Real-time visualization of perforin nanopore assembly

Leung, Carl; Hodel, Adrian W; Brennan, A. J.; Lukoyanova, Natalya; Tran, Sharon; House, Colin M.; Kondos, S.C.; Whisstock, James C.; Dunstone, Michelle Anne; Trapani, Joseph A.; Voskoboinik, Ilia; Saibil, Helen R.; Hoogenboom, Bart W.

doi:10.1038/nnano.2016.303

Cited by 93 publications

(148 citation statements)

References 48 publications

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“…[22] In order to efficiently kill a bacterium, the MAC needs to be generated by surface-bound C5 convertases. [55,56] The shape of MAC pores that were visualized by AFM seemed consistent with the cryo-EM structures that were generated with purified MAC components. Whereas C5 convertases were thought to only be essential to trigger MAC formation via the conversion of C5 into C5b, this study revealed that the role of C5 convertases extends beyond this conversion of C5.…”

Section: Bacterial Killing By the Mac Requires Local Mac Assembly By supporting

confidence: 61%

How the Membrane Attack Complex Damages the Bacterial Cell Envelope and Kills Gram‐Negative Bacteria

2019

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The human immune system can directly lyse invading micro-organisms and aberrant host cells by generating pores in the cell envelope, called membrane attack complexes (MACs). Recent studies using single-particle cryoelectron microscopy have revealed that the MAC is an asymmetric, flexible pore and have provided a structural basis on how the MAC ruptures single lipid membranes. Despite these insights, it remains unclear how the MAC ruptures the composite cell envelope of Gram-negative bacteria. Recent functional studies on Gram-negative bacteria elucidate that local assembly of MAC pores by surface-bound C5 convertase enzymes is essential to stably insert these pores into the bacterial outer membrane (OM). These convertase-generated MAC pores can subsequently efficiently damage the bacterial inner membrane (IM), which is essential for bacterial killing. This review summarizes these recent insights of MAC assembly and discusses how MAC pores kill Gramnegative bacteria. Furthermore, this review elaborates on how MAC-dependent OM damage could lead to IM destabilization, which is currently not well understood. A better understanding on how MAC pores kill bacteria could facilitate the future development of novel strategies to treat infections with Gram-negative bacteria.

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Section: Bacterial Killing By the Mac Requires Local Mac Assembly By supporting

confidence: 61%

How the Membrane Attack Complex Damages the Bacterial Cell Envelope and Kills Gram‐Negative Bacteria

2019

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“…In the neutral pH of the extracellular space perforin can bind to the target cell membrane and in the presence of Ca 2+ the monomers aggregate and form pores in the membrane (Figure ) . These ring‐shaped pores have a structure equivalent to a transmembrane channel with a pore lumen of 13–20 nm that may even coalesce into larger pores by recruiting additional perforin oligomers . As granzyme B has a stokes radius of 5 nm, it should be able to enter the target cell directly through the perforin pores, which was demonstrated in recent studies .…”

Section: Nk Cell Degranulation and Entry Of Granzymes Into The Targetmentioning

confidence: 90%

“…91 These ring-shaped pores have a structure equivalent to a transmembrane channel with a pore lumen of 13-20 nm 17 that may even coalesce into larger pores by recruiting additional perforin oligomers. 92 As granzyme B has a stokes radius of 5 nm, it should be able to enter the target cell directly through the perforin pores, 93 which was demonstrated in recent studies. 94,95 As an alternative mechanism of granzyme entry into target cells, it was proposed that granzymes are taken up by the target cell via endocytosis.…”

Section: Nk Cell Degranulation and Entry Of Granzymes Into The Targetmentioning

confidence: 93%

Mechanisms of natural killer cell-mediated cellular cytotoxicity

Prager

Watzl

2019

Journal of Leukocyte Biology

388

315

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Cellular cytotoxicity, the ability to kill other cells, is an important effector mechanism of the immune system to combat viral infections and cancer. Cytotoxic T cells and natural killer (NK) cells are the major mediators of this activity. Here, we summarize the cytotoxic mechanisms of NK cells. NK cells can kill virally infected of transformed cells via the directed release of lytic granules or by inducing death receptor‐mediated apoptosis via the expression of Fas ligand or TRAIL. The biogenesis of perforin and granzymes, the major components of lytic granules, is a highly regulated process to prevent damage during the synthesis of these cytotoxic molecules. Additionally, NK cells have developed several strategies to protect themselves from the cytotoxic activity of granular content upon degranulation. While granule‐mediated apoptosis is a fast process, death receptor‐mediated cytotoxicity requires more time. Current data suggest that these 2 cytotoxic mechanisms are regulated during the serial killing activity of NK cells. As many modern approaches of cancer immunotherapy rely on cellular cytotoxicity for their effectiveness, unraveling these pathways will be important to further progress these therapeutic strategies.

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“…Pore growing mechanisms without the vertical collapse of poreforming proteins have been described for members of the membrane attack complex/perforin (MACPF) family (Lukoyanova et al, 2015;Metkar et al, 2015;Leung et al, 2017), whereas a prepore-to-pore transitions associated with a vertical collapse have been reported for cholesterol-dependent cytolysins (CDCs; Czajkowsky et al, 2004;Tilley et al, 2005;Leung et al, 2014;van Pee et al, 2016). To see whether a vertical collapse is associated with the assembly and pore-forming of GSDMD Nterm oligomers, we analyzed the time-lapse AFM topographs for the heights of the oligomers protruding from the surface of the lipid membrane.…”

Section: Gsdmd Nterm Oligomers Grow Pores Without Vertical Collapsementioning

confidence: 99%

Mechanism of membrane pore formation by human gasdermin‐D

et al. 2018

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Gasdermin‐D (GSDMD), a member of the gasdermin protein family, mediates pyroptosis in human and murine cells. Cleaved by inflammatory caspases, GSDMD inserts its N‐terminal domain (GSDMDNterm) into cellular membranes and assembles large oligomeric complexes permeabilizing the membrane. So far, the mechanisms of GSDMDNterm insertion, oligomerization, and pore formation are poorly understood. Here, we apply high‐resolution (≤ 2 nm) atomic force microscopy (AFM) to describe how GSDMDNterm inserts and assembles in membranes. We observe GSDMDNterm inserting into a variety of lipid compositions, among which phosphatidylinositide (PI(4,5)P2) increases and cholesterol reduces insertion. Once inserted, GSDMDNterm assembles arc‐, slit‐, and ring‐shaped oligomers, each of which being able to form transmembrane pores. This assembly and pore formation process is independent on whether GSDMD has been cleaved by caspase‐1, caspase‐4, or caspase‐5. Using time‐lapse AFM, we monitor how GSDMDNterm assembles into arc‐shaped oligomers that can transform into larger slit‐shaped and finally into stable ring‐shaped oligomers. Our observations translate into a mechanistic model of GSDMDNterm transmembrane pore assembly, which is likely shared within the gasdermin protein family.

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Real-time visualization of perforin nanopore assembly

Cited by 93 publications

References 48 publications

How the Membrane Attack Complex Damages the Bacterial Cell Envelope and Kills Gram‐Negative Bacteria

How the Membrane Attack Complex Damages the Bacterial Cell Envelope and Kills Gram‐Negative Bacteria

Mechanisms of natural killer cell-mediated cellular cytotoxicity

Mechanism of membrane pore formation by human gasdermin‐D

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