RESUMO
The need for therapeutics to treat a plethora of medical conditions and diseases is on the rise and the demand for alternative approaches to mammalian-based production systems is increasing. Plant-based strategies provide a safe and effective alternative to produce biological drugs but have yet to enter mainstream manufacturing at a competitive level. Limitations associated with batch consistency and target protein production levels are present; however, strategies to overcome these challenges are underway. In this study, we apply state-of-the-art mass spectrometry-based proteomics to define proteome remodelling of the plant following agroinfiltration with bacteria grown under shake flask or bioreactor conditions. We observed distinct signatures of bacterial protein production corresponding to the different growth conditions that directly influence the plant defence responses and target protein production on a temporal axis. Our integration of proteomic profiling with small molecule detection and quantification reveals the fluctuation of secondary metabolite production over time to provide new insight into the complexities of dual system modulation in molecular pharming. Our findings suggest that bioreactor bacterial growth may promote evasion of early plant defence responses towards Agrobacterium tumefaciens (updated nomenclature to Rhizobium radiobacter). Furthermore, we uncover and explore specific targets for genetic manipulation to suppress host defences and increase recombinant protein production in molecular pharming.
Assuntos
Agrobacterium tumefaciens , Reatores Biológicos , Nicotiana , Proteômica , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologia , Nicotiana/crescimento & desenvolvimento , Reatores Biológicos/microbiologia , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Agricultura Molecular/métodos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteoma/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
While much research has examined the use of glucose and glutamine by tumor cells, many cancers instead prefer to metabolize fats. Despite the pervasiveness of this phenotype, knowledge of pathways that drive fatty acid oxidation (FAO) in cancer is limited. Prolyl hydroxylase domain proteins hydroxylate substrate proline residues and have been linked to fuel switching. Here, we reveal that PHD3 rapidly triggers repression of FAO in response to nutrient abundance via hydroxylation of acetyl-coA carboxylase 2 (ACC2). We find that PHD3 expression is strongly decreased in subsets of cancer including acute myeloid leukemia (AML) and is linked to a reliance on fat catabolism regardless of external nutrient cues. Overexpressing PHD3 limits FAO via regulation of ACC2 and consequently impedes leukemia cell proliferation. Thus, loss of PHD3 enables greater utilization of fatty acids but may also serve as a metabolic and therapeutic liability by indicating cancer cell susceptibility to FAO inhibition.
Assuntos
Acetil-CoA Carboxilase/metabolismo , Ácidos Graxos/metabolismo , Regulação Neoplásica da Expressão Gênica , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Leucemia Mieloide Aguda/metabolismo , Prolina/metabolismo , Acetil-CoA Carboxilase/antagonistas & inibidores , Acetil-CoA Carboxilase/química , Acetil-CoA Carboxilase/genética , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Hidroxilação , Prolina Dioxigenases do Fator Induzível por Hipóxia/química , Prolina Dioxigenases do Fator Induzível por Hipóxia/genética , Células K562 , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Masculino , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Endogâmicos NOD , Modelos Moleculares , Transplante de Neoplasias , Oxirredução , Prolina/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Homologia Estrutural de Proteína , Análise de SobrevidaRESUMO
The combination cancer immunotherapies with oncolytic virus (OV) and immune checkpoint blockade (ICB) reinstate otherwise dysfunctional antitumor CD8 T cell responses. One major mechanism that aids such reinstatement of antitumor CD8 T cells involves the availability of new class I major histocompatibility complex (MHC-I)-bound tumor epitopes following therapeutic intervention. Thus, therapy-induced changes within the MHC-I peptidome hold the key to understanding the clinical implications for therapy-reinstated CD8 T cell responses. Here, using mass spectrometry-based immuno-affinity methods and tumor-bearing animals treated with OV and ICB (alone or in combination), we captured the therapy-induced alterations within the tumor MHC-I peptidome, which were then tested for their CD8 T cell response-stimulating activity. We found that the oncolytic reovirus monotherapy drives up- as well as downexpression of tumor MHC-I peptides in a cancer type and oncolysis susceptibility dependent manner. Interestingly, the combination of reovirus + ICB results in higher numbers of differentially expressed MHC-I-associated peptides (DEMHCPs) relative to either monotherapies. Most importantly, OV+ICB-driven DEMHCPs contain biologically active epitopes that stimulate interferon-gamma responses in cognate CD8 T cells, which may mediate clinically desired antitumor attack and cancer immunoediting. These findings highlight that the therapy-induced changes to the MHC-I peptidome contribute toward the reinstated antitumor CD8 T cell attack established following OV + ICB combination cancer immunotherapy.
Assuntos
Neoplasias , Vírus Oncolíticos , Animais , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Inibidores de Checkpoint Imunológico , Imunoterapia/métodos , Complexo Principal de Histocompatibilidade , Neoplasias/genética , Vírus Oncolíticos/genéticaRESUMO
Cellular senescence is the irreversible arrest of normally dividing cells and is driven by the cell cycle inhibitors Cdkn2a, Cdkn1a, and Trp53. Senescent cells are implicated in chronic diseases and tissue repair through their increased secretion of pro-inflammatory factors known as the senescence-associated secretory phenotype (SASP). Here, we use spatial transcriptomics and single-cell RNA sequencing (scRNAseq) to demonstrate that cells displaying senescent characteristics are "transiently" present within regenerating skeletal muscle and within the muscles of D2-mdx mice, a model of Muscular Dystrophy. Following injury, multiple cell types including macrophages and fibrog-adipogenic progenitors (FAPs) upregulate senescent features such as senescence pathway genes, SASP factors, and senescence-associated beta-gal (SA-ß-gal) activity. Importantly, when these cells were removed with ABT-263, a senolytic compound, satellite cells are reduced, and muscle fibers were impaired in growth and myonuclear accretion. These results highlight that an "acute" senescent phenotype facilitates regeneration similar to skin and neonatal myocardium.
Assuntos
Senescência Celular , Senoterapia , Animais , Senescência Celular/fisiologia , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético , Células-Tronco/metabolismoRESUMO
INTRODUCTION: Aldehyde dehydrogenase 1A3 (ALDH1A3) is a cancer stem cell (CSC) marker and in breast cancer it is associated with triple-negative/basal-like subtypes and aggressive disease. Studies on the mechanisms of ALDH1A3 in cancer have primarily focused on gene expression changes induced by the enzyme; however, its effects on metabolism have thus far been unstudied and may reveal novel mechanisms of pathogenesis. OBJECTIVE: Determine how ALDH1A3 alters the metabolite profile in breast cancer cells and assess potential impacts. METHOD: Triple-negative MDA-MB-231 tumors and cells with manipulated ALDH1A3 levels were assessed by HPLC-MS metabolomics and metabolite data was integrated with transcriptome data. Mice harboring MDA-MB-231 tumors with or without altered ALDH1A3 expression were treated with γ-aminobutyric acid (GABA) or placebo. Effects on tumor growth, and lungs and brain metastasis were quantified by staining of fixed thin sections and quantitative PCR. Breast cancer patient datasets from TCGA, METABRIC and GEO were used to assess the co-expression of GABA pathway genes with ALDH1A3. RESULTS: Integrated metabolomic and transcriptome data identified GABA metabolism as a primary dysregulated pathway in ALDH1A3 expressing breast tumors. Both ALDH1A3 and GABA treatment enhanced metastasis. Patient dataset analyses revealed expression association between ALDH1A3 and GABA pathway genes and corresponding increased risk of metastasis. CONCLUSION: This study revealed a novel pathway affected by ALDH1A3, GABA metabolism. Like ALDH1A3 expression, GABA treatment promotes metastasis. Given the clinical use of GABA mimics to relieve chemotherapy-induced peripheral nerve pain, further study of the effects of GABA in breast cancer progression is warranted.
Assuntos
Neoplasias da Mama , Aldeído Desidrogenase/genética , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metabolômica , Camundongos , Camundongos SCID , Ácido gama-Aminobutírico/genética , Ácido gama-Aminobutírico/metabolismoRESUMO
The efficacy of oncolytic viruses (OVs), such as reovirus, is dictated by host immune responses, including those mediated by the pro- versus anti-inflammatory macrophages. As such, a detailed understanding of the interaction between reovirus and different macrophage types is critical for therapeutic efficacy. To explore reovirus-macrophage interactions, we performed tandem mass tag (TMT)-based quantitative temporal proteomics on mouse bone marrow-derived macrophages (BMMs) generated with two cytokines, macrophage colony stimulating factor (M-CSF) and granulocytic-macrophage colony stimulating factor (GM-CSF), representing anti- and proinflammatory macrophages, respectively. We quantified 6863 proteins across five time points in duplicate, comparing M-CSF (M-BMM) and GM-CSF (GM-BMM) in response to OV. We find that GM-BMMs have lower expression of key intrinsic proteins that facilitate an antiviral immune response, express higher levels of reovirus receptor protein JAM-A, and are more susceptible to oncolytic reovirus infection compared to M-BMMs. Interestingly, although M-BMMs are less susceptible to reovirus infection and subsequent cell death, they initiate an antireovirus adaptive T cell immune response comparable to that of GM-BMMs. Taken together, these data describe distinct proteome differences between these two macrophage populations in terms of their ability to mount antiviral immune responses.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos , Fator Estimulador de Colônias de Macrófagos , Animais , Medula Óssea , Células da Medula Óssea , Células Cultivadas , Camundongos , ProteomaRESUMO
MHC class I (MHC-I)-bound ligands play a pivotal role in CD8 T cell immunity and are hence of major interest in understanding and designing immunotherapies. One of the most commonly utilized approaches for detecting MHC ligands is LC-MS/MS. Unfortunately, the effectiveness of current algorithms to identify MHC ligands from LC-MS/MS data is limited because the search algorithms used were originally developed for proteomics approaches detecting tryptic peptides. Consequently, the analysis often results in inflated false discovery rate (FDR) statistics and an overall decrease in the number of peptides that pass FDR filters. Andreatta et al. describe a new scoring tool (MS-rescue) for peptides from MHC-I immunopeptidome datasets. MS-rescue incorporates the existence of MHC-I peptide motifs to rescore peptides from ligandome data. The tool is demonstrated here using peptides assigned from LC-MS/MS data with PEAKs software but can be deployed on data from any search algorithm. This new approach increased the number of peptides identified by up to 20-30% and promises to aid the discovery of novel MHC-I ligands with immunotherapeutic potential.
Assuntos
Peptídeos , Espectrometria de Massas em Tandem , Algoritmos , Cromatografia Líquida , Ligantes , ProteômicaRESUMO
Oncolytic viruses (OVs), known for their cancer-killing characteristics, also overturn tumor-associated defects in antigen presentation through the MHC class I pathway and induce protective neo-antitumor CD8 T cell responses. Nonetheless, whether OVs shape the tumor MHC-I ligandome remains unknown. Here, we investigated if an OV induces the presentation of novel MHC I-bound tumor antigens (termed tumor MHC-I ligands). Using comparative mass spectrometry (MS)-based MHC-I ligandomics, we determined differential tumor MHC-I ligand expression following treatment with oncolytic reovirus in a murine ovarian cancer model. In vitro, we found that reovirus changes the tumor ligandome of cancer cells. Concurrent multiplexed quantitative proteomics revealed that the reovirus-induced changes in tumor MHC-I ligand presentation were mostly independent of their source proteins. In an in vivo model, tumor MHC-I ligands induced by reovirus were detectable not only in tumor tissues but also the spleens (a source of antigen-presenting cells) of tumor-bearing mice. Most importantly, therapy-induced MHC-I ligands stimulated antigen-specific IFNγ responses in antitumor CD8 T cells from mice treated with reovirus. These data show that therapy-induced MHC-I ligands may shape underlying neo-antitumor CD8 T cell responses. As such, they should be considered in strategies promoting the efficacy of OV-based cancer immunotherapies.
Assuntos
Neoplasias/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Proteômica/métodos , Animais , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Células Dendríticas/patologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoterapia , Interferon gama/genética , Interferon gama/imunologia , Ligantes , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/virologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
MHC-I peptides are intracellular-cleaved peptides, usually 8-11 amino acids in length, which are presented on the cell surface and facilitate CD8+ T cell responses. Despite the appreciation of CD8+ T-cell antitumor immune responses toward improvement in patient outcomes, the MHC-I peptide ligands that facilitate the response are poorly described. Along these same lines, although many therapies have been recognized for their ability to reinvigorate antitumor CD8+ T-cell responses, whether these therapies alter the MHC-I peptide repertoire has not been fully assessed due to the lack of quantitative strategies. We develop a multiplexing platform for screening therapy-induced MHC-I ligands by employing tandem mass tags (TMTs). We applied this approach to measuring responses to doxorubicin, which is known to promote antitumor CD8+ T-cell responses during its therapeutic administration in cancer patients. Using both in vitro and in vivo systems, we show successful relative quantitation of MHC-I ligands using TMT-based multiplexing and demonstrate that doxorubicin induces MHC-I peptide ligands that are largely derived from mitotic progression and cell-cycle proteins. This high-throughput MHC-I ligand discovery approach may enable further explorations to understand how small molecules and other therapies alter MHC-I ligand presentation that may be harnessed for CD8+ T-cell-based immunotherapies.
Assuntos
Antibióticos Antineoplásicos/análise , Neoplasias do Colo/terapia , Doxorrubicina/análise , Antígenos de Histocompatibilidade Classe I/análise , Linfoma/terapia , Animais , Antibióticos Antineoplásicos/farmacologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/imunologia , Doxorrubicina/farmacologia , Descoberta de Drogas , Células HCT116 , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoterapia , Ligantes , Linfoma/imunologia , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Células Tumorais CultivadasRESUMO
Reticulon-4 (RTN4), commonly known as a neurite outgrowth inhibitor (Nogo), is emerging as an important player in human cancers. Clinically, we found lower RTN4 expression in patient-derived tumors was associated with significantly better survival in lung, breast, cervical, and renal cancer patients. To identify the role of RTN4 in cancer biology, we performed mass spectrometry-based quantitative proteomic analysis on cancer cells following RTN4 knockdown and found its link with pro-survival as well as cytoskeleton-related processes. Subsequent mechanistic investigations revealed that RTN4 regulates lipid homeostasis, AKT signaling, and cytoskeleton modulation. In particular, downregulation of RTN4 reduced sphingomyelin synthesis and impaired plasma membrane localization of AKT, wherein AKT phosphorylation, involved in many cancers, was significantly reduced without any comparable effect on AKT-related upstream kinases, in a sphingolipid-dependent manner. Furthermore, knockdown of RTN4 retarded proliferation of cancer cells in vitro as well as tumor xenografts in mice. Finally, RTN4 knockdown affected tubulin stability and promoted higher cytotoxic effects with chemotherapeutic paclitaxel in cancer cells both in vitro and in vivo. In summary, RTN4 is involved in carcinogenesis and represents a molecular candidate that may be targeted to achieve desired antitumor effects in clinics.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Citoesqueleto/metabolismo , Técnicas de Silenciamento de Genes/métodos , Proteínas Nogo/genética , Paclitaxel/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Paclitaxel/farmacologia , Proteômica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world's most devastating pathogens.
Assuntos
Mycobacterium tuberculosis/genética , Complexo de Endopeptidases do Proteassoma/química , Virulência , Trifosfato de Adenosina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Sítios de Ligação , Escherichia coli/metabolismo , Feminino , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mycobacterium tuberculosis/patogenicidade , Peptídeos/química , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA/química , Proteínas Recombinantes/química , Tuberculose/microbiologia , Ubiquitina/químicaRESUMO
Class I major histocompatibility complex (MHC-I)-bound peptide ligands dictate the activation and specificity of CD8+ T cells and thus are important for devising T-cell immunotherapies. In recent times, advances in mass spectrometry (MS) have enabled the precise identification of these MHC-I peptides, wherein MS spectra are compared against a reference proteome. Unfortunately, matching these spectra to reference proteome databases is hindered by inflated search spaces attributed to a lack of enzyme restriction in the searches, limiting the efficiency with which MHC ligands are discovered. Here we offer a solution to this problem whereby we developed a targeted database search approach and accompanying tool SpectMHC, that is based on a priori-predicted MHC-I peptides. We first validated the approach using MS data from two different allotype-specific immunoprecipitates for the C57BL/6 mouse background. We then developed allotype-specific HLA databases to search previously published MS data sets of human peripheral blood mononuclear cells (PBMCs). This targeted search strategy improved peptide identifications for both mouse and human ligandomes by greater than 2-fold and is superior to traditional "no enzyme" searches of reference proteomes. Our targeted database search promises to uncover otherwise missed novel T-cell epitopes of therapeutic potential.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Espectrometria de Massas/métodos , Peptídeos/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Epitopos de Linfócito T/genética , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Imunoterapia , Ligantes , Camundongos , Peptídeos/genéticaRESUMO
PURPOSE: Open dismembered pyeloplasty is the preferred repair for ureteropelvic junction obstruction. Minimally invasive techniques have been applied to the original open approach but no clear advantage has been demonstrated for these technological advances. We evaluate outcomes between transperitoneal laparoscopic and open pyeloplasty in children. MATERIALS AND METHODS: All children 1 to 18 years old with ureteropelvic junction obstruction requiring operative repair were offered enrollment in the study. Patients were prospectively randomized to either laparoscopic or open pyeloplasty through a flank incision. RESULTS: A total of 50 patients in the laparoscopic group and 48 in the open group were enrolled from 2005 to 2014. Mean followup was similar between the groups (13.7 months in the laparoscopic group vs 12.3 months in the open group, p = 0.54). The only significantly different outcomes were for mean operative time, which was 139.5 minutes (range 94 to 213) in the laparoscopic group and 122.5 minutes (83 to 239) in the open group (p <0.01), and mean length of stay, which was 25.9 hours (18 to 143) in the laparoscopic group and 28.2 hours (16 to 73) in the open group (p = 0.02). Analgesic usage, success rate, total charges and all parameters in children older than 11 years were similar between the groups. CONCLUSIONS: Open and laparoscopic dismembered pyeloplasty are comparable and effective methods for repair of ureteropelvic junction obstruction. Although operative time was statistically shorter in the open group and length of stay was shorter in the laparoscopic group, the clinical significance of these variables is questionable. The approach to repair may best be based on family preference for incision aesthetics and surgeon comfort with either approach, rather than more classically objective outcome measures.
Assuntos
Pelve Renal/cirurgia , Laparoscopia , Obstrução Ureteral/cirurgia , Procedimentos Cirúrgicos Urológicos/métodos , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Estudos Prospectivos , Resultado do TratamentoRESUMO
Yeast (Saccharomyces cerevisiae) has served as a key model system in biology and as a benchmark for "omics" technology. Although near-complete proteomes of log phase yeast have been measured, protein abundance in yeast is dynamic, particularly during the transition from log to stationary phase. Defining the dynamics of proteomic changes during this transition, termed the diauxic shift, is important to understand the basic biology of proliferative versus quiescent cells. Here, we perform temporal quantitative proteomics to fully capture protein induction and repression during the diauxic shift. Accurate and sensitive quantitation at a high temporal resolution and depth of proteome coverage was achieved using TMT10 reagents and LC-MS3 analysis on an Orbitrap Fusion tribrid mass spectrometer deploying synchronous precursor selection. Triplicate experiments were analyzed using the time-course R package and a simple template matching strategy was used to reveal groups of proteins with similar temporal patterns of protein induction and repression. Within these groups are functionally distinct types of proteins such as those of glyoxylate metabolism and many proteins of unknown function not previously associated with the diauxic shift (e.g. YNR034W-A and FMP16). We also perform a dual time-course experiment to determine Hap2-dependent proteins during the diauxic shift. These data serve as an important basic model for fermentative versus respiratory growth of yeast and other eukaryotes and are a benchmark for temporal quantitative proteomics.
Assuntos
Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Cromatografia Líquida/métodos , Regulação Fúngica da Expressão Gênica , Espectrometria de Massas/métodos , Saccharomyces cerevisiae/metabolismoRESUMO
Quantitative metabolomics and proteomics technologies are powerful approaches to explore cellular metabolic regulation. Unfortunately, combining the two technologies typically requires different LC-MS setups for sensitive measurement of metabolites and peptides. One approach to enhance the analysis of certain classes of metabolites is by derivatization with various types of tags to increase ionization and chromatographic efficiency. We demonstrate here that derivatization of amine metabolites with tandem mass tags (TMT), typically used in multiplexed peptide quantitation, facilitates amino acid analysis by standard nanoflow reversed-phase LC-MS setups used for proteomics. We demonstrate that this approach offers the potential to perform experiments at the MS1-level using duplex tags or at the MS2-level using novel 10-plex reporter ion-containing isobaric tags for multiplexed amine metabolite analysis. We also demonstrate absolute quantitative measurements of amino acids conducted in parallel with multiplexed quantitative proteomics, using similar LC-MS setups to explore cellular amino acid regulation. We further show that the approach can also be used to determine intracellular metabolic labeling of amino acids from glucose carbons.
Assuntos
Aminas/metabolismo , Metabolômica , Neoplasias/patologia , Proteômica , Humanos , Neoplasias/metabolismoRESUMO
BACKGROUND: The efficacy of irrigating the peritoneal cavity during appendectomy for perforated appendicitis has been debated extensively. To date, prospective comparative data are lacking. Therefore, we conducted a prospective, randomized trial comparing peritoneal irrigation to suction alone during laparoscopic appendectomy in children. METHODS: Children younger than 18 years with perforated appendicitis were randomized to peritoneal irrigation with a minimum of 500 mL normal saline, or suction only during laparoscopic appendectomy. Perforation was defined as a hole in the appendix or fecalith in the abdomen. The primary outcome variable was postoperative abscess. Using a power of 0.8 and alpha of 0.05, a sample size of 220 patients was calculated. A battery-powered laparoscopic suction/irrigator was used in all cases. Pre- and postoperative management was controlled. Data were analyzed on an intention-to-treat basis. RESULTS: A total of 220 patients were enrolled between December 2008 and July 2011. There were no differences in patient characteristics at presentation. There was no difference in abscess rate, which was 19.1% with suction only and 18.3% with irrigation (P = 1.0). Duration of hospitalization was 5.5 ± 3.0 with suction only and 5.4 ± 2.7 days with group (P = 0.93). Mean hospital charges was $48.1K in both groups (P = 0.97). Mean operative time was 38.7 ± 14.9 minutes with suction only and 42.8 ± 16.7 minutes with irrigation (P = 0.056). Irrigation was felt to be necessary in one case (0.9%) randomized to suction only. In the patients who developed an abscess, there was no difference in duration of hospitalization, days of intravenous antibiotics, duration of home health care, or abscess-related charges. CONCLUSIONS: There is no advantage to irrigation of the peritoneal cavity over suction alone during laparoscopic appendectomy for perforated appendicitis. The study was registered with clinicaltrials.gov at the inception of enrollment (NCT00981136).
Assuntos
Abscesso Abdominal/prevenção & controle , Apendicectomia/métodos , Apendicite/cirurgia , Laparoscopia , Lavagem Peritoneal , Complicações Pós-Operatórias/prevenção & controle , Sucção , Abscesso Abdominal/epidemiologia , Abscesso Abdominal/etiologia , Adolescente , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Análise de Intenção de Tratamento , Masculino , Complicações Pós-Operatórias/epidemiologia , Estudos Prospectivos , Resultado do TratamentoRESUMO
Dysregulated metabolism in cancers is, by now, well established. Although metabolic adaptations provide cancers with the ability to synthesize the precursors required for rapid biosynthesis, some metabolites have direct functional, or bioactive, effects in human cells. Here we summarize recently identified metabolites that have bioactive roles either as post-translational modifications (PTMs) on proteins or in, yet unknown ways. We propose that these metabolites could play a bioactive role in promoting or inhibiting cancer cell phenotypes in a manner that is mostly unexplored. To study these potentially important bioactive roles, we discuss several novel metabolomic and proteomic approaches aimed at defining novel PTMs and metabolite-protein interactions. Understanding metabolite PTMs and protein interactors of bioactive metabolites may provide entirely new therapeutic targets for cancer.
RESUMO
Comparing cancer proteomes across many samples offers a window into cancer cell biology and may reveal new treatment options for specific subsets of cancer. Here we describe a method using tandem mass tag (TMT) technology to multiplex up to 18 samples in a single analysis, paving the way for the analysis of large cohorts of tumors, cell lines, and perturbations thereof. The procedure we describe will result in samples ready for in-depth LC-MS/MS analysis in 3-4 days.
Assuntos
Neoplasias , Proteômica , Cromatografia Líquida/métodos , Proteoma/análise , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
CD8 T cells play a central role in antiviral immunity. Type I interferons are among the earliest responders after virus exposure and can cause extensive reprogramming and antigen-independent bystander activation of CD8 T cells. Although bystander activation of pre-existing memory CD8 T cells is known to play an important role in host defense and immunopathology, its impact on naïve CD8 T cells remains underappreciated. Here we report that exposure to reovirus, both in vitro or in vivo, promotes bystander activation of naïve CD8 T cells within 24 hours and that this distinct subtype of CD8 T cell displays an innate, antiviral, type I interferon sensitized signature. The induction of bystander naïve CD8 T cells is STAT1 dependent and regulated through nicotinamide phosphoribosyl transferase (NAMPT)-mediated enzymatic actions within NAD+ salvage metabolic biosynthesis. These findings identify a novel aspect of CD8 T cell activation following virus infection with implications for human health and physiology.
Assuntos
NAD , Viroses , Humanos , Linfócitos T CD8-Positivos , Antígenos , AntiviraisRESUMO
Cancer cell energy metabolism plays an important role in dictating the efficacy of oncolysis by oncolytic viruses. To understand the role of multiple myeloma metabolism in reovirus oncolysis, we performed semi-targeted mass spectrometry-based metabolomics on 12 multiple myeloma cell lines and revealed a negative correlation between NAD+ levels and susceptibility to oncolysis. Likewise, a negative correlation was observed between the activity of the rate-limiting NAD+ synthesis enzyme NAMPT and oncolysis. Indeed, depletion of NAD+ levels by pharmacological inhibition of NAMPT using FK866 sensitized several myeloma cell lines to reovirus-induced killing. The myelomas that were most sensitive to this combination therapy expressed a functional p53 and had a metabolic and transcriptomic profile favoring mitochondrial metabolism over glycolysis, with the highest synergistic effect in KMS12 cells. Mechanistically, U-13C-labeled glucose flux, extracellular flux analysis, multiplex proteomics, and cell death assays revealed that the reovirus + FK866 combination caused mitochondrial dysfunction and energy depletion, leading to enhanced autophagic cell death in KMS12 cells. Finally, the combination of reovirus and NAD+ depletion achieved greater antitumor effects in KMS12 tumors in vivo and patient-derived CD138+ multiple myeloma cells. These findings identify NAD+ depletion as a potential combinatorial strategy to enhance the efficacy of oncolytic virus-based therapies in multiple myeloma.