMG2 receptor (Santelli et al. Nature, 2004)

33 (modified from Bann and Hultgren, N&V, Nature 2004)

34 Channel forming loop Protective antigen Von Wildebrand domain of the CMG2 receptor (Lacy et al. PNAS, 2004) 1 histidine in CMG 2 implicated in domain II binding Domain II-domain IV interface contains 7 histidines (Santelli et al. Nature, 2004)

35 NalP (Santelli et al. Nature, 2004) (Oomen et al. EMBO, 2004)

36 The protective antigen has to time its entry How does it do it? Acidic ph (Prince, J Clin Invest. 2003)

37 PA heptamers are spatially segregated from monomers Raft like domain Clathrin dependent entry (Abrami et al., J Cell Biol. 2003)

38 Labile molecules that associate in to labile structures: structural aggregates that form functional networks

39 Spatial segregation of PA83 and PA63 Solubilize cells 1% Triton 4 C DRMs L DRM 0% PA83 +PA63 30% Caveolin-1 40% Cell lysat Top Bottom L Density gradient (Abrami et al., J. Cell Biol., 2003)

40 Raft associated (Abrami et al. JCB 2003) No endocytosis Clathrin dependent entry Surface behavior of ATRs is regulated by at least 3 post-translationnal modifications: 1. S-Palmitoylation 2. Ubiquination 3. Phosphorylation

41 What are the signaling events that take place in rafts to induce endocytosis?

42 Interaction between a cargo receptor and clathrin requires adaptor Clathrin adaptors or accessory proteins AP-1 (Adaptor Protein complex 1) AP-2 ß-arrestins 1 & 2 Dab2 Calm/AP180? Epsin/Eps15 Cargos (Golgi) Listeria entry Transferin receptor, LAMP1 GPCRs LDL receptors EGFR, ENaC And adapters need signals to be recruited

43 Molecular players involved in anthrax toxin endocytosis Phosphorylation deficient receptors src P ß-arrestin cbl Ub IP: TEM8-HA AP1 clathrin (Abrami et,pnas 2010, PLoS Patho. 2010)

44 At what location do toxin enter the cytoplasm? (Gruenberg, 2001)

45 Endosomes contain intraluminal vesicles Murk et al., PNAS 2003

46 The unconventional anthrax toxin entry route Also followed by certain viruses One way Early endosomes Back fusion Late endosomes

47 All (almost) AB toxins must cross a membrane This can occur at a variety of intracellular sites which depend on the toxin Diphtheria toxin Exotoxin A Anthrax toxin