RESUMO
Natural killer cells are innate lymphocytes with the ability to lyse tumour cells depending on the balance of their activating and inhibiting receptors. Growing numbers of clinical trials show promising results of NK cell-based immunotherapies. Unlike T cells, NK cells can lyse tumour cells independent of antigen presentation, based simply on their activation and inhibition receptors. Various strategies to improve NK cell-based therapies are being developed, all with one goal: to shift the balance to activation. In this review, we discuss the current understanding of ways NK cells can lyse tumour cells and all the inhibitory signals stopping their cytotoxic potential.
Assuntos
Imunoterapia Adotiva , Neoplasias , Humanos , Imunoterapia , Imunoterapia Adotiva/métodos , Células Matadoras Naturais , Neoplasias/terapia , Linfócitos TRESUMO
RNA variants that emerge from editing and alternative splicing form important regulatory stages in protein signalling. In this report, we apply an integrated DNA and RNA variant detection workbench to define the range of RNA variants that deviate from the reference genome in a human melanoma cell model. The RNA variants can be grouped into (i) classic ADAR-like or APOBEC-like RNA editing events and (ii) multiple-nucleotide variants (MNVs) including three and six base pair in-frame non-canonical unmapped exons. We focus on validating representative genes of these classes. First, clustered non-synonymous RNA edits (A-I) in the CDK13 gene were validated by Sanger sequencing to confirm the integrity of the RNA variant detection workbench. Second, a highly conserved RNA variant in the MAP4K5 gene was detected that results most likely from the splicing of a non-canonical three-base exon. The two RNA variants produced from the MAP4K5 locus deviate from the genomic reference sequence and produce V569E or V569del isoform variants. Low doses of splicing inhibitors demonstrated that the MAP4K5-V569E variant emerges from an SF3B1-dependent splicing event. Mass spectrometry of the recombinant SBP-tagged MAP4K5V569E and MAP4K5V569del proteins pull-downs in transfected cell systems was used to identify the protein-protein interactions of these two MAP4K5 isoforms and propose possible functions. Together these data highlight the utility of this integrated DNA and RNA variant detection platform to detect RNA variants in cancer cells and support future analysis of RNA variant detection in cancer tissue.
Assuntos
Processamento Alternativo , DNA/genética , Éxons , Proteínas Serina-Treonina Quinases/genética , RNA/genética , Humanos , Isoenzimas , Edição de RNARESUMO
RNA editing is one of the most prevalent and abundant forms of post-transcriptional RNA modification observed in normal physiological processes and often aberrant in diseases including cancer. RNA editing changes the sequences of mRNAs, making them different from the source DNA sequence. Edited mRNAs can produce editing-recoded protein isoforms that are functionally different from the corresponding genome-encoded protein isoforms. The major type of RNA editing in mammals occurs by enzymatic deamination of adenosine to inosine (A-to-I) within double-stranded RNAs (dsRNAs) or hairpins in pre-mRNA transcripts. Enzymes that catalyse these processes belong to the adenosine deaminase acting on RNA (ADAR) family. The vast majority of knowledge on the RNA editing landscape relevant to human disease has been acquired using in vitro cancer cell culture models. The limitation of such in vitro models, however, is that the physiological or disease relevance of results obtained is not necessarily obvious. In this review we focus on discussing in vivo occurring RNA editing events that have been identified in human cancer tissue using samples surgically resected or clinically retrieved from patients. We discuss how RNA editing events occurring in tumours in vivo can identify pathological signalling mechanisms relevant to human cancer physiology which is linked to the different stages of cancer progression including initiation, promotion, survival, proliferation, immune escape and metastasis.
Assuntos
Adenosina/genética , Carcinogênese/patologia , Inosina/genética , Neoplasias/patologia , Edição de RNA , RNA de Cadeia Dupla/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Carcinogênese/genética , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
A diverse family of cytoskeletal dynein motors powers various cellular transport systems, including axonemal dyneins generating the force for ciliary and flagellar beating essential to movement of extracellular fluids and of cells through fluid. Multisubunit outer dynein arm (ODA) motor complexes, produced and preassembled in the cytosol, are transported to the ciliary or flagellar compartment and anchored into the axonemal microtubular scaffold via the ODA docking complex (ODA-DC) system. In humans, defects in ODA assembly are the major cause of primary ciliary dyskinesia (PCD), an inherited disorder of ciliary and flagellar dysmotility characterized by chronic upper and lower respiratory infections and defects in laterality. Here, by combined high-throughput mapping and sequencing, we identified CCDC151 loss-of-function mutations in five affected individuals from three independent families whose cilia showed a complete loss of ODAs and severely impaired ciliary beating. Consistent with the laterality defects observed in these individuals, we found Ccdc151 expressed in vertebrate left-right organizers. Homozygous zebrafish ccdc151(ts272a) and mouse Ccdc151(Snbl) mutants display a spectrum of situs defects associated with complex heart defects. We demonstrate that CCDC151 encodes an axonemal coiled coil protein, mutations in which abolish assembly of CCDC151 into respiratory cilia and cause a failure in axonemal assembly of the ODA component DNAH5 and the ODA-DC-associated components CCDC114 and ARMC4. CCDC151-deficient zebrafish, planaria, and mice also display ciliary dysmotility accompanied by ODA loss. Furthermore, CCDC151 coimmunoprecipitates CCDC114 and thus appears to be a highly evolutionarily conserved ODA-DC-related protein involved in mediating assembly of both ODAs and their axonemal docking machinery onto ciliary microtubules.
Assuntos
Dineínas do Axonema/metabolismo , Cílios/patologia , Síndrome de Kartagener/genética , Proteínas Associadas aos Microtúbulos/fisiologia , Mutação/genética , Animais , Dineínas do Axonema/genética , Axonema/genética , Células Cultivadas , Cílios/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Exoma/genética , Feminino , Imunofluorescência , Humanos , Immunoblotting , Imunoprecipitação , Hibridização In Situ , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Linhagem , Fenótipo , Técnicas do Sistema de Duplo-Híbrido , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismoRESUMO
Defects of motile cilia cause primary ciliary dyskinesia (PCD), characterized by recurrent respiratory infections and male infertility. Using whole-exome resequencing and high-throughput mutation analysis, we identified recessive biallelic mutations in ZMYND10 in 14 families and mutations in the recently identified LRRC6 in 13 families. We show that ZMYND10 and LRRC6 interact and that certain ZMYND10 and LRRC6 mutations abrogate the interaction between the LRRC6 CS domain and the ZMYND10 C-terminal domain. Additionally, ZMYND10 and LRRC6 colocalize with the centriole markers SAS6 and PCM1. Mutations in ZMYND10 result in the absence of the axonemal protein components DNAH5 and DNALI1 from respiratory cilia. Animal models support the association between ZMYND10 and human PCD, given that zmynd10 knockdown in zebrafish caused ciliary paralysis leading to cystic kidneys and otolith defects and that knockdown in Xenopus interfered with ciliogenesis. Our findings suggest that a cytoplasmic protein complex containing ZMYND10 and LRRC6 is necessary for motile ciliary function.
Assuntos
Cílios/genética , Síndrome de Kartagener/genética , Proteínas/genética , Sistema Respiratório/metabolismo , Proteínas Supressoras de Tumor/genética , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Dineínas do Axonema/genética , Dineínas do Axonema/metabolismo , Biomarcadores/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cílios/metabolismo , Cílios/patologia , Proteínas do Citoesqueleto , Exoma , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patologia , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Linhagem , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas/metabolismo , Ratos , Sistema Respiratório/patologia , Proteínas Supressoras de Tumor/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Primary ciliary dyskinesia (PCD) is a rare genetically heterogeneous disorder caused by the abnormal structure and/or function of motile cilia. The PCD diagnosis is challenging and requires a well-described clinical phenotype combined with the identification of abnormalities in ciliary ultrastructure and/or beating pattern as well as the recognition of genetic cause of the disease. Regarding the pace of identification of PCD-related genes, a rapid acceleration during the last 2-3â years is notable. This is the result of new technologies, such as whole-exome sequencing, that have been recently applied in genetic research. To date, PCD-causative mutations in 29 genes are known and the number of causative genes is bound to rise. Even though the genetic causes of approximately one-third of PCD cases still remain to be found, the current knowledge can already be used to create new, accurate genetic tests for PCD that can accelerate the correct diagnosis and reduce the proportion of unexplained cases. This review aims to present the latest data on the relations between ciliary structure aberrations and their genetic basis.
Assuntos
Dineínas do Axonema/genética , Axonema/ultraestrutura , Cílios/fisiologia , Testes Genéticos/métodos , Síndrome de Kartagener/diagnóstico , Síndrome de Kartagener/genética , Dineínas do Axonema/metabolismo , Cílios/ultraestrutura , Humanos , Síndrome de Kartagener/patologia , Microscopia Eletrônica de TransmissãoRESUMO
The SARS-CoV-2 pandemic was defined by the emergence of new variants formed through virus mutation originating from random errors not corrected by viral proofreading and/or the host antiviral response introducing mutations into the viral genome. While sequencing information hints at cellular RNA editing pathways playing a role in viral evolution, here, we use an in vitro human cell infection model to assess RNA mutation types in two SARS-CoV-2 strains representing the original and the alpha variants. The variants showed both different cellular responses and mutation patterns with alpha showing higher mutation frequency with most substitutions observed being C-U, indicating an important role for apolipoprotein B mRNA editing catalytic polypeptide-like editing. Knockdown of select APOBEC3s through RNAi increased virus production in the original virus, but not in alpha. Overall, these data suggest a deaminase-independent anti-viral function of APOBECs in SARS-CoV-2 while the C-U editing itself might function to enhance genetic diversity enabling evolutionary adaptation.
RESUMO
Introduction: Preeclampsia is responsible for more than 70 000 and 500 000 maternal and fetal deaths, respectively each year. Incomplete remodelling of the spiral arteries in placenta is the most accepted theory of preeclampsia pathogenesis. However, the process is complexed with immunological background, as pregnancy resembles allograft transplantation. Fetus expresses human leukocyte antigens (HLA) inherited from both parents, thus is semiallogeneic to the maternal immune system. Therefore, induction of fetal tolerance is crucial for physiological outcome of pregnancy. Noteworthy, the immunogenicity of discordant HLA antigens is determined by functional epitopes called eplets, which are continuous and discontinuous short sequences of amino acids. This way various HLA molecules may express the same eplet and some HLA incompatibilities can be more immunogenic due to different eplet combination. Therefore, we hypothesized that maternal- fetal HLA incompatibility may be involved in the pathogenesis of gestational hypertension and its progression to preeclampsia. We also aimed to test if particular maternal-fetal eplet mismatches are more prone for induction of anti- fetal HLA antibodies in gestational hypertension and preeclampsia. Methods: High resolution next-generation sequencing of HLA-A, -B, -C, -DQB1 and -DRB1 antigens was performed in mothers and children from physiological pregnancies (12 pairs) and from pregnancies complicated with gestational hypertension (22 pairs) and preeclampsia (27 pairs). In the next step HLA eplet identification and analysis of HLA eplet incompatibilities was performed with in silico approach HLAMatchmaker algorithm. Simultaneously maternal sera were screened for anti-fetal HLA class I, class II and anti-MICA antibodies with Luminex, and data were analyzed with HLA-Fusion software. Results: We observed that high HLA-C, -B, and DQB1 maternal-fetal eplet compatibility was associated with severe preeclampsia (PE) manifestation. Both quantity and quality of HLA epletmismatches affected the severity of PE. Mismatches in HLA-B eplets: 65QIA+76ESN, 70IAO, 180E, HLA-C eplets: 193PL3, 267QE, and HLA-DRB1 eplet: 16Y were associated with a mild outcome of preeclampsia if the complication occurred. Conclusions: High HLA-C, HLA-DQB1 and HLA-B eplet compatibility between mother and child is associated with severe manifestation of preeclampsia. Both quantity and quality of maternal-fetal HLA eplet mismatches affects severity of preeclampsia.
Assuntos
Hipertensão Induzida pela Gravidez , Pré-Eclâmpsia , Gravidez , Feminino , Criança , Humanos , Antígenos HLA-C , Antígenos HLA , Feto , Antígenos HLA-BRESUMO
Autoimmune disease results from the immune response against self-antigens, while cancer develops when the immune system does not respond to malignant cells. Thus, for years, autoimmunity and cancer have been considered as two separate fields of research that do not have a lot in common. However, the discovery of immune checkpoints and the development of anti-cancer drugs targeting PD-1 (programmed cell death receptor 1) and CTLA-4 (cytotoxic T lymphocyte antigen 4) pathways proved that studying autoimmune diseases can be extremely helpful in the development of novel anti-cancer drugs. Therefore, autoimmunity and cancer seem to be just two sides of the same coin. In the current review, we broadly discuss how various regulatory cell populations, effector molecules, genetic predisposition, and environmental factors contribute to the loss of self-tolerance in autoimmunity or tolerance induction to cancer. With the current paper, we also aim to convince the readers that the pathways involved in cancer and autoimmune disease development consist of similar molecular players working in opposite directions. Therefore, a deep understanding of the two sides of immune tolerance is crucial for the proper designing of novel and selective immunotherapies.
Assuntos
Antineoplásicos , Doenças Autoimunes , Neoplasias , Doenças Autoimunes/etiologia , Autoimunidade , Humanos , Imunoterapia , Neoplasias/terapiaRESUMO
Triple-negative breast cancer (TNBC) is an aggressive form of mammary malignancy currently without satisfactory systemic treatment options. Agents generating reactive oxygen species (ROS), such as ascorbate (Asc) and menadione (Men), especially applied in combination, have been proposed as an alternative anticancer modality. However, their effectiveness can be hampered by the cytoprotective effects of elevated antioxidant enzymes (e.g., peroxiredoxins, PRDX) in cancer. In this study, PRDX1 mRNA and protein expression were assessed in TNBC tissues by analysis of the online RNA-seq datasets and immunohistochemical staining of tissue microarray, respectively. We demonstrated that PRDX1 mRNA expression was markedly elevated in primary TNBC tumors as compared to non-malignant controls, with PRDX1 protein staining intensity correlating with favorable survival parameters. Subsequently, PRDX1 functionality in TNBC cell lines or non-malignant mammary cells was targeted by genetic silencing or chemically by auranofin (AUR). The PRDX1-knockdown or AUR treatment resulted in inhibition of the growth of TNBC cells in vitro. These cytotoxic effects were further synergistically potentiated by the incubation with a combination of the prooxidant agents, Asc and Men. In conclusion, we report that the PRDX1-related antioxidant system is essential for maintaining redox homeostasis in TNBC cells and can be an attractive therapeutic target in combination with ROS-generating agents.
RESUMO
In recent years, much research has been focused on the field of adoptive cell therapies (ACT) that use native or genetically modified T cells as therapeutic tools. Immunotherapy with T cells expressing chimeric antigen receptors (CARs) demonstrated great success in the treatment of haematologic malignancies, whereas adoptive transfer of autologous tumour infiltrating lymphocytes (TILs) proved to be highly effective in metastatic melanoma. These encouraging results initiated many studies where ACT was tested as a treatment for various solid tumours. In this review, we provide an overview of the challenges of T cell-based immunotherapies of solid tumours. We describe alternative approaches for choosing the most efficient T cells for cancer treatment in terms of their tumour-specificity and phenotype. Finally, we present strategies for improvement of anti-tumour potential of T cells, including combination therapies.
RESUMO
BACKGROUND: The identification of mutated proteins in human cancer cells-termed proteogenomics, requires several technologically independent research methodologies including DNA variant identification, RNA sequencing, and mass spectrometry. Any one of these methodologies are not optimized for identifying potential mutated proteins and any one output fails to cover completely a specific landscape. METHODS: An isogenic melanoma cell with a p53-null genotype was created by CRISPR/CAS9 system to determine how p53 gene inactivation affects mutant proteome expression. A mutant peptide reference database was developed by comparing two distinct DNA and RNA variant detection platforms using these isogenic cells. Chemically fractionated tryptic peptides from lysates were processed using a TripleTOF 5600+ mass spectrometer and their spectra were identified against this mutant reference database. RESULTS: Approximately 190 mutated peptides were enriched in wt-p53 cells, 187 mutant peptides were enriched in p53-null cells, with an overlap of 147 mutated peptides. STRING analysis highlighted that the wt-p53 cell line was enriched for mutant protein pathways such as CDC5L and POLR1B, whilst the p53-null cell line was enriched for mutated proteins comprising EGF/YES, Ubiquitination, and RPL26/5 nodes. CONCLUSION: Our study produces a well annotated p53-dependent and p53-independent mutant proteome of a common melanoma cell line model. Coupled to the application of an integrated DNA and RNA variant detection platform (CLCbio) and software for identification of proteins (ProteinPilot), this pipeline can be used to detect high confident mutant proteins in cells. GENERAL SIGNIFICANCE: This pipeline forms a blueprint for identifying mutated proteins in diseased cell systems.
Assuntos
Inativação Gênica , Melanoma/genética , Proteoma/genética , Proteína Supressora de Tumor p53/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , ProteogenômicaRESUMO
Spinocerebellar ataxia type 3 (SCA3/MJD) is a polyQ neurodegenerative disease where the presymptomatic phase of pathogenesis is unknown. Therefore, we investigated the molecular network of transcriptomic and proteomic triggers in young presymptomatic SCA3/MJD brain from Ki91 knock-in mouse. We found that transcriptional dysregulations resulting from mutant ataxin-3 are not occurring in young Ki91 mice, while old Ki91 mice and also postmitotic patient SCA3 neurons demonstrate the late transcriptomic changes. Unlike the lack of early mRNA changes, we have identified numerous early changes of total proteins and phosphoproteins in 2-month-old Ki91 mouse cortex and cerebellum. We discovered the network of processes in presymptomatic SCA3 with three main groups of disturbed processes comprising altered proteins: (I) modulation of protein levels and DNA damage (Pabpc1, Ddb1, Nedd8), (II) formation of neuronal cellular structures (Tubb3, Nefh, p-Tau), and (III) neuronal function affected by processes following perturbed cytoskeletal formation (Mt-Co3, Stx1b, p-Syn1). Phosphoproteins downregulate in the young Ki91 mouse brain and their phosphosites are associated with kinases that interact with ATXN3 such as casein kinase, Camk2, and kinases controlled by another Atxn3 interactor p21 such as Gsk3, Pka, and Cdk kinases. We conclude that the onset of SCA3 pathology occurs without altered transcript level and is characterized by changed levels of proteins responsible for termination of translation, DNA damage, spliceosome, and protein phosphorylation. This disturbs global cellular processes such as cytoskeleton and transport of vesicles and mitochondria along axons causing energy deficit and neurodegeneration also manifesting in an altered level of transcripts at later ages.
Assuntos
Ataxina-3/metabolismo , Encéfalo/metabolismo , Doença de Machado-Joseph/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Transcrição Gênica/fisiologia , Fatores Etários , Animais , Ataxina-3/genética , Encéfalo/patologia , Células Cultivadas , Humanos , Doença de Machado-Joseph/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Fosfoproteínas/genéticaRESUMO
Primary ciliary dyskinesia (PCD) is a rare recessive disease with a prevalence of 1/10,000; its symptoms are caused by a kinetic dysfunction of motile cilia in the respiratory epithelium, flagella in spermatozoids, and primary cilia in the embryonic node. PCD is genetically heterogeneous: genotyping the already known PCD-related genes explains the genetic basis in 60-65% of the cases, depending on the population. While identification of new genes involved in PCD pathogenesis remains crucial, the search for new, population-specific mutations causative for PCD is equally important. The Slavs remain far less characterized in this respect compared to West European populations, which significantly limits diagnostic capability. The main goal of this study was to characterize the profile of causative genetic defects in one of the PCD-causing genes, ZMYND10, in the cohort of PCD patients of Slavic origin. The study was carried out using biological material from 172 unrelated PCD individuals of Polish origin, with no causative mutation found in nine major PCD genes. While none of the previously described mutations was found using the HRM-based screening, a novel frameshift mutation (c.367delC) in ZMYND10, unique for Slavic PCD population, was found in homozygous state in two unrelated PCD patients. Immunofluorescence analysis confirmed the absence of outer and inner dynein arms from the ciliary axoneme, consistent with the already published ZMYND10-mutated phenotype; cDNA analysis revealed the lack of ZMYND10 mRNA, indicating nonsense-mediated decay of the truncated transcript.
Assuntos
Cílios/metabolismo , Mutação da Fase de Leitura , Síndrome de Kartagener/etnologia , Síndrome de Kartagener/genética , RNA Mensageiro/genética , Proteínas Supressoras de Tumor/genética , Sequência de Bases , Cílios/patologia , Proteínas do Citoesqueleto , Dineínas/genética , Dineínas/metabolismo , Feminino , Expressão Gênica , Genes Recessivos , Heterogeneidade Genética , Homozigoto , Humanos , Síndrome de Kartagener/patologia , Masculino , Dados de Sequência Molecular , Linhagem , Polônia , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas Supressoras de Tumor/metabolismo , População BrancaRESUMO
BACKGROUND: The polyglutamine (polyQ) family of disorders comprises 9 genetic diseases, including several types of ataxia and Huntington disease. Approximately two decades of investigation and the creation of more than 130 mouse models of polyQ disorders have revealed many similarities between these diseases. The disorders share common mutation types, neurological characteristics and certain aspects of pathogenesis, including morphological and physiological neuronal alterations. All of the diseases still remain incurable. DESCRIPTION: The large volume of information collected as a result of the investigation of polyQ models currently represents a great potential for searching, comparing and translating pathogenesis and therapeutic information between diseases. Therefore, we generated a public database comprising the polyQ mouse models, phenotypes and therapeutic interventions tested in vivo. The database is available at http://conyza.man.poznan.pl/ . CONCLUSION: The use of the database in the field of polyQ diseases may accelerate research on these and other neurodegenerative diseases and provide new perspectives for future investigation.