Annette Ciccone

The structural basis for membrane binding and pore formation by lymphocyte perforin.

Natural killer cells and cytotoxic T lymphocytes accomplish the critically important function of killing virus-infected and neoplastic cells. They do this by releasing the pore-forming protein perforin and granzyme proteases from cytoplasmic granules into the cleft formed between the abutting killer and target cell membranes. Perforin, a 67-kilodalton multidomain protein, oligomerizes to form pores that deliver the pro-apoptopic granzymes into the cytosol of the target cell. The importance of perforin is highlighted by the fatal consequences of congenital perforin deficiency, with more than 50 different perforin mutations linked to familial haemophagocytic lymphohistiocytosis (type 2 FHL). Here we elucidate the mechanism of perforin pore formation by determining the X-ray crystal structure of monomeric murine perforin, together with a cryo-electron microscopy reconstruction of the entire perforin pore. Perforin is a thin ‘key-shaped’ molecule, comprising an amino-terminal membrane attack complex perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain followed by an epidermal growth factor (EGF) domain that, together with the extreme carboxy-terminal sequence, forms a central shelf-like structure. A C-terminal C2 domain mediates initial, Ca2+-dependent membrane binding. Most unexpectedly, however, electron microscopy reveals that the orientation of the perforin MACPF domain in the pore is inside-out relative to the subunit arrangement in CDCs. These data reveal remarkable flexibility in the mechanism of action of the conserved MACPF/CDC fold and provide new insights into how related immune defence molecules such as complement proteins assemble into pores.

The Molecular Basis for Perforin Oligomerization and Transmembrane Pore Assembly

Perforin, a pore-forming protein secreted by cytotoxic lymphocytes, is indispensable for destroying virus-infected cells and for maintaining immune homeostasis. Perforin polymerizes into transmembrane channels that inflict osmotic stress and facilitate target cell uptake of proapoptotic granzymes. Despite this, the mechanism through which perforin monomers self-associate remains unknown. Our current study establishes the molecular basis for perforin oligomerization and pore assembly. We show that after calcium-dependent membrane binding, direct ionic attraction between the opposite faces of adjacent perforin monomers was necessary for pore formation. By using mutagenesis, we identified the opposing charges on residues Arg213 (positive) and Glu343 (negative) to be critical for intermolecular interaction. Specifically, disrupting this interaction had no effect on perforin synthesis, folding, or trafficking in the killer cell, but caused a marked kinetic defect of oligomerization at the target cell membrane, severely disrupting lysis and granzyme B-induced apoptosis. Our study provides important insights into perforins mechanism of action.

Residual active granzyme B in cathepsin C-null lymphocytes is sufficient for perforin-dependent target cell apoptosis.

Cathepsin C activates serine proteases expressed in hematopoietic cells by cleaving an N-terminal dipeptide from the proenzyme upon granule packaging. The lymphocytes of cathepsin C–null mice are therefore proposed to totally lack granzyme B activity and perforin-dependent cytotoxicity. Surprisingly, we show, using live cell microscopy and other methodologies, that cells targeted by allogenic CD8+ cytotoxic T lymphocyte (CTL) raised in cathepsin C–null mice die through perforin-dependent apoptosis indistinguishable from that induced by wild-type CTL. The cathepsin C–null CTL expressed reduced but still appreciable granzyme B activity, but minimal granzyme A activity. Also, in contrast to mice with inactivation of both their granzyme A/B genes, cathepsin C deficiency did not confer susceptibility to ectromelia virus infection in vivo. Overall, our results indicate that although cathepsin C clearly generates the majority of granzyme B activity, some is still generated in its absence, pointing to alternative mechanisms for granzyme B processing and activation. Cathepsin C deficiency also results in considerably milder immune deficiency than perforin or granzyme A/B deficiency.

Residual active granzyme B in cathepsin C–null lymphocytes is sufficient for perforin-dependent target cell apoptosis

Sutton et al. 2007. J. Cell Biol. doi:10.1083/jcb.200609077 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1083%252Fjcb.200609077%26rft_id%253Dinfo%253Apmid%252F17283185%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%

Protection from Endogenous Perforin: Glycans and the C Terminus Regulate Exocytic Trafficking in Cytotoxic Lymphocytes

Cytotoxic lymphocyte-mediated apoptosis is dependent on the delivery of perforin to secretory granules and its ability to form calcium-dependent pores in the target cell after granule exocytosis. It is unclear how cytotoxic lymphocytes synthesize and store perforin without incurring damage or death. We discovered that the extreme C terminus of perforin was essential for rapid trafficking from the endoplasmic reticulum to the Golgi compartment. Substitution of the C-terminal tryptophan residue resulted in retention of perforin in the ER followed by calcium-dependent toxic activity that eliminated host cells. We also found that N-linked glycosylation of perforin was critical for transport from the Golgi to secretory granules. Overall, an intact C terminus and N-linked glycosylation provide accurate and efficient export of perforin from the endoplasmic reticulum to the secretory granules and are critical for cytotoxic lymphocyte survival.

Cytotoxic T Lymphocytes from Cathepsin B-deficient Mice Survive Normally in Vitro and in Vivo after Encountering and Killing Target Cells

The lysosomal protease cathepsin B has been proposed to protect cytotoxic T lymphocytes from the membrane-disruptive effects of perforin secreted during the execution phase of target cell death. Accordingly, cathepsin B that translocates to the lymphocyte surface upon degranulation has been postulated to cleave and inactivate perforin molecules that diffuse back to the killer cell. We have found that recombinant perforin is cleaved inefficiently by cathepsin B and shows no significant reduction in its lytic activity following co-incubation. Furthermore, purified CD8+ cytotoxic T lymphocytes of cathepsin B-null gene-targeted mice were able to induce normal death of target cells both in vitro and in vivo and to survive the encounter with target cells as efficiently as cathepsin B-expressing killer cells. We conclude that cathepsin B is not essential for protection of cytotoxic lymphocytes from the toxic effects of their secreted perforin.

Blocking granule-mediated death by primary human NK cells requires both protection of mitochondria and inhibition of caspase activity

Human GraB (hGraB) preferentially induces apoptosis via Bcl-2-regulated mitochondrial damage but can also directly cleave caspases and caspase substrates in cell-free systems. How hGraB kills cells when it is delivered by cytotoxic lymphocytes (CL) and the contribution of hGraB to CL-induced death is still not clear. We show that primary human natural killer (hNK) cells, which specifically used hGraB to induce target cell death, were able to induce apoptosis of cells whose mitochondria were protected by Bcl-2. Purified hGraB also induced apoptosis of Bcl-2-overexpressing targets but only when delivered at 5- to 10-fold the concentration required to kill cells expressing endogenous Bcl-2. Caspases were critical in this process as inhibition of caspase activity permitted clonogenic survival of Bcl-2-overexpressing cells treated with hGraB or hNK cells but did not protect cells that only expressed endogenous Bcl-2. Our data therefore show that hGraB triggers caspase activation via mitochondria-dependent and mitochondria-independent mechanisms that are activated in a hierarchical manner, and that the combined effects of Bcl-2 and direct caspase inhibition can block cell death induced by hGraB and primary hNK cells.

Dihydrofuro[3,4-c]pyridinones as inhibitors of the cytolytic effects of the pore-forming glycoprotein perforin

Dihydrofuro[3,4-c]pyridinones are the first class of small molecules reported to inhibit the cytolytic effects of the lymphocyte toxin perforin. A lead structure was identified from a high throughput screen, and a series of analogues were designed and prepared to explore structure-activity relationships around the core bicyclic thioxofuropyridinone and pendant furan ring. This resulted in the identification of a submicromolar inhibitor of the perforin-induced lysis of Jurkat T-lymphoma cells.

Inhibition of the cellular function of perforin by 1-amino-2,4-dicyanopyrido[1,2-a]benzimidazoles.

A high throughput screen showed the ability of a 1-amino-2,4-dicyanopyrido[1,2-a]benzimidazole analogue to directly inhibit the lytic activity of the pore-forming protein perforin. A series of analogues were prepared to study structure-activity relationships (SAR) for the this activity, either directly added to cells or released in situ by KHYG-1 NK cells, at non-toxic concentrations. These studies showed that the pyridobenzimidazole moiety was required for effective activity, with strongly basic centres disfavoured. This class of compounds was relatively unaffected by the addition of serum, which was not the case for a previous class of direct inhibitors.

Exploration of a series of 5-arylidene-2-thioxoimidazolidin-4-ones as inhibitors of the cytolytic protein perforin.

A series of novel 5-arylidene-2-thioxoimidazolidin-4-ones were investigated as inhibitors of the lymphocyte-expressed pore-forming protein perforin. Structure–activity relationships were explored through variation of an isoindolinone or 3,4-dihydroisoquinolinone subunit on a fixed 2-thioxoimidazolidin-4-one/thiophene core. The ability of the resulting compounds to inhibit the lytic activity of both isolated perforin protein and perforin delivered in situ by natural killer cells was determined. A number of compounds showed excellent activity at concentrations that were nontoxic to the killer cells, and several were a significant improvement on previous classes of inhibitors, being substantially more potent and soluble. Representative examples showed rapid and reversible binding to immobilized mouse perforin at low concentrations (≤2.5 μM) by surface plasmon resonance and prevented formation of perforin pores in target cells despite effective target cell engagement, as determined by calcium influx studies. Mouse PK studies of two analogues showed T1/2 values of 1.1–1.2 h (dose of 5 mg/kg iv) and MTDs of 60–80 mg/kg (ip).