The Functional Basis for Hemophagocytic Lymphohistiocytosis in a Patient with Co-inherited Missense Mutations in the Perforin (PFN1) Gene

About 30% of cases of the autosomal recessive immunodeficiency disorder hemophagocytic lymphohistiocytosis are believed to be caused by inactivating mutations of the perforin gene. We expressed perforin in rat basophil leukemia cells to define the basis of perforin dysfunction associated with two mutations, R225W and G429E, inherited by a compound heterozygote patient. Whereas RBL cells expressing wild-type perforin (67 kD) efficiently killed Jurkat target cells to which they were conjugated, the substitution to tryptophan at position 225 resulted in expression of a truncated (∼45 kD) form of the protein, complete loss of cytotoxicity, and failure to traffic to rat basophil leukemia secretory granules. By contrast, G429E perforin was correctly processed, stored, and released, but the rat basophil leukemia cells possessed reduced cytotoxicity. The defective function of G429E perforin mapped downstream of exocytosis and was due to its reduced ability to bind lipid membranes in a calcium-dependent manner. This study elucidates the cellular basis for perforin dysfunctions in hemophagocytic lymphohistiocytosis and provides the means for studying structure–function relationships for lymphocyte perforin

Molecular study of the perforin gene in familial hematological malignancies

Perforin gene (PRF1) mutations have been identified in some patients diagnosed with the familial form of hemophagocytic lymphohistiocytosis (HLH) and in patients with lymphoma. The aim of the present study was to determine whether patients with a familial aggregation of hematological malignancies harbor germline perforin gene mutations. For this purpose, 81 unrelated families from Tunisia and France with aggregated hematological malignancies were investigated. The variants detected in the PRF1 coding region amounted to 3.7% (3/81). Two of the three variants identified were previously described: the p.Ala91Val pathogenic mutation and the p.Asn252Ser polymorphism. A new p.Ala 211Val missense substitution was identified in two related Tunisian patients. In order to assess the pathogenicity of this new variation, bioinformatic tools were used to predict its effects on the perforin protein structure and at the mRNA level. The segregation of the mutant allele was studied in the family of interest and a control population was screened. The fact that this variant was not found to occur in 200 control chromosomes suggests that it may be pathogenic. However, overexpression of mutated PRF1 in rat basophilic leukemia cells did not affect the lytic function of perforin differently from the wild type protein

Conformational changes during pore formation by the perforin-related protein pleurotolysin

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ~70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function

Immune surveillance of lymphoma in humans?

Perforin mutations may predispose some individuals to lymphoma, supporting the concept that key immune molecules may act as extrinsic tumor suppressors

Perforin-mediated target-cell death and immune homeostasis

The granule exocytosis pathway of cytotoxic lymphocytes is crucial for immune surveillance and homeostasis. The trafficking of granule components, including the membrane-disruptive protein perforin, to the immunological synapse leads to the delivery of granule proteases (granzymes) into the target cell and its destruction through apoptosis. Several independent molecular abnormalities associated with defects of either granule trafficking or perforin function can cause cytotoxic lymphocyte dysfunction. In humans, inherited perforin mutations result in severe immune dysregulation that manifests as familial haemophagocytic lymphohistiocytosis. This Review describes recent progress in defining the structure, function, biochemistry and cell biology of perforin

Copper stimulates trafficking of a distinct pool of the Menkes copper ATPase (ATP7A) to the plasma membrane and diverts it into a rapid recycling pool.

MNK (Menkes copper-translocating P-type ATPase, or the Menkes protein; ATP7A) plays a key role in regulating copper homoeostasis in humans. MNK has been shown to have a dual role in the cell: it delivers copper to cuproenzymes in the Golgi compartment and effluxes excess copper from the cell. These roles can be achieved through copper-regulated trafficking of MNK. It has previously been shown to undergo trafficking from the trans -Golgi network to the plasma membrane in response to elevated copper concentrations, and to be endocytosed from the plasma membrane to the trans -Golgi network upon the removal of elevated copper. However, the fundamental question as to whether copper influences trafficking of MNK to or from the plasma membrane remained unanswered. In this study we utilized various methods of cell-surface biotinylation to attempt to resolve this issue. These studies suggest that copper induces trafficking of MNK to the plasma membrane but does not affect its rate of internalization from the plasma membrane. We also found that only a specific pool of MNK can traffic to the plasma membrane in response to elevated copper. Significantly, copper appeared to divert MNK into a fast-recycling pool and prevented it from recycling to the Golgi compartment, thus maintaining a high level of MNK in the proximity of the plasma membrane. These findings shed new light on the cell biology of MNK and the mechanism of copper homoeostasis in general

Apoptosis induced by the lymphocyte effector molecule perforin

Recent progress has been made in discovering structure/function relationships of the cytotoxic lymphocyte pore-forming protein - perforin - and its role in immune regulation. Long recognized as a key component of the granule exocytosis mechanism of lymphocyte-mediated apoptosis and in host defence against a variety of pathogens and tumors, more recent attention has focused upon mutations of the perforin gene in some patients with hemophagocytic lymphohistiocytosis. This clinical picture has prompted further work to uncover the molecular structure of perforin and to define its precise role in immune regulation and immunopathology

Cytotoxic granules house potent proapoptotic toxins critical for antiviral responses and immune homeostasis

General Introduction Cytotoxic lymphocytes and apoptosis The immune system of high-order organisms is a highly specialized compartment that eliminates transformed cells and cells infected with viruses or bacteria through a controlled process of cell-mediated cytotoxicity. The immune cells responsible for mediating cell death are collectively called cytotoxic lymphocytes (CLs) and are made up of natural killer (NK) cells and cytotoxic T lymphocytes (CTL). CLs are distinguished primarily by their respective mechanism of antigen recognition. NK cells form part of the innate immune response, a generalized first line of defense. NK cells are generally CD3aeCD56+lymphocytes that recognize and respond to abnormal cells through an imbalance of facilitatory and inhibitory receptors (Bottino et al., 2004; Moretta et al., 2004). CTLs formpart of the adaptive immune response, a more specific response that is generated subsequent to and as a consequence of the innate response. These cells use their clonotypic T-cell receptors (TcRs) to recognize a peptide antigen presented on the major histocompatability complex (MHC) proteins on the surface of the target cell. CTLs can be identified on the basis of expression of CD3 and CD8 (CD3+CD8+) on their cell surface. In addition, some CD4+T cells (typically T-helper cells) can have limited cytotoxic capacity