Dimensión 1: Liderazgo en costo
1.3. Justificación Teórica
Conclusions and Outlook
This dissertation examined the activity of a variety of peptides and proteins that are involved in biological processes relevant to human health. Specifically, we investigated how these peptides and proteins induce membrane curvature to result in the topological changes that underlie different membrane-remodeling events. The first several chapters studied synthetic analogs of AMPs and CPPs, and the AMP-like activity of a natural protein. In chapter 2, we found that a series of nylon-3-based polymeric antimicrobials engages two interdependent mechanisms of action: permeabilization of bacterial membranes and binding to intracellular targets such as DNA. Chapters 3 and 4 explored α-helical polypeptides characterized by distinct shape-changing, adaptable architectures that allow for efficient membrane permeation. Like natural AMPs and CPPs, both the nylon-3-based polymers and α-helical polypeptides were shown to induce topologically active NGC that correlated with their ability to permeate membranes. In chapter 5, we identified previously unrecognized direct antimicrobial activity of cytokine IFN-β. Further examination attributed the antimicrobial activity of the full IFN-β protein to a component helix that resembled AMPs in terms of structural and compositional properties, and ability to induce membrane curvature. As these synthetic analogs and component helix of IFN-β are characterized with hydrophobic and cationic domains, we expect their generated membrane curvature to result from the combination of hydrophobic insertion and membrane-wrapping mechanisms, which is in fact, not unlike natural AMPs and CPPs.
fusion protein and found that it adopts a β-strand-rich conformation in membranes and can oligomerize into β-sheets. Secondly, we observed that sperm protein IZUMO1 can facilitate gamete membrane fusion through Ca2+-mediated IZUMO1–membrane interactions. In both of
these systems, the protein-induced membrane curvatures, which result from a coordination of mechanisms that likely include hydrophobic insertion and molecular crowding, cooperate with the membrane properties to induce the topological changes that enable the formation of hemifusion intermediates and fusion pores.
In chapter 8, we examined two yeast mitochondrial fission machinery proteins, Dnm1 and Fis1. Dnm1 is believed to mediate mitochondrial fission through self-assembly into spirals around the mitochondrial membrane, which can undergo subsequent mechanochemical constriction. However, this constriction of the membrane has been found to be insufficient for fission. In this arrangement, Dnm1 oligomers apply the scaffolding mechanism to constrain the membrane into a cylindrical shape. Our experiments found that Dnm1–membrane interactions also result in NGC generation, which can function synergistically with membrane scaffolding effects to efficiently catalyze mitochondrial fission. In contrast, we observed that Fis1 attenuates the pro-fission membrane curvature induced by Dnm1, which suggests that the two proteins operate in concert to regulate mitochondrial fission.
Here we have highlighted several examples of peptides and proteins that coordinate membrane curvature generation mechanisms to induce the topological changes necessary for a variety of membrane-remodeling processes. Furthermore, the studies presented in this dissertation collectively emphasize the importance of membrane curvature in enabling membrane restructuring in general. Our finding that Fis1 antagonizes the Dnm1-induced membrane curvature effects to potentially reduce or inhibit mitochondrial fission introduces the concept of peptides or proteins
that can actively counteract the topological changes resulting from membrane curvature generation. Thus, this general approach may be applied toward the design of new therapeutics to prevent undesired membrane-remodeling events, such as virus–cell fusion during viral infection and gamete fusion during fertilization.
References
1. Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P., Baltimore, D. & Darnell, J. Molecular
Cell Biology, 4th Edn. (W. H. Freeman and Co., 2000).
2. van Meer, G., Voelker, D.R. & Feigenson, G.W. Membrane lipids: Where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112–124 (2008).
3. Sackmann, E. Biological Membranes Architecture and Function. In Handbook of
Biological Physics, Vol. 1. (eds. Lipowsky, R. & Sackmann, E.) 1–63 (North-Holland,
1995).
4. Saita, E.A. & de Mendoza, D. Thermosensing via transmembrane protein–lipid interactions. Biochim. Biophys. Acta, Biomembr. 1848, 1757–1764 (2015).
5. Epand, R.M. & Epand, R.F. Bacterial membrane lipids in the action of antimicrobial agents. J. Pept. Sci. 17, 298–305 (2011).
6. Shearman, G.C., Ces, O., Templer, R.H. & Seddon, J.M. Inverse lyotropic phases of lipids and membrane curvature. J. Phys.: Condens. Matter 18, S1105–S1124 (2006).
7. Helfrich, W. Elastic properties of lipid bilayers: Theory and possible experiments. Z.
Naturforsch. C 28, 693–703 (1973).
8. Siegel, D.P. & Kozlov, M.M. The Gaussian curvature elastic modulus of N- monomethylated dioleoylphosphatidylethanolamine: Relevance to membrane fusion and lipid phase behavior. Biophys. J. 87, 366–374 (2004).
9. Israelachvili, J.N., Marčelja, S. & Horn, R.G. Physical principles of membrane organization. Q. Rev. Biophys. 13, 121–200 (1980).
10. Israelachvili, J.N. Intermolecular and Surface Forces, 3rd Edn. (Academic Press, 2011). 11. Seddon, J.M. & Templer, R.H. Polymorphism of Lipid–Water Systems. In Handbook of
Biological Physics, Vol. 1. (eds. Lipowsky, R. & Sackmann, E.) 97–160 (North-Holland,
1995).
12. Zasloff, M. Antimicrobial peptides of multicellular organisms. Nature 415, 389–395
(2002).
13. Shai, Y. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by α-helical antimicrobial and cell non-selective membrane-lytic peptides.
14. White, S.H. & Wimley, W.C. Membrane protein folding and stability: Physical principles.
Annu. Rev. Biophys. Biomol. Struct. 28, 319–365 (1999).
15. Bechinger, B. Rationalizing the membrane interactions of cationic amphipathic antimicrobial peptides by their molecular shape. Curr. Opin. Colloid Interface Sci. 14,
349–355 (2009).
16. Dathe, M., Schümann, M., Wieprecht, T., Winkler, A., Beyermann, M., Krause, E., Matsuzaki, K., Murase, O. & Bienert, M. Peptide helicity and membrane surface charge modulate the balance of electrostatic and hydrophobic interactions with lipid bilayers and biological membranes. Biochemistry 35, 12612–12622 (1996).
17. Segrest, J.P., De Loof, H., Dohlman, J.G., Brouillette, C.G. & Anantharamaiah, G.M. Amphipathic helix motif: Classes and properties. Proteins: Struct., Funct., Bioinf. 8, 103–
117 (1990).
18. Tytler, E.M., Segrest, J.P., Epand, R.M., Nie, S.Q., Epand, R.F., Mishra, V.K., Venkatachalapathi, Y.V. & Anantharamaiah, G.M. Reciprocal effects of apolipoprotein and lytic peptide analogs on membranes. Cross-sectional molecular shapes of amphipathic α helixes control membrane stability. J. Biol. Chem. 268, 22112–22118 (1993).
19. Zimmerberg, J. & Gawrisch, K. The physical chemistry of biological membranes. Nat.
Chem. Biol. 2, 564–567 (2006).
20. Campelo, F., McMahon, H.T. & Kozlov, M.M. The hydrophobic insertion mechanism of membrane curvature generation by proteins. Biophys. J. 95, 2325–2339 (2008).
21. Zemel, A., Ben-Shaul, A. & May, S. Modulation of the spontaneous curvature and bending rigidity of lipid membranes by interfacially adsorbed amphipathic peptides. J. Phys. Chem.
B 112, 6988–6996 (2008).
22. Epand, R.M., Shai, Y., Segrest, J.P. & Anantharamiah, G.M. Mechanisms for the modulation of membrane bilayer properties by amphipathic helical peptides. Biopolymers
37, 319–338 (1995).
23. Chen, F.-Y., Lee, M.-T. & Huang, H.W. Evidence for membrane thinning effect as the mechanism for peptide-induced pore formation. Biophys. J. 84, 3751–3758 (2003).
24. Tristram-Nagle, S., Chan, R., Kooijman, E., Uppamoochikkal, P., Qiang, W., Weliky, D.P. & Nagle, J.F. HIV fusion peptide penetrates, disorders, and softens T-cell membrane mimics. J. Mol. Biol. 402, 139–153 (2010).
26. Safinya, C.R., Sirota, E.B., Roux, D. & Smith, G.S. Universality in interacting membranes: The effect of cosurfactants on the interfacial rigidity. Phys. Rev. Lett. 62, 1134–1137
(1989).
27. Szleifer, I., Kramer, D., Ben-Shaul, A., Roux, D. & Gelbart, W.M. Curvature elasticity of pure and mixed surfactant films. Phys. Rev. Lett. 60, 1966–1969 (1988).
28. Wieprecht, T., Dathe, M., Epand, R.M., Beyermann, M., Krause, E., Maloy, W.L., MacDonald, D.L. & Bienert, M. Influence of the angle subtended by the positively charged helix face on the membrane activity of amphipathic, antibacterial peptides. Biochemistry
36, 12869–12880 (1997).
29. Drin, G. & Antonny, B. Amphipathic helices and membrane curvature. FEBS Lett. 584,
1840–1847 (2010).
30. Kik, R.A., Leermakers, F.A.M. & Kleijn, J.M. Molecular modeling of protein-like inclusions in lipid bilayers: Lipid-mediated interactions. Phys. Rev. E 81, 021915 (2010).
31. Johannsson, A., Keightley, C.A., Smith, G.A., Richards, C.D., Hesketh, T.R. & Metcalfe, J.C. The effect of bilayer thickness and n-alkanes on the activity of the (Ca2+ + Mg2+)-
dependent ATPase of sarcoplasmic reticulum. J. Biol. Chem. 256, 1643–1650 (1981).
32. Montecucco, C., Smith, G.A., Dabbeni-sala, F., Johannsson, A., Galante, Y.M. & Bisson, R. Bilayer thickness and enzymatic activity in the mitochondrial cytochrome c oxidase and ATPase complex. FEBS Lett. 144, 145–148 (1982).
33. Michelangeli, F., Grimes, E.A., East, J.M. & Lee, A.G. Effects of phospholipids on the function of the (Ca2+–Mg2+)-ATPase. Biochemistry 30, 342–351 (1991).
34. Cornea, R.L. & Thomas, D.D. Effects of membrane thickness on the molecular dynamics and enzymic activity of reconstituted Ca-ATPase. Biochemistry 33, 2912–2920 (1994).
35. de Planque, M.R.R., Goormaghtigh, E., Greathouse, D.V., Koeppe, R.E., Kruijtzer, J.A.W., Liskamp, R.M.J., de Kruijff, B. & Killian, J.A. Sensitivity of single membrane- spanning α-helical peptides to hydrophobic mismatch with a lipid bilayer: Effects on backbone structure, orientation, and extent of membrane incorporation. Biochemistry 40,
5000–5010 (2001).
36. Botelho, A.V., Huber, T., Sakmar, T.P. & Brown, M.F. Curvature and hydrophobic forces drive oligomerization and modulate activity of rhodopsin in membranes. Biophys. J. 91,
4464–4477 (2006).
37. Lundbæk, J.A., Maer, A.M. & Andersen, O.S. Lipid bilayer electrostatic energy, curvature stress, and assembly of gramicidin channels. Biochemistry 36, 5695–5701 (1997).
38. Lee, A.G. How lipids affect the activities of integral membrane proteins. Biochim. Biophys.
Acta, Biomembr. 1666, 62–87 (2004).
39. Killian, J.A. Hydrophobic mismatch between proteins and lipids in membranes. Biochim.
Biophys. Acta, Rev. Biomembr. 1376, 401–416 (1998).
40. Watnick, P.I., Chan, S.I. & Dea, P. Hydrophobic mismatch in gramicidin A'/lecithin systems. Biochemistry 29, 6215–6221 (1990).
41. de Planque, M.R.R., Greathouse, D.V., Koeppe, R.E., Schäfer, H., Marsh, D. & Killian, J.A. Influence of lipid/peptide hydrophobic mismatch on the thickness of diacylphosphatidylcholine bilayers. A 2H NMR and ESR study using designed transmembrane α-helical peptides and gramicidin A. Biochemistry 37, 9333–9345 (1998).
42. Morein, S., Koeppe Ii, R.E., Lindblom, G., de Kruijff, B. & Antoinette Killian, J. The effect of peptide/lipid hydrophobic mismatch on the phase behavior of model membranes mimicking the lipid composition in Escherichia coli membranes. Biophys. J. 78, 2475–
2485 (2000).
43. Schmidt, N.W., Mishra, A., Wang, J., DeGrado, W.F. & Wong, G.C.L. Influenza virus A M2 protein generates negative gaussian membrane curvature necessary for budding and scission. J. Am. Chem. Soc. 135, 13710–13719 (2013).
44. Zimmerberg, J. & Kozlov, M.M. How proteins produce cellular membrane curvature. Nat.
Rev. Mol. Cell Biol. 7, 9–19 (2006).
45. Farsad, K. & Camilli, P.D. Mechanisms of membrane deformation. Curr. Opin. Cell Biol.
15, 372–381 (2003).
46. McMahon, H.T. & Gallop, J.L. Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature 438, 590–596 (2005).
47. Baumgart, T., Capraro, B.R., Zhu, C. & Das, S.L. Thermodynamics and mechanics of membrane curvature generation and sensing by proteins and lipids. Annu. Rev. Phys. Chem.
62, 483–506 (2011).
48. Morlot, S. & Roux, A. Mechanics of dynamin-mediated membrane fission. Annu. Rev.
Biophys. 42, 629–649 (2013).
49. Takei, K., Haucke, V., Slepnev, V., Farsad, K., Salazar, M., Chen, H. & De Camilli, P. Generation of coated intermediates of clathrin-mediated endocytosis on protein-free liposomes. Cell 94, 131–141 (1998).