Perforin is a 67KD protein released by most cytotoxic lymphocytes (including NK cells, CTLs, NKT cells and γδ+T cells) It has the ability in forming pores in the cell membrane of transformed host cells or virus-infected cells. It collaborate with a family of structurally related serine proteases (granzymes). Granzymes are the mediators of many cell-death pathways, they are packaged in the granules, but they all seem to depend on perforin for their effective delivery.
They are secreted by exocytosis and together induce apoptosis of the target cell. Apoptosis could be caspases and non-caspases mediate.
CHEMICAL STRUCTURE AND IMAGES
Protein Aminoacid Percentage:
Secondary structure and tridimensional structure:
Perforin is made up of different functional domains. An amino-terminal membrane attack complex perforin-like (MACPF)/cholesterol dependent cytolysin (CDC) domain is followed by an epidermal growth factor (EGF) domain. These two moieties, together with the extreme carboxy-terminal sequence, form a central shelf-like structure. Finally, the A C-terminal C2 domain mediates the initial Ca2+-dependent membrane binding.
MACPF perforin domain (Red, the central β -sheet). A central feature of the perforin MACPF domain is a bent and twisted four-stranded β-sheet flanked by two clusters of α-helices, termed CH1 and CH2 (orange). CH1 is loosely held between the central sheet (the remainder of this domain is blue).
EGF-like domain (green): this is made up by the C-terminal α-helix of the MACPF domain and the disulphide constrained EGF-like fold that follows the MACPF domain. At the end of the EGF domain, a conserved disulphide bond (C407–C241) is formed with the first helix of CH2. The EGF domain is associated with the C-terminal sequence (residues 524–551 in magenta).
Together, these structures form a continuous shelf on which the MACPF sits, and beneath which it hangs the C2 domain (figure c). Several FHL-associated mutations map to this region.
C2 domain. (yellow) The C2 domain mediates calcium-dependent binding to lipid membranes, which happens in neutral PH conditions. In fact, premature activation of perforin is prevented by both low calcium concentration and acidic PH. The C2 fold can coordinate up to four calcium atoms (site I to IV in the figure). Law et al observed that one Ca2+atom is canonically coordinated in the site I position between the CBR1–3 regions of the C2 domain. A second Ca2+ atom is coordinated outside of CBR3 by D490, but this site is not conserved among species. Therefore, this last site it is probably not essential for perforin function.
A subsequent conformational change leads to the insertion of the beta-hairpin structures into the membrane, and finally to pore formation. The pore is formed by transmembrane beta-strands.
Law et al work showed by electron microscopy that the EGFR region is extremely flexible , and it is extremely important in the conformational change of the CH1 and CH2 structures. The C2 domain interacts with the upper leaflet of the membrane bilayer, (as we can see in figure-B),and the CH2 region results to be the trans-membrane domain.
Post-traslational modifications: N-glicosilation
General information about the mouse gene:
General information about the human gene:
Perforin plays a key role in secretory granule-dependent cell death, which is of crucial importance in the defense against both virus-infections and the growth of neoplastic cells. As perforin plays an important role in killing cells that are recognized as non-self by the immune system, it is also involved in transplant rejection, and in some autoimmune diseases. Perforin insert itself into the membrane of target cells while in its calcium-bound form. If can then oligomerize, finally forming large pores. Perforin promotes cytolysis and apoptosis of target cells by facilitating the uptake of cytotoxic granzymes. It belongs to the complement C6/C7/C8/C9 family.
- Monomer when in its soluble form,
- Oligomer during pore formation.
- Cytoplasmic granule lumen of cytotoxic T- linfocites/ Natural killer cells/ granulocites
- Cell membrane;
- Multi-pass membrane protein
- Endosome lumen
Note: Perforin is stored in cytoplasmic granules of cytolytic T-lymphocytes and secreted into the cleft between T-lymphocyte and target cell. It inserts itself into the cell membrane of target cells and forms pores. Membrane insertion and pore formation require a major conformational change. Perforin may be taken up via endocytosis, which involves clathrin-coated vesicles, and initial accumulation in large early endosomes.
The molecular basis for the relative resistance of CTLs and NK cells to perforin-mediated lysis has been explained recently. Cathepsin B, a lysosomal protease that is also present in cytolytic granules, becomes sequestered on the effector-cell membrane after degranulation, and can inactivate perforin molecules that diffuse back to the effector cell.
- Ubiquitary expression
- expression increase in blood, lymphoid organs, bone marrow and thymus
- expression decrease in case of cancer
- protein binding;
- calcium ion binding;
- wide pore channel activity.
Studies in transgenic knoc-out mice indicate that perforin is vital for cytotoxic effector function of immune cells. Perforin-deficient mice are abnormally susceptible to many viruses, and other intracellular pathogens. These mice fail to reject some types of allografts, and they are highly susceptible to spontaneous B-cell lymphomas while ageing.
- circadian rhythm;
- response to ethanol;
- defense response to tumor cell;
- cellular defense response;
- ADCC mechanism (antibody mediated).
- immune response to tumor cell;
- defense response to virus;
- protein homooligomerization
See page URL
Involvement in desease:
- Perforin is also important for the control of intracellular bacterial infections, such as Mycobacterium tuberculosis.
- Perforin plays an important role in NK cell-mediated suppression of tumor initiation and metastasis. IL-12 and IL-21 activate NK cells to destroy a variety of tumors in a perforin-dependent manner.
- NK cells also mediate the acute rejection of incompatible bone-marrow cell (BMC) grafts. The perforin and FasL pathways have been shown to have an important role in NK-cell-mediated rejection of both allogeneic and MHC class-I-deficient BMCs .
Es. See pathway involved in graft versus host desease
- Role in B cell homeostasis and T cell memory
- Role for perforin in immunoregulation in conditions of immune-system perturbation caused by microbial infection, autoimmunity, or loss of other cell-death.
Es. see pathway of autoimmune tiroiditis
SYNTHESIS AND TURNOVER:
Image of PFP-1 gene and splicing:
The role of perforin and granzymes in the pathophysiology of many diseases is usually dissected using gene-targeted mice that lack these molecules and in humans who have perforin mutations. Also, the involvement of death-receptor-mediated killing has been assessed in humans and mice with mutations in FasL (gld mice) or Fas (lpr mice), and in mice deficient in tumour-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL).
Perforin Synthesis and invesciculation:
CD8+ T cells follow a program of proliferation and differentiation into CTL armed with effector functions that facilitate pathogen clearance or containment. Viral infections and tumors stimulate memory T cells to proliferate, secrete cytokines, and exhibit increased cytolytic activity against virus-infected cells. The dominant mechanism that CTL use to kill virus-infected cells is the granule exocytosis pathway, with perforin and two serine proteases, granzyme A (GrA)3 and granzyme B (GrB), forming the major lines of defense.
Perforin and granzymes are stoared in Cytotoxic granules, specialized secretory lysosomes that are present only in cells with cytolytic capability Their expression is induced by immunization. But The finding that circulating VV-specific CD8+ T cells express GrA and GrB more frequently than perforin further suggests that these effector molecules are differentially regulated. Rock et al suggest that Tight control of perforin expression is not unexpected given the cytotoxic potential of this molecule. Expression regulation of this protein is now partially not yet known.
It seemed that expression mechanism of cytotoxic molecules (expecialy for granzymes) is inhibited by some viruses.
Interestingly, poxviruses have evolved various mechanisms to interfere with the activity of host immune responses and possess large numbers of viral homologues of cytokines, chemokines, and their receptors. Poxviruses also encode proteins related to the serpin family of proteinase inhibitors, termed SPI-1, -2, and -3 . The cysteine response modifier, SPI-2, has been shown to inhibit serine and cysteine proteases, including in vitro inhibition of GrB. However, cell death induced by CTL occurs in the presence of complete caspase blockade, using peptide caspase inhibitors, including crmA. This finding indicates that CTL also activate pathways causing caspase-independent cell death. Thus, although the specific cellular pathways responsible for control of poxvirus infections remain to be elucidated.
Cytotoxic T lymphocytes (CTLs) and NK cells employ similar target cell killing methods, involving the polarized release of perforin and granzymes from lytic granules toward the target cell.
- the formation of an immunological synapse (IS) at the site of cell–cell contact,This synapse is organized into a central supramolecular activation complex (cSMAC) (yellow) containing signaling molecules and the TCR, surrounded by a peripheral integrin-rich ring, the pSMAC (red).
- the movement of cytotoxic granules toward the microtubule organizing center (MTOC)
- the reorientation of the MTOC toward the target cell
- Release of cytolytic proteins is confined to a secretory cleft that forms in an area of otherwise tight membrane contact and provides a confined space in which perforin and other lytic proteins can be kept concentrated for attack on the target. The mechanisms that trigger cleft formation are not understood. However, Mutations in FHL tried to explain exocitosis induction (not well known): it seems to be involved Munc13-4, which interacts with Rab27a in cytotoxic cells. This molecular interaction is probably involved in coordination of the final step of exocytosis, between the docking and priming of lytic granules.
Previous studies of CTLs have revealed that not only does the centrosome direct polarisation of the secretory lysosomes to the immunological synapse, but the Golgi complex and the recycling endosomes also polarise to the point of centrosomal contact with the plasma membrane. In this way, centrosome positioning directs membrane traffic, and the synapse becomes a focal point for both exocytosis and endocytosis. The immunological synapse bears a number of striking similarities to primary cilia where the centrosome also migrates right up to the plasma membrane and forms the site of initiation for cilia formation. Curiously, the Golgi complex and recycling endosomes also polarise to the site of cilia formation and to the flagella pocket, and the plasma membrane around the cilia also becomes specialised in exocytosis and endocytosis.
While the role of the centrosome in directing polarised secretion has been well documented in CTLs, little is known about whether the centrosome contacts the plasma membrane in other cytolytic cell types of the immune system with perforin-containing secretory lysosomes.
This is the hypothesis they’re propose:
- Cytotoxic molecules are recovered by the coated vesicles of an immunological synapse after NK cells have secreted their toxic granules.
- At the end of a classical endocytosis of the cytotoxic molecules, they are stored in new vesicles (that constitute the ‘reserve pool’) that have transformed from late endosomes which have, in turn, matured from early endosomes formed from a clathrin-coated pit.
- Nascent secretory lysosomes in NK cells are transformed from their “reserve pool” when target-cell recognition has triggered rapid biogenesis and the sensitization of secretory lysosomes.
To understand Perforin activity, are important KO mice models, but in order to understand human phisiology is important familiar hemophagocytic lymphohistiocytosis: see FLH desease