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| A complete oligomerization results in a prepore. To determine the structure by X-ray or Cryo-EM of the prepore is a challenging process that till the date did not produce any result. The only available information about the prepore structure was provided by Atomic Force Microscopy (AFM).] The measured prepore height was 90 Å]<sup>,]</sup> and the width 118 Å, with an inner pore of 50 Å (Figure 3).<sup>40,56</sup> Interestingly, a recent study in Aerolysin points out that the currently accepted model for the Lysenin prepore should be revisited, according to the new available data about the Aerolysin insertion.] | A complete oligomerization results in a prepore. To determine the structure by X-ray or Cryo-EM of the prepore is a challenging process that till the date did not produce any result. The only available information about the prepore structure was provided by Atomic Force Microscopy (AFM).] The measured prepore height was 90 Å]<sup>,]</sup> and the width 118 Å, with an inner pore of 50 Å (Figure 3).<sup>40,56</sup> Interestingly, a recent study in Aerolysin points out that the currently accepted model for the Lysenin prepore should be revisited, according to the new available data about the Aerolysin insertion.] | ||
| ] | ] | ||
| A conformational change transforms the PFM in the transmembrane β-barrel, leading to the pore state. Such a conformational change produces a decrease of the oligomer height of , according to AFM measurements.] Using X-ray structure, they were measured the main dimensions, being height 97 Å, width 115 Å and the inner pore of 30 Å (Figure |
A conformational change transforms the PFM in the transmembrane β-barrel, leading to the pore state. Such a conformational change produces a decrease of the oligomer height of , according to AFM measurements.] Using X-ray structure, they were measured the main dimensions, being height 97 Å, width 115 Å and the inner pore of 30 Å (Figure 2).] Interestingly, the complete oligomerization is not a requisite for the insertion. It is usual to observe incomplete oligomers in prepore and pore states ]<sup>,]</sup>. However, the incomplete pores did not evolve to complete, a phenomenon that was observed in CDCs family.] The triggering mechanism for the prepore to pore transition in Lysenin depend in three glutamic acids (E92, E94 and E97), and is activate by a decreased of pH<sup>25</sup>, from physiological conditions. This residues are located in an α-helix that form part of the PFM. | ||
| ⚫ | A conformational change transforms the PFM in the transmembrane β-barrel, leading to the pore state. Such a conformational change produces a decrease of the oligomer height of , according to AFM measurements.] Using X-ray structure, they were measured the main dimensions, being height 97 Å, width 115 Å and the inner pore of 30 Å (Figure 2).] Interestingly, the complete oligomerization is not a requisite for the insertion. It is usual to observe incomplete oligomers in prepore and pore states ]<sup>,]</sup>. However, the incomplete pores did not evolve to complete, a phenomenon that was observed in CDCs family.] The triggering mechanism for the prepore to pore transition in Lysenin depend in three glutamic acids (E92, E94 and E97), and is activate by a decreased of pH<sup>25</sup>, from physiological conditions. This residues are located in an α-helix that form part of the PFM. | ||
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| |'''<nowiki>Figure 3 | Lysenin mechanism of action Scheme. a)</nowiki>''' Lysenin monomers (PDB ID 3ZXD) are segregated as soluble proteins that bind specifically to sphingomyelin by its receptor binding domain. After binding, and subsequently condensation, the oligomerization starts. '''b)''' After a complete oligomerization, the prepore is formed. The prepore model shown here was assembled from the monomer structure and aligned with the pore structure (PDB ID 5GAQ) by their receptor-binding domains (residues 160 to 297). The height of the prepore was set to agree with the Atomic Force Microscopy measurements. '''c)''' Membrane inserted Lysenin assembly (PDB ID 5GAQ). The height of the pore was measured from the detergent belt to the last residue, assuming that the detergent belt corresponds part of the pore surrounded by the membrane. The membrane was placed in the β-barrel of the pore to match with the detergent belt, that englobe all the hydrophobic residues of the β-barrel. The hydrophobic surface colour scale is according to the hydrophobicity scale of Kyte and Doolittle.] | |||
| |} | |||
| ⚫ | A conformational change transforms the PFM in the transmembrane β-barrel, leading to the pore state. Such a conformational change produces a decrease of the oligomer height of , according to AFM measurements.] Using X-ray structure, they were measured the main dimensions, being height 97 Å, width 115 Å and the inner pore of 30 Å (Figure |
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| ==Lysenin insertion consequences== | ==Lysenin insertion consequences== | ||
Revision as of 08:19, 10 August 2018
Lysenin is a Pore-forming toxin in the coelomic fluid of the earthworm Eisenia fetida. Template:New unreviewed article
Lysenin Monomer
Lysenin, is a protein produced in the coelomocytes-Leucocyte of animals with coelom-of Eisenia fetida. The protein was first isolated from the coelomic fluid by Yoshiyuki Sekizawa from Kobayashi group in 1996. They also named the protein Lysenin (lysis+Eisenia). Lysenin is a relatively small protein with a molecular weight of 33 kDa (Figure 2). The X-ray structure was first determined by de Colibus et al., and hence classified as a member of Aerolysin family, with which shares structure and function. Lysenin monomers, from a structural point of view, comprises the receptor binding domain and Pore Forming Module (PFM). Actually, this classification is shared with the rest of the Aerolysin family. De Colibus et al. obtained the first X-ray structure of the monomer, which showed that PFM is the motive that specifically interacts with sphingomyelin. Two recently published works revealed that the receptor binding domain is the part in contact with sphingomyelin, where it was even shown the fit of the phosphatidylcholine headgroups of sphingomyelin.
The definition of PFM changed after extracting the Lysenin pore structure. The domain that forms the β-barrel, the so-called β-hairpin, was alleged to be a small region in the vicinity of the PFM (shown in green in Figure 2). Nowadays, it is known that the β-barrel is formed by a larger region, also including regions in the middle of the PFM (illustrated in yellow in Figure 1).
Lysenin membrane receptors
The natural target of Lysenin is an animal plasma membrane lipid called Sphingomyelin. Sphingomyelin is synthesized in the endoplasmic reticulum, and is mostly present in the outer leaflet of the animal plasma membrane, where it plays an important role as a secondary messenger.
In the raft, lipids are in liquid-ordered phase, meaning more lateral dense packed, rigid, low chain motion and high lateral mobility. This rigidity is in part due to the high transition temperature of sphingolipids, as well as the interactions of these lipids with Cholesterol. Cholesterol is a relatively small, amphipathic molecule that can accommodate between the large acyl chains of sphingolipids, resulting in the so-called liquid ordered phase.
Cholesterol plays an important role in the oligomerization of Lysenin. This sterol provides a stable platform with high lateral mobility were the monomer-monomer encounters are more probable.
PFT have been shown to be able to remodel the membrane structure, even mixing lipid phases. The detergent belt-the part of the β-barrel occupied by detergent in the Cryogenic Electron Microscopy (Cryo-EM) study- of Lysenin pore is 32 Å in height. This height implies a bend of the membrane in the region surrounding the pore, call negative mismatch.
Lysenin binding, oligomerization and insertion
Membrane attachment is a requisite to initiate oligomerization. Lysenin monomers bind specifically to sphingomyelin via the receptor binding domain. How the oligomerization process is remains as an open question, due to the current technical limitations for recording the monomer diffusion.
The final Lysenin oligomer is constituted by nine monomers without quantified deviations, as shown for the first time by Munguira et al.
Lysenin monomers bind to sphingomyelin-enriched domains, which provide a stable platform with a high lateral mobility, hence favouring the oligomerization. Lysenin oligomerization occurs in a two-step process, as for proteins in general.
First, the monomers adsorb to the membrane by specific interactions, resulting in an increased concentration. Lipid rafts are believed to play a key role contributing to the increasing of the monomer concentration. After a critical concentration is reached, several oligomers are formed simultaneously. The second step consists in the growth of new oligomers. It takes places in the borders of the oligomers cluster.
A complete oligomerization results in a prepore. To determine the structure by X-ray or Cryo-EM of the prepore is a challenging process that till the date did not produce any result. The only available information about the prepore structure was provided by Atomic Force Microscopy (AFM). The measured prepore height was 90 Å and the width 118 Å, with an inner pore of 50 Å (Figure 3). Interestingly, a recent study in Aerolysin points out that the currently accepted model for the Lysenin prepore should be revisited, according to the new available data about the Aerolysin insertion.
A conformational change transforms the PFM in the transmembrane β-barrel, leading to the pore state. Such a conformational change produces a decrease of the oligomer height of , according to AFM measurements. Using X-ray structure, they were measured the main dimensions, being height 97 Å, width 115 Å and the inner pore of 30 Å (Figure 2). Interestingly, the complete oligomerization is not a requisite for the insertion. It is usual to observe incomplete oligomers in prepore and pore states . However, the incomplete pores did not evolve to complete, a phenomenon that was observed in CDCs family. The triggering mechanism for the prepore to pore transition in Lysenin depend in three glutamic acids (E92, E94 and E97), and is activate by a decreased of pH, from physiological conditions. This residues are located in an α-helix that form part of the PFM.
A conformational change transforms the PFM in the transmembrane β-barrel, leading to the pore state. Such a conformational change produces a decrease of the oligomer height of , according to AFM measurements. Using X-ray structure, they were measured the main dimensions, being height 97 Å, width 115 Å and the inner pore of 30 Å (Figure 2). Interestingly, the complete oligomerization is not a requisite for the insertion. It is usual to observe incomplete oligomers in prepore and pore states . However, the incomplete pores did not evolve to complete, a phenomenon that was observed in CDCs family. The triggering mechanism for the prepore to pore transition in Lysenin depend in three glutamic acids (E92, E94 and E97), and is activate by a decreased of pH, from physiological conditions. This residues are located in an α-helix that form part of the PFM.
Lysenin insertion consequences
The ultimate consequences caused by the formation of the Lysenin pore are not well documented, however three are the plausible consequences after the formation of the Lysenin pore:
· To punch the membrane breaks the sphingomyelin asymmetry between the two leaflets of the lipid bilayer, which leads to apoptosis. PFT are supposed to induce lipid flip-flop that can also break the sphingomyelin asymmetry.
· The presence of the pore increases the calcium concentration in the cytoplasm, which drives to apoptosis.
· A decrease in the potassium concentration in the cytoplasm causes apoptosis.
Biological role of Lysenin
The biological role of Lysenin still remains unknown. It was suggested that Lysenin can play a role as a defeat mechanism against attackers such as bacteria, fungi or small invertebrates. Nevertheless, sphingomyelin starts to appear in the plasma membrane of chordates. Therefore, Lysenin, being dependent on sphingomyelin as a binding factor, cannot affect bacteria, fungi or in general invertebrates. Another hypothesis is that the earthworm, which is able to expel coelomic fluid under stress, generates an avoidance behaviour to its vertebrate predators (like birds, hedgehogs or moles). In that case, the expelled Lysenin could be more effective if the coelomic fluid reaches the eye, where the concentration of sphingomyelin is ten times higher than in other body organs.
Related to this, coelomic fluid, exuded under stressful situations by the earthworm, has a pungent smell -that gives name to the earthworm- was suggested to be an antipredator adaptation. In any case, if Lysenin plays a role generating an avoiding behaviour, remains unknown.
References
1. Sekizawa, Y., Hagiwara, K., Nakajima, T. & Kobayashi, H. A Novel Protein, Lysenin, That Causes Contraction of the Isolated Rat Aorta : Its Puriification from the Coleomic Fluid of the Earthworm, Eisenia Foetida. Biomedical research 17, 197-203 (1996).
2. De Colibus, L. et al. Structures of Lysenin Reveal a Shared Evolutionary Origin for Pore-Forming Proteins And Its Mode of Sphingomyelin Recognition. Structure 20, 1498-1507 (2012).
3. BERNHEIMER, A.W. & AVIGAD, L.S. Partial Characterization of Aerolysin, a Lytic Exotoxin from Aeromonas hydrophila. INFECTION AND IMMUNITY, 1016-1021 (1974).
4. Parker, M.W. et al. Structure of the Aeromonas toxin in its water-soluble and membrane-channel states. Nature 367, 292-295 (1994).
5. Szczesny, P. et al. Extending the Aerolysin Family: From Bacteria to Vertebrates. PLoS ONE 6, 1-10 (2011).
6. Bokori-Brown, M. et al. Cryo-EM structure of lysenin pore elucidates membrane insertion by an aerolysin family protein. Nat Commun 7, 11293 (2016).
7. Podobnik, M. et al. Crystal structure of an invertebrate cytolysin pore reveals unique properties and mechanism of assembly. Nat Commun 7, 11598 (2016).
8. Yamaji, A. et al. Lysenin, a Novel Sphingomyelin-specific Binding Protein. THE JOURNAL OF BIOLOGICAL CHEMISTRY 273, 5300–5306 (1998).
9. Murate, M. & Kobayashi, T. Revisiting transbilayer distribution of lipids in the plasma membrane. Chem Phys Lipids 194, 58-71 (2016).
10. Sphingomyelin breakdown and cell fate. TIBS 21, 468-471 (1996).
11. Slotte, J.P. The importance of hydrogen bonding in sphingomyelin's membrane interactions with co-lipids. Biochim Biophys Acta 1858, 304-10 (2016).
12. Ishitsuka, R. & Kobayashi, T. Cholesterol and lipid/protein ratio control the oligomerization of a sphingomyelin-specific toxin, lysenin. Biochemistry 46, 1495-1502 (2007).
13. Yamaji-Hasegawa, A. et al. Oligomerization and Pore Formation of a Sphingomyelin-specific Toxin, Lysenin. Journal of Biological Chemistry 278, 22762-22770 (2003).
14. Rojko, N. & Anderluh, G. How Lipid Membranes Affect Pore Forming Toxin Activity. Acc Chem Res 48, 3073-9 (2015).
15. Ros, U. & Garcia-Saez, A.J. More Than a Pore: The Interplay of Pore-Forming Proteins and Lipid Membranes. J Membr Biol 248, 545-61 (2015).
16. Guigas, G. & Weiss, M. Effects of protein crowding on membrane systems. Biochim Biophys Acta 1858, 2441-50 (2016).
17. Phillips, R., Ursell, T., Wiggins, P. & Sens, P. Emerging roles for lipids in shaping membrane-protein function. Nature 459, 379-385 (2009).
18. Munguira, I. et al. Glasslike Membrane Protein Diffusion in a Crowded Membrane. ACS Nano 10, 2584-90 (2016).
19. Wolde, P.R.t. Enhancement of Protein Crystal Nucleation by Critical Density Fluctuations. Science 277, 1975-1978 (1997).
20. Chunga, S., Shin, S.-H., Bertozzi, C.R. & Yoreo, J.J.D. Self-catalyzed growth of S layers via an amorphousto-crystalline transition limited by folding kinetics. PNAS 107, 16536–16541 (2010).
21. Yilmaz, N. et al. Real-Time Visualization of Assembling of a Sphingomyelin-Specific Toxin on Planar Lipid Membranes. Biophysical Journal 105, 1397-1405 (2013).
22. Iacovache, I. et al. Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process. Nat Commun 7, 12062 (2016).
23. Kyte, J. & Doolittle, R.F. A simple method for displaying the hydropathic character of a protein. Journal of molecular biology 157, 105-132 (1982).
24. Mulvihill, E., van Pee, K., Mari, S.A., Muller, D.J. & Yildiz, O. Directly Observing the Lipid-Dependent Self-Assembly and Pore-Forming Mechanism of the Cytolytic Toxin Listeriolysin O. Nano Lett 15, 6965-73 (2015).
25. Munguira, I. L. B., Takahashi, H., Casuso, I. & Scheuring, S. Lysenin Toxin Membrane Insertion Is pH-Dependent but Independent of Neighboring Lysenins. Biophysical Journal 113, 2029–2036 (2017).
26. Green, D.R. Apoptosis and Sphingomyelin Hydrolysis: The Flip Side. The Journal of Cell Biology 150, F5–F7 (2000).
27. Orrenius, S., Zhivotovsky, B. & Nicotera, P. Regulation of cell death: the calcium-apoptosis link. Nat Rev Mol Cell Biol 4, 552-65 (2003).
28. Yu, S.P. Regulation and critical role of potassium homeostasis in apoptosis. Progress in Neurobiology 70, 363-386 (2003).
29. Ballarin, L. & Cammarata, M. Lessons in Immunity: From Single-cell Organisms to Mammals. (Academic Press, 2016).
30. KOBAYASHI, H., SEKIZAWA, Y., AIZU, M. & UMEDA, M. Lethal and Non-Lethal Responses of Spermatozoa From a Wide Variety of Vertebrates and Invertebrates to Lysenin, a Protein From the Coelomic Fluid of the Earthworm Eisenia foetida. JOURNAL OF EXPERIMENTAL ZOOLOGY 286, 538–549 (2000).
31. Sukumwang, N. & Umezawa, K. Earthworm-derived pore-forming toxin lysenin and screening of its inhibitors. Toxins (Basel) 5, 1392-401 (2013).
32. Kobayashi, H., Ohta, N. & Umeda, M. Biology of Lysenin, a Protein in the Coelomic Fluid of the Earthworm Eisenia foetida. International Review of Cytology 236, 45-99 (2004).
33. Swiderska, B. et al. Lysenin family proteins in earthworm coelomocytes - Comparative approach. Dev Comp Immunol 67, 404-412 (2017).
34. Berman, E.R. Biochemistry of the eye. (1991).
35. Edwards, C.A. & Bohlen, P.J. Biology and Ecology of Earthworms, Volume 3. Springer Science & Business Media, 426 (1996).
External links
https://www.theses.fr/2017AIXM0124
Pore Forming Toxins