Methods for investigating STAT3 regulation of lysosomal function in mammary epithelial cells.

The transcription factor STAT3 is activated by multiple cytokines and other extrinsic factors. It plays a key role in immune and inflammatory responses and, when dysregulated, in tumourigenesis. STAT3 is also an indispensable mediator of the cell death process that occurs during post-lactational regression of the mammary gland, one of the most dramatic examples of physiological cell death in adult mammals. During this involution of the gland, STAT3 powerfully enhances the lysosomal system to efficiently remove superfluous milk-producing mammary epithelial cells via a lysosomal-mediated programmed cell death pathway. The lysosome is a membrane-enclosed  cytoplasmic organelle that digests and recycles cellular waste, with an important role as a signalling centre that monitors cellular metabolism. Here, we describe key strategies for investigating the role of STAT3 in regulating lysosomal function using a mammary epithelial cell culture model system. These include protocols for lysosome enrichment and enzyme activity assays, in addition to microscopic analyses of the vesicular compartment in cell lines. Collectively, these approaches provide the tools to investigate multiple aspects of lysosome biogenesis and function, and to define both direct and indirect roles for STAT3.

Stat3-mediated alterations in lysosomal membrane protein composition.

Lysosome function is essential in cellular homeostasis. In addition to its recycling role, the lysosome has recently been recognized as a cellular signaling hub. We have shown in mammary epithelial cells, both in vivo and in vitro, that signal transducer and activator of transcription 3 (Stat3) modulates lysosome biogenesis and can promote the release of lysosomal proteases that culminates in cell death. To further investigate the impact of Stat3 on lysosomal function, we conducted a proteomic screen of changes in lysosomal membrane protein components induced by Stat3 using an iron nanoparticle enrichment strategy. Our results show that Stat3 activation not only elevates the levels of known membrane proteins but results in the appearance of unexpected factors, including cell surface proteins such as annexins and flotillins. These data suggest that Stat3 may coordinately regulate endocytosis, intracellular trafficking, and lysosome biogenesis to drive lysosome-mediated cell death in mammary epithelial cells. The methodologies described in this study also provide significant improvements to current techniques used for the purification and analysis of the lysosomal proteome.

Analysis of Perforin Assembly by Quartz Crystal Microbalance Reveals a Role for Cholesterol and Calcium-independent Membrane Binding.

Perforin is an essential component in the cytotoxic lymphocyte-mediated cell death pathway. The traditional view holds that perforin monomers assemble into pores in the target cell membrane via a calcium-dependent process and facilitate translocation of cytotoxic proteases into the cytoplasm to induce apoptosis. Although many studies have examined the structure and role of perforin, the mechanics of pore assembly and granzyme delivery remain unclear. Here we have employed quartz crystal microbalance with dissipation monitoring (QCM-D) to investigate binding and assembly of perforin on lipid membranes, and show that perforin monomers bind to the membrane in a cooperative manner. We also found that cholesterol influences perforin binding and activity on intact cells and model membranes. Finally, contrary to current thinking, perforin efficiently binds membranes in the absence of calcium. When calcium is added to perforin already on the membrane, the QCM-D response changes significantly, indicating that perforin becomes membranolytic only after calcium binding.

Assembly of streptolysin O pores assessed by quartz crystal microbalance and atomic force microscopy provides evidence for the formation of anchored but incomplete oligomers.

Streptolysin O (SLO) is a bacterial pore forming protein that is part of the cholesterol dependent cytolysin (CDC) family. We have used quartz crystal microbalance with dissipation monitoring (QCM-D) to examine SLO membrane binding and pore formation. In this system, SLO binds tightly to cholesterol-containing membranes, and assembles into partial and complete pores confirmed by atomic force microscopy. SLO binds to the lipid bilayer at a single rate consistent with the Langmuir isotherm model of adsorption. Changes in dissipation illustrate that SLO alters the viscoelastic properties of the bilayer during pore formation, but there is no loss of material from the bilayer as reported for small membrane-penetrating peptides. SLO mutants were used to further dissect the assembly and insertion processes by QCM-D. This shows the signature of SLO in QCM-D changes when pore formation is inhibited, and that bound and inserted SLO forms can be distinguished. Furthermore a pre-pore locked SLO mutant binds reversibly to lipid, suggesting that the partially complete wtSLO forms observed by AFM are anchored to the membrane.

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, Ca(2+)-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 perforin pore facilitates the delivery of cationic cargos.

Cytotoxic lymphocytes eliminate virally infected or neoplastic cells through the action of cytotoxic proteases (granzymes). The pore-forming protein perforin is essential for delivery of granzymes into the cytoplasm of target cells; however the mechanism of this delivery is incompletely understood. Perforin contains a membrane attack complex/perforin (MACPF) domain and oligomerizes to form an aqueous pore in the plasma membrane; therefore the simplest (and best supported) model suggests that granzymes passively diffuse through the perforin pore into the cytoplasm of the target cell. Here we demonstrate that perforin preferentially delivers cationic molecules while anionic and neutral cargoes are delivered inefficiently. Furthermore, another distantly related pore-forming MACPF protein, pleurotolysin (from the oyster mushroom), also favors the delivery of cationic molecules, and efficiently delivers human granzyme B. We propose that this facilitated diffusion is due to conserved features of oligomerized MACPF proteins, which may include an anionic lumen.

Intercellular communication via the endo-lysosomal system: translocation of granzymes through membrane barriers.

Cytotoxic lymphocytes (CLs) are responsible for the clearance of virally infected or neoplastic cells. CLs possess specialised lysosome-related organelles called granules which contain the granzyme family of serine proteases and perforin. Granzymes may induce apoptosis in the target cell when delivered by the pore forming protein, perforin. Here we follow the perforin-granzyme pathway from synthesis and storage in the granule, to exocytosis and finally delivery into the target cell. This review focuses on the controversial subject of perforin-mediated translocation of granzymes into the target cell cytoplasm. It remains unclear whether this occurs at the cell surface with granzymes moving through a perforin pore in the plasma membrane, or if it involves internalisation of perforin and granzymes and subsequent release from an endocytic compartment. The latter mechanism would represent an example of cross talk between the endo-lysosomal pathways of individual cells. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome.

Perforin evolved from a gene duplication of MPEG1, followed by a complex pattern of gene gain and loss within Euteleostomi.

BACKGROUND: The pore-forming protein perforin is central to the granule-exocytosis pathway used by cytotoxic lymphocytes to kill abnormal cells. Although this mechanism of killing is conserved in bony vertebrates, cytotoxic cells are present in other chordates and invertebrates, and their cytotoxic mechanism has not been elucidated. In order to understand the evolution of this pathway, here we characterize the origins and evolution of perforin. RESULTS: We identified orthologs and homologs of human perforin in all but one species analysed from Euteleostomi, and present evidence for an earlier ortholog in Gnathostomata but not in more primitive chordates. In placental mammals perforin is a single copy gene, but there are multiple perforin genes in all lineages predating marsupials, except birds. Our comparisons of these many-to-one homologs of human perforin show that they mainly arose from lineage-specific gene duplications in multiple taxa, suggesting acquisition of new roles or different modes of regulation. We also present evidence that perforin arose from duplication of the ancient MPEG1 gene, and that it shares a common ancestor with the functionally related complement proteins. CONCLUSIONS: The evolution of perforin in vertebrates involved a complex pattern of gene, as well as intron, gain and loss. The primordial perforin gene arose at least 500 million years ago, at around the time that the major histocompatibility complex-T cell receptor antigen recognition system was established. As it is absent from primitive chordates and invertebrates, cytotoxic cells from these lineages must possess a different effector molecule or cytotoxic mechanism.

Cathepsin H is an additional convertase of pro-granzyme B.

The serine protease granzyme B (GrB) is the most potent proapoptotic cytotoxin of the granule exocytosis pathway of cytotoxic lymphocytes. GrB is synthesized as a zymogen (proGrB) and activated in cytotoxic granules by the lysosomal cysteine protease cathepsin C (CatC) which removes the N-terminal dipeptide Gly-Glu. It has been shown recently that mice lacking CatC nonetheless express significant residual GrB activity, indicating the presence of additional proGrB convertases. Here, we describe an assay to assess activation of proGrB and show that the amino-peptidase cathepsin H (CatH) has proGrB convertase activity in vitro, whereas dipeptidylpeptidase II does not. We generated mice lacking both CatC and CatH expression (CatCH(-/-)) and found that their lymphocytes have reduced convertase activity compared with those from CatC-deficient mice. Despite this, cytotoxic lymphocytes from CatCH(-/-) mice retain cytotoxic activity and some residual GrB activity. We conclude that CatH can act as an additional proGrB convertase and that other protease/s (apart from dipeptidylpeptidase II) must also possess convertase activity. This indicates a great deal of functional redundancy in GrB maturation, which would prevent pathogen-mediated immune suppression by via convertase inhibition.

Highly efficient multiplex genetic engineering of porcine primary fetal fibroblasts.

BACKGROUND: Genetically engineered porcine donors are a potential solution for the shortage of human organs for transplantation. Incompatibilities between humans and porcine donors are largely due to carbohydrate xenoantigens on the surface of porcine cells, provoking an immune response which leads to xenograft rejection. MATERIALS AND METHODS: Multiplex genetic knockout of GGTA1, ß4GalNT2, and CMAH is predicted to increase the rate of xenograft survival, as described previously for GGTA1. In this study, the clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 system was used to target genes relevant to xenotransplantation, and a method for highly efficient editing of multiple genes in primary porcine fibroblasts was described. RESULTS: Editing efficiencies greater than 85% were achieved for knockout of GGTA1, ß4GalNT2, and CMAH. CONCLUSION: The high-efficiency protocol presented here reduces scale and cost while accelerating the production of genetically engineered primary porcine fibroblast cells for in vitro studies and the production of animal models.

Cloning and characterising an unusual perforin from chicken (Gallus gallus).

In mammals the 67 kDa pore-forming protein perforin is essential to the granule exocytosis pathway used by cytotoxic lymphocytes to eliminate virally infected and malignant cells. There is indirect evidence that this pathway exists in lower vertebrates such as teleost fish and birds, although in genome databases for the chicken and other bird species the perforin gene is incomplete and no full length expressed sequence tag has been reported. We present here the full gene and transcript sequence of chicken perforin. The inferred protein product contains an extended C-terminus that is at least 90 amino acids longer than any mammalian perforin, which is also evident in partial genomic sequences from other birds. To determine whether this extension is present in the translated protein, we raised two polyclonal antisera. The antisera identified a protein of just less than 80 kDa in both transfected COS-1 cells and concanavalin A stimulated chicken splenocytes, indicating that the extended C-terminus is present in the mature protein. Our findings confirm that perforin exists in birds, and show that it is considerably longer than perforin of non-avian vertebrates.