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1.
Blood ; 139(12): 1833-1849, 2022 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-35081253

RESUMO

Niemann-Pick disease type C1 (NP-C1) is a rare lysosomal storage disorder resulting from mutations in an endolysosomal cholesterol transporter, NPC1. Despite typically presenting with pronounced neurological manifestations, NP-C1 also resembles long-term congenital immunodeficiencies that arise from impairment of cytotoxic T lymphocyte (CTL) effector function. CTLs kill their targets through exocytosis of the contents of lysosome-like secretory cytotoxic granules (CGs) that store and ultimately release the essential pore-forming protein perforin and proapoptotic serine proteases, granzymes, into the synapse formed between the CTL and target cell. We discovered that NPC1 deficiency increases CG lipid burden, impairs autophagic flux through stalled trafficking of the transcription factor EB (TFEB), and dramatically reduces CTL cytotoxicity. Using a variety of immunological and cell biological techniques, we found that the cytotoxic defect arises specifically from impaired perforin pore formation. We demonstrated defects of CTL function of varying severity in patients with NP-C1, with the greatest losses of function associated with the most florid and/or earliest disease presentations. Remarkably, perforin function and CTL cytotoxicity were restored in vitro by promoting lipid clearance with therapeutic 2-hydroxypropyl-ß-cyclodextrin; however, restoration of autophagy through TFEB overexpression was ineffective. Overall, our study revealed that NPC1 deficiency has a deleterious impact on CTL (but not natural killer cell) cytotoxicity that, in the long term, may predispose patients with NP-C1 to atypical infections and impaired immune surveillance more generally.


Assuntos
Doença de Niemann-Pick Tipo A , Doença de Niemann-Pick Tipo C , Colesterol/metabolismo , Granzimas , Humanos , Doença de Niemann-Pick Tipo C/metabolismo , Perforina/genética , Linfócitos T Citotóxicos/metabolismo
2.
Immunity ; 34(6): 879-92, 2011 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-21658975

RESUMO

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.


Assuntos
Movimento Celular , Exocitose , Perforina/imunologia , Polissacarídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Autólise/imunologia , Linhagem Celular , Retículo Endoplasmático/imunologia , Glicosilação , Humanos , Camundongos , Camundongos Knockout , Mutação , Perforina/deficiência , Ratos
3.
EMBO Rep ; 18(10): 1775-1785, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28808112

RESUMO

Perforin is a highly cytotoxic pore-forming protein essential for immune surveillance by cytotoxic lymphocytes. Prior to delivery to target cells by exocytosis, perforin is stored in acidic secretory granules where it remains functionally inert. However, how cytotoxic lymphocytes remain protected from their own perforin prior to its export to secretory granules, particularly in the Ca2+-rich endoplasmic reticulum, remains unknown. Here, we show that N-linked glycosylation of the perforin C-terminus at Asn549 within the endoplasmic reticulum inhibits oligomerisation of perforin monomers and thus protects the host cell from premature pore formation. Subsequent removal of this glycan occurs through proteolytic processing of the C-terminus within secretory granules and is imperative for perforin activation prior to secretion. Despite evolutionary conservation of the C-terminus, we found that processing is carried out by multiple proteases, which we attribute to the unstructured and exposed nature of the region. In sum, our studies reveal a post-translational regulatory mechanism essential for maintaining perforin in an inactive state until its secretion from the inhibitory acidic environment of the secretory granule.


Assuntos
Sinapses Imunológicas , Perforina/química , Perforina/metabolismo , Animais , Grânulos Citoplasmáticos/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilação , Humanos , Interleucina-2/imunologia , Células Matadoras Naturais/imunologia , Glicoproteínas de Membrana , Camundongos , Perforina/genética , Processamento de Proteína Pós-Traducional , Proteólise
4.
J Virol ; 89(15): 7991-8002, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26018154

RESUMO

UNLABELLED: There are 3 to 4 million new hepatitis C virus (HCV) infections annually around the world, but no vaccine is available. Robust T-cell mediated responses are necessary for effective clearance of the virus, and DNA vaccines result in a cell-mediated bias. Adjuvants are often required for effective vaccination, but during natural lytic viral infections damage-associated molecular patterns (DAMPs) are released, which act as natural adjuvants. Hence, a vaccine that induces cell necrosis and releases DAMPs will result in cell-mediated immunity (CMI), similar to that resulting from natural lytic viral infection. We have generated a DNA vaccine with the ability to elicit strong CMI against the HCV nonstructural (NS) proteins (3, 4A, 4B, and 5B) by encoding a cytolytic protein, perforin (PRF), and the antigens on a single plasmid. We examined the efficacy of the vaccines in C57BL/6 mice, as determined by gamma interferon enzyme-linked immunosorbent spot assay, cell proliferation studies, and intracellular cytokine production. Initially, we showed that encoding the NS4A protein in a vaccine which encoded only NS3 reduced the immunogenicity of NS3, whereas including PRF increased NS3 immunogenicity. In contrast, the inclusion of NS4A increased the immunogenicity of the NS3, NS4B, andNS5B proteins, when encoded in a DNA vaccine that also encoded PRF. Finally, vaccines that also encoded PRF elicited similar levels of CMI against each protein after vaccination with DNA encoding NS3, NS4A, NS4B, and NS5B compared to mice vaccinated with DNA encoding only NS3 or NS4B/5B. Thus, we have developed a promising "multiantigen" vaccine that elicits robust CMI. IMPORTANCE: Since their development, vaccines have reduced the global burden of disease. One strategy for vaccine development is to use commercially viable DNA technology, which has the potential to generate robust immune responses. Hepatitis C virus causes chronic liver infection and is a leading cause of liver cancer. To date, no vaccine is currently available, and treatment is costly and often results in side effects, limiting the number of patients who are treated. Despite recent advances in treatment, prevention remains the key to efficient control and elimination of this virus. Here, we describe a novel DNA vaccine against hepatitis C virus that is capable of inducing robust cell-mediated immune responses in mice and is a promising vaccine candidate for humans.


Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Linfócitos T/imunologia , Vacinas de DNA/imunologia , Vacinas contra Hepatite Viral/imunologia , Animais , Anticorpos Antivirais/imunologia , Feminino , Hepacivirus/genética , Hepatite C/virologia , Humanos , Imunidade Celular , Imunização , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas contra Hepatite Viral/administração & dosagem , Vacinas contra Hepatite Viral/genética , Proteínas não Estruturais Virais/administração & dosagem , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia
5.
Crit Rev Immunol ; 35(4): 325-47, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26757394

RESUMO

A synapse is a specialized structure that forms when the plasma membrane of two cells come into close contact to facilitate communication and signaling. Cells of the immune system form 'immunological' synapses that have an ordered structure and are essential for immune cell activation, function and homeostasis. Optimal synapse formation is not only critical for the generation of effective immunity against pathogens but is also essential for immune surveillance against cancer and for the prevention of immune disorders. Not surprisingly, defective synapse formation can therefore have severe consequences for human health, culminating in poor immune function leading to immunodeficiency disease or failure to detect and control infected or cancerous cells. Here, we discuss the immunological synapse formed by cytotoxic lymphocytes in both immunodeficiency diseases and anticancer immunity and touch on novel therapies that may alter or enhance synapse formation.


Assuntos
Síndromes de Imunodeficiência/imunologia , Sinapses Imunológicas/metabolismo , Imunoterapia , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos de Neoplasias/imunologia , Homeostase , Humanos , Imunidade Celular , Síndromes de Imunodeficiência/terapia , Vigilância Imunológica , Neoplasias/terapia
6.
J Immunol ; 193(11): 5744-50, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25348626

RESUMO

Cytotoxic lymphocytes destroy pathogen-infected and transformed cells through the cytotoxic granule exocytosis death pathway, which is dependent on the delivery of proapoptotic granzymes into the target cell cytosol by the pore-forming protein, perforin. Despite the importance of mouse models in understanding the role of cytotoxic lymphocytes in immune-mediated disease and their role in cancer immune surveillance, no reliable intracellular detection method exists for mouse perforin. Consequently, rapid, flow-based assessment of cytotoxic potential has been problematic, and complex assays of function are generally required. In this study, we have developed a novel method for detecting perforin in primary mouse cytotoxic T lymphocytes by immunofluorescence and flow cytometry. We used this new technique to validate perforin colocalization with granzyme B in cytotoxic granules polarized to the immunological synapse, and to assess the expression of perforin in cytotoxic T lymphocytes at various stages of activation. The sensitivity of this technique also allowed us to distinguish perforin levels in Prf1(+/+) and Prf1(+/-) mice. This new methodology will have broad applications and contribute to advances within the fields of lymphocyte biology, infectious disease, and cancer.


Assuntos
Granzimas/metabolismo , Sinapses Imunológicas/metabolismo , Espaço Intracelular/metabolismo , Perforina/metabolismo , Linfócitos T Citotóxicos/imunologia , Animais , Separação Celular , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Perforina/genética , Transporte Proteico
7.
Immunol Cell Biol ; 93(6): 575-80, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25776844

RESUMO

The production and delivery of functional perforin (PRF; PRF1 gene) by cytotoxic lymphocytes maintains immune homeostasis and tumour immune surveillance. In humans, inheritance of the common PRF1 polymorphism, p.A91V, (c.272C>T) found in 8-9% of the Caucasian population, with another mutated allele resulting in reduced PRF function or trafficking, has been shown to result in hyperinflammatory diseases and/or haematological cancers. In this study, we sought to investigate the function of p.A91V on a wild-type (WT) perforin background. We first developed an assay that distinguishes the relative levels of transcription of individual PRF1 alleles, including p.A91V. The p.A91V allele was seen to be expressed at similar levels as the WT allele in primary human natural killer (NK) cells, ruling out that allelic expression imbalance influenced their function. We then demonstrated that the p.A91V mutation results in protein misfolding and an appreciable reduction in NK-cell cytotoxicity in healthy carriers of p.A91V. We propose that this level of cytotoxic dysfunction may readily account for the predisposition to immune-mediated disease in individuals homozygous for p.A91V. Also, the fact that monoallelic mutations of PRF1 decrease NK-cell cytotoxicity should be considered in individuals presenting with the manifestations of immune deficiency states that impinge on NK-cell cytotoxicity.


Assuntos
Citotoxicidade Imunológica/genética , Heterozigoto , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Mutação , Perforina/genética , Alelos , Substituição de Aminoácidos , Códon , Expressão Gênica , Genes Dominantes , Voluntários Saudáveis , Humanos , Perforina/química , Polimorfismo de Nucleotídeo Único , Dobramento de Proteína , RNA Mensageiro/genética
8.
Trends Immunol ; 33(8): 406-12, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22608996

RESUMO

Considerable progress has been made in understanding how cytotoxic lymphocytes use the highly toxic pore-forming protein perforin to eliminate dangerous cells, while remaining refractory to lysis. At least two mechanisms jointly preserve the killer cell: the C-terminal residues of perforin dictate its rapid export from the endoplasmic reticulum (ER), whose milieu otherwise favours pore formation; perforin is then stored in secretory granules whose acidity prevent its oligomerisation. Following exocytosis, perforin delivers the proapoptotic protease, granzyme B, into the target cell by disrupting its plasma membrane. Although the precise mechanism of perforin/granzyme synergy remains controversial, the recently defined crystal structure of the perforin monomer and cryo-electron microscopy (EM) of the entire pore suggest that passive transmembrane granzyme diffusion is the dominant proapoptotic mechanism.


Assuntos
Perforina/imunologia , Animais , Morte Celular , Humanos , Perforina/química , Perforina/metabolismo , Fagocitose , Transporte Proteico , Linfócitos T Citotóxicos/imunologia
9.
J Immunol ; 191(5): 2328-34, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23885110

RESUMO

The effective engagement of cytotoxic lymphocytes (CLs) with their target cells is essential for the removal of virus-infected and malignant cells from the body. The spatiotemporal properties that define CL engagement and killing of target cells remain largely uncharacterized due to a lack of biological reporters. We have used a novel live cell microscopy technique to visualize the engagement of primary human and mouse CL with their targets and the subsequent delivery of the lethal hit. Extensive quantitative real-time analysis of individual effector-target cell conjugates demonstrated that a single effector calcium flux event was sufficient for the degranulation of human CLs, resulting in the breach of the target cell membrane by perforin within 65-100 s. In contrast, mouse CLs demonstrated distinct calcium signaling profiles leading to degranulation: whereas mouse NKs required a single calcium flux event, CD8(+) T cells typically required several calcium flux events before perforin delivery. Irrespective of their signaling profile, every target cell that was damaged by perforin died by apoptosis. To our knowledge, we demonstrate for the first time that perforin pore delivery is unidirectional, occurring exclusively on the target cell membrane, but sparing the killer cell. Despite this, the CTL membrane was not intrinsically perforin resistant, as intact CTLs presented as targets to effector CTLs were capable of being killed by perforin-dependent mechanisms. Our results highlight the remarkable efficiency and specificity of perforin pore delivery by CLs.


Assuntos
Sinapses Imunológicas/imunologia , Células Matadoras Naturais/imunologia , Microscopia Confocal/métodos , Perforina/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Degranulação Celular/imunologia , Células Cultivadas , Humanos , Sinapses Imunológicas/metabolismo , Células Matadoras Naturais/metabolismo , Camundongos , Linfócitos T Citotóxicos/metabolismo
10.
Blood ; 119(7): 1713-6, 2012 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-22186995

RESUMO

Mutations in the perforin gene (PRF1) are a common cause of the fatal immune dysregulation disorder, familial hemophagocytic lymphohistiocytosis (type 2 FHL, FHL2). Here we report a female infant born with biallelic PRF1 mutations: a novel substitution, D49N, and a previously identified in-frame deletion, K285del. We assessed the effects of each mutation on the cytotoxicity of human NK cells in which the expression of endogenous perforin was ablated with miR30-based short hairpin (sh) RNAs. Both mutations were detrimental for function, thereby explaining the clinically severe presentation and rapidly fatal outcome. We demonstrate that D49N exerts its deleterious effect by generating an additional (third) N-linked glycosylation site, resulting in protein misfolding and degradation in the killer cell. Our data provide a rationale for treating some cases of type 2 familial hemophagocytic lymphohistiocytosis, based on the pharmacologic inhibition or modification of glycosylation.


Assuntos
Doenças do Sistema Imunitário/genética , Linfócitos/metabolismo , Mutação de Sentido Incorreto/fisiologia , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Sequência de Bases , Células Cultivadas , Análise Mutacional de DNA , Evolução Fatal , Feminino , Glicosilação , Células HEK293 , Humanos , Doenças do Sistema Imunitário/imunologia , Doenças do Sistema Imunitário/patologia , Recém-Nascido , Linfócitos/imunologia , Linfócitos/patologia , Insuficiência de Múltiplos Órgãos/genética , Insuficiência de Múltiplos Órgãos/imunologia , Linhagem , Perforina , Proteínas Citotóxicas Formadoras de Poros/fisiologia
11.
Biochem J ; 456(3): 323-35, 2013 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24070258

RESUMO

Following its secretion from cytotoxic lymphocytes into the immune synapse, perforin binds to target cell membranes through its Ca(2+)-dependent C2 domain. Membrane-bound perforin then forms pores that allow passage of pro-apoptopic granzymes into the target cell. In the present study, structural and biochemical studies reveal that Ca(2+) binding triggers a conformational change in the C2 domain that permits four key hydrophobic residues to interact with the plasma membrane. However, in contrast with previous suggestions, these movements and membrane binding do not trigger irreversible conformational changes in the pore-forming MACPF (membrane attack complex/perforin-like) domain, indicating that subsequent monomer-monomer interactions at the membrane surface are required for perforin pore formation.


Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Fosfolipídeos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Animais , Cálcio/química , Membrana Celular/química , Membrana Celular/genética , Humanos , Células Jurkat , Células K562 , Camundongos , Camundongos Knockout , Fosfolipídeos/química , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Estrutura Terciária de Proteína , Ratos
12.
Cell Death Differ ; 2024 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-39261596

RESUMO

Tumour immune evasion presents a significant challenge to the effectiveness of cancer immunotherapies. Recent advances in high-throughput screening techniques have uncovered that loss of antigen presentation and cytokine signalling pathways are central mechanisms by which tumours evade T cell immunity. To uncover additional vulnerabilities in tumour cells beyond the well-recognized antigen presentation pathway, we conducted a genome-wide CRISPR/Cas9 screen to identify genes that mediate resistance to chimeric-antigen receptor (CAR)-T cells, which function independently of classical antigen presentation. Our study revealed that loss of core-binding factor subunit beta (CBFß) enhances tumour cell resistance to T cell killing, mediated through T cell-derived TNF. Mechanistically, RNA-sequencing and elemental analyses revealed that deletion of CBFß disrupts numerous pathways including those involved in zinc homoeostasis. Moreover, we demonstrated that modulation of cellular zinc, achieved by supplementation or chelation, significantly altered tumour cell susceptibility to TNF by regulating the levels of inhibitor of apoptosis proteins. Consistent with this, treatment of tumour cells with a membrane-permeable zinc chelator had no impact on tumour cell viability alone, but significantly increased tumour cell lysis by CD8+ T cells in a TNF-dependent but perforin-independent manner. These results underscore the crucial role of intracellular zinc in regulating tumour cell susceptibility to T cell-mediated killing, revealing a novel vulnerability in tumour cells that might be exploited for the development of future cancer immunotherapeutics.

13.
Front Immunol ; 13: 931820, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36618385

RESUMO

When killing through the granule exocytosis pathway, cytotoxic lymphocytes release key effector molecules into the immune synapse, perforin and granzymes, to initiate target cell killing. The pore-forming perforin is essential for the function of cytotoxic lymphocytes, as its pores disrupt the target cell membrane and allow diffusion of pro-apoptotic serine proteases, granzyme, into the target cell, where they initiate various cell death cascades. Unlike human perforin, the detection of its murine counterpart in a live cell system has been problematic due its relatively low expression level and the lack of sensitive antibodies. The lack of a suitable methodology to visualise murine perforin secretion into the synapse hinders the study of the cytotoxic lymphocyte secretory machinery in murine models of human disease. Here, we describe a novel recombinant technology, whereby a short ALFA-tag sequence has been fused with the amino-terminus of a mature murine perforin, and this allowed its detection by the highly specific FluoTag®-X2 anti-ALFA nanobodies using both Total Internal Reflection Fluorescence (TIRF) microscopy of an artificial synapse, and confocal microscopy of the physiological immune synapse with a target cell. This methodology can have broad application in the field of cytotoxic lymphocyte biology and for the many models of human disease.


Assuntos
Sinapses Imunológicas , Perforina , Linfócitos T Citotóxicos , Animais , Camundongos , Morte Celular , Membrana Celular/metabolismo , Granzimas/metabolismo , Perforina/metabolismo
14.
Front Immunol ; 13: 931630, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35874669

RESUMO

Cytotoxic lymphocytes are essential for anti-tumor immunity, and for effective responses to cancer immunotherapy. Natural killer cell granule protein 7 (NKG7) is expressed at high levels in cytotoxic lymphocytes infiltrating tumors from patients treated with immunotherapy, but until recently, the role of this protein in cytotoxic lymphocyte function was largely unknown. Unexpectedly, we found that highly CD8+ T cell-immunogenic murine colon carcinoma (MC38-OVA) tumors grew at an equal rate in Nkg7+/+ and Nkg7-/- littermate mice, suggesting NKG7 may not be necessary for effective CD8+ T cell anti-tumor activity. Mechanistically, we found that deletion of NKG7 reduces the ability of CD8+ T cells to degranulate and kill target cells in vitro. However, as a result of inefficient cytotoxic activity, NKG7 deficient T cells form a prolonged immune synapse with tumor cells, resulting in increased secretion of inflammatory cytokines, including tumor necrosis factor alpha (TNF). By deleting the TNF receptor, TNFR1, from MC38-OVA tumors, we demonstrate that this hyper-secretion of TNF compensates for reduced synapse-mediated cytotoxic activity against MC38-OVA tumors in vivo, via increased TNF-mediated tumor cell death. Taken together, our results demonstrate that NKG7 enhances CD8+ T cell immune synapse efficiency, which may serve as a mechanism to accelerate direct cytotoxicity and limit potentially harmful inflammatory responses.


Assuntos
Linfócitos T CD8-Positivos , Sinapses Imunológicas , Proteínas de Membrana , Neoplasias , Animais , Imunoterapia/métodos , Inflamação/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Neoplasias/terapia , Fator de Necrose Tumoral alfa/metabolismo
15.
Cell Death Differ ; 25(8): 1517-1529, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29416110

RESUMO

The pore forming, Ca2+-dependent protein, perforin, is essential for the function of cytotoxic lymphocytes, which are at the frontline of immune defence against pathogens and cancer. Perforin is a glycoprotein stored in the secretory granules prior to release into the immune synapse. Congenital perforin deficiency causes fatal immune dysregulation, and is associated with various haematological malignancies. At least 50% of pathological missense mutations in perforin result in protein misfolding and retention in the endoplasmic reticulum. However, the regulation of perforin proteostasis remains unexplored. Using a variety of biochemical assays that assess protein stability and acquisition of complex glycosylation, we demonstrated that the binding of Ca2+ to the C2 domain stabilises perforin and regulates its export from the endoplasmic reticulum to the secretory granules. As perforin is a thermo-labile protein, we hypothesised that by altering its C2 domain it may be possible to improve protein stability. On the basis of the X-ray crystal structure of the perforin C2 domain, we designed a mutation (T431D) in the Ca2+ binding loop. Mutant perforin displayed markedly enhanced thermal stability and lytic function, despite its trafficking from the endoplasmic reticulum remaining unchanged. Furthermore, by introducing the T431D mutation into A90V perforin, a pathogenic mutation, which results in protein misfolding, we corrected the A90V folding defect and completely restored perforin's cytotoxic function. These results revealed an unexpected role for the Ca2+-dependent C2 domain in maintaining perforin proteostasis and demonstrated the possibility of designing perforin with supra-physiological cytotoxic function through stabilisation of the C2 domain.


Assuntos
Apoptose , Perforina/metabolismo , Animais , Cálcio/química , Cálcio/metabolismo , Linhagem Celular Tumoral , Cristalografia por Raios X , Retículo Endoplasmático/metabolismo , Camundongos , Mutagênese Sítio-Dirigida , Perforina/genética , Domínios Proteicos , Dobramento de Proteína , Estabilidade Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Temperatura de Transição
16.
Cell Signal ; 33: 86-97, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28193539

RESUMO

α-lactalbumin is a protein of dual function found in milk of most mammals. α-lactalbumin binds ß-1,4-galactosyltransferase to form the regulatory subunit for lactose synthesis and has also been shown to cause cell death. This study shows, for the first time, that α-lactalbumin isolated in a rare 28kDa dimeric form induces cell death, while 14kDa monomeric α-lactalbumin is inactive. In contrast to the casein derived and chemically induced α-lactalbumin variants, MAL and HAMLET/BAMLET, the effects of 28kDa α-lactalbumin are calcium independent and, unlike MAL and HAMLET, 28kDa α-lactalbumin dimer causes cell death of primary mammary cells and a variety of immortalised cell lines, which are committed to cell death pathways within 1-4h of exposure. Microarray analysis confirmed that cell death was the result of an apoptotic response. Functional assays determined that the mechanism by which 28kDa α-lactalbumin kills cells involved inhibition of histone deacetylase activity mediated by NF-kB. We also show that 28kDa α-lactalbumin occurs naturally in the milk of cows, goats and sheep, is low in concentration during mid-lactation, but accumulates during milk stasis, consistent with a role in involution.


Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Histona Desacetilases/metabolismo , Lactalbumina/farmacologia , Regulação para Cima/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular Transformada , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Cromatografia em Gel , Cabras , Humanos , Lactalbumina/química , Células MCF-7 , Camundongos , Peso Molecular , NF-kappa B/metabolismo , Multimerização Proteica , Ovinos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
17.
Nat Nanotechnol ; 12(5): 467-473, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28166206

RESUMO

Perforin is a key protein of the vertebrate immune system. Secreted by cytotoxic lymphocytes as soluble monomers, perforin can self-assemble into oligomeric pores of 10-20 nm inner diameter in the membranes of virus-infected and cancerous cells. These large pores facilitate the entry of pro-apoptotic granzymes, thereby rapidly killing the target cell. To elucidate the pathways of perforin pore assembly, we carried out real-time atomic force microscopy and electron microscopy studies. Our experiments reveal that the pore assembly proceeds via a membrane-bound prepore intermediate state, typically consisting of up to approximately eight loosely but irreversibly assembled monomeric subunits. These short oligomers convert to more closely packed membrane nanopore assemblies, which can subsequently recruit additional prepore oligomers to grow the pore size.


Assuntos
Membrana Eritrocítica , Nanoporos/ultraestrutura , Proteínas Citotóxicas Formadoras de Poros , Animais , Membrana Eritrocítica/química , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/ultraestrutura , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Ovinos
18.
FEBS J ; 283(5): 947-61, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26756195

RESUMO

The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+) CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means.


Assuntos
Células Matadoras Naturais/metabolismo , Proteoglicanas/metabolismo , Vesículas Secretórias/metabolismo , Linfócitos T Citotóxicos/citologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Antígeno CD11b/metabolismo , Linfócitos T CD8-Positivos/citologia , Separação Celular , Células Cultivadas , Cruzamentos Genéticos , Feminino , Citometria de Fluxo , Granzimas/metabolismo , Masculino , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Proteólise , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismo
19.
J Mol Endocrinol ; 41(3): 103-16, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18535120

RESUMO

Mammary explants can be hormonally stimulated to mimic the biochemical changes that occur during lactogenesis. Previous studies using mammary explants concluded that the addition of exogenous macromolecules were required for mammary epithelial cells to remain viable in culture. The present study examines the survival of mammary explants from the dairy cow using milk protein gene expression as a functional marker of lactation and cell viability. Mammary explants cultured from late pregnant cows mimicked lactogenesis and showed significantly elevated milk protein gene expression after 3 days of culture with lactogenic hormones. The subsequent removal of exogenous hormones from the media for 10 days resulted in the down-regulation of milk protein genes. During this time, the mammary explants remained hormone responsive, the alveolar architecture was maintained and the expression of milk protein genes was re-induced after a second challenge with lactogenic hormones. We report that a population of bovine mammary epithelial cells have an intrinsic capacity to remain viable and hormone responsive for extended periods in chemically defined media without any exogenous macromolecules. In addition, we found mammary explant viability was dependent on de novo protein and RNA synthesis. Global functional microarray analysis showed that differential expression of genes involved in energy production, immune responses, oxidative stress and apoptosis signalling might contribute to cell survival. As the decline in milk production in dairy cattle after peak lactation results in considerable economic loss, the identification of novel survival genes may be used as genetic markers for breeding programmes to improve lactational persistency in dairy cows.


Assuntos
Hormônios/deficiência , Glândulas Mamárias Animais/citologia , Análise de Variância , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Glândulas Mamárias Animais/efeitos dos fármacos , Proteínas do Leite/genética , Proteínas do Leite/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Biossíntese de Proteínas/efeitos dos fármacos , RNA/biossíntese , Ovinos
20.
J Endocrinol ; 196(3): 483-96, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18310444

RESUMO

Hormonal stimulation of mammary explants mimics many of the biochemical changes observed during lactogenesis. Previous studies using eutherian species conclude that mammary explants require addition of exogenous macromolecules to remain hormone responsive in culture. The present study examines the survival of mammary explants from the wallaby and mouse using milk protein gene expression as a functional marker of lactation and cell viability. Mammary explants from pregnant tammars and mice showed that milk protein gene expression was significantly elevated after 3 days of culture with lactogenic hormones. The subsequent removal of exogenous hormones from the media for 10 days resulted in the down-regulation of milk protein genes. Surprisingly, mammary explants remained hormone responsive and expression of milk protein genes was re-induced after a second challenge with lactogenic hormones. Furthermore, the alveolar architecture was maintained. Global functional microarray analysis showed that classic involution markers were not differentially expressed, although two stress-induced survival genes were significantly up-regulated. We report that a population of mammary epithelial cells have an intrinsic capacity to remain viable and hormone responsive for extended periods in chemically defined media without any exogenous macromolecules. We propose that the mammary explant culture model uncouples the first phase of involution, as milk accumulation that normally provides involution stimuli is absent in this culture model allowing a population of cells to survive.


Assuntos
Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Lactação/fisiologia , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Caseínas/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Lactoglobulinas/genética , Macropodidae , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Prolactina/farmacologia , Prolactina/fisiologia
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