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1.
Front Oncol ; 13: 1110916, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36776330

RESUMO

Multiple Myeloma (MM) is an incurable neoplasm of mature B cells and the second most prevalent hematological malignancy worldwide. While combinations of proteasome inhibitors like bortezomib (Bz) and immunomodulators (IMiDs) like lenalinomide (Len) are generally effective in newly diagnosed patients, some do not respond to this first-line therapy, and all others will eventually become drug resistant. We previously reported that inhibiting the Sec61 translocon with mycolactone synergizes with Bz to induce terminal unfolded protein response in MM cells, irrespective of their resistance to proteasome inhibition. Here, we examined how Sec61 blockade interferes with IMiD action and whether it overrides resistance to Len. With this aim, we knocked out the IMiD target CRBN in the MM1S cell line and a Bz-resistant subclone to generate Len- and Len/Bz-resistant daughters, respectively. Both the Len- and Len/Bz-resistant clones were susceptible to mycolactone toxicity, especially the doubly resistant one. Notably, the synergy between mycolactone and Bz was maintained in these two clones, and mycolactone also synergized with Len in the two Len-susceptible ones. Further, mycolactone enhanced the therapeutic efficacy of the Bz/Len combination in both mice engrafted with parental or double drug resistant MM1S. Together, these data consolidate the interest of Sec61 blockers as new anti-MM agents and reveal their potential for treatment of refractory or relapsed MM.

2.
EMBO Mol Med ; 14(3): e14740, 2022 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-35014767

RESUMO

Multiple myeloma (MM) is an incurable malignancy characterized by the uncontrolled expansion of plasma cells in the bone marrow. While proteasome inhibitors like bortezomib efficiently halt MM progression, drug resistance inevitably develop, and novel therapeutic approaches are needed. Here, we used a recently discovered Sec61 inhibitor, mycolactone, to assess the interest of disrupting MM proteostasis via protein translocation blockade. In human MM cell lines, mycolactone caused rapid defects in secretion of immunoglobulins and expression of pro-survival interleukin (IL)-6 receptor and CD40, whose activation stimulates IL-6 production. Mycolactone also triggered pro-apoptotic endoplasmic reticulum stress responses synergizing with bortezomib for induction of MM cell death and overriding acquired resistance to the proteasome inhibitor. Notably, the mycolactone-bortezomib combination rapidly killed patient-derived MM cells ex vivo, but not normal mononuclear cells. In immunodeficient mice engrafted with MM cells, it demonstrated superior therapeutic efficacy over single drug treatments, without inducing toxic side effects. Collectively, these findings establish Sec61 blockers as novel anti-MM agents and reveal the interest of targeting both the translocon and the proteasome in proteostasis-addicted tumors.


Assuntos
Antineoplásicos , Mieloma Múltiplo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático , Humanos , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Transporte Proteico , Canais de Translocação SEC/metabolismo
3.
Elife ; 102021 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-34951591

RESUMO

Successful control of Mycobacterium tuberculosis (Mtb) infection by macrophages relies on immunometabolic reprogramming, where the role of fatty acids (FAs) remains poorly understood. Recent studies unraveled Mtb's capacity to acquire saturated and monounsaturated FAs via the Mce1 importer. However, upon activation, macrophages produce polyunsaturated fatty acids (PUFAs), mammal-specific FAs mediating the generation of immunomodulatory eicosanoids. Here, we asked how Mtb modulates de novo synthesis of PUFAs in primary mouse macrophages and whether this benefits host or pathogen. Quantitative lipidomics revealed that Mtb infection selectively activates the biosynthesis of ω6 PUFAs upstream of the eicosanoid precursor arachidonic acid (AA) via transcriptional activation of Fads2. Inhibiting FADS2 in infected macrophages impaired their inflammatory and antimicrobial responses but had no effect on Mtb growth in host cells nor mice. Using a click-chemistry approach, we found that Mtb efficiently imports ω6 PUFAs via Mce1 in axenic culture, including AA. Further, Mtb preferentially internalized AA over all other FAs within infected macrophages by mechanisms partially depending on Mce1 and supporting intracellular persistence. Notably, IFNγ repressed de novo synthesis of AA by infected mouse macrophages and restricted AA import by intracellular Mtb. Together, these findings identify AA as a major FA substrate for intracellular Mtb, whose mobilization by innate immune responses is opportunistically hijacked by the pathogen and downregulated by IFNγ.


Assuntos
Ácidos Graxos Insaturados/farmacologia , Fatores Imunológicos/farmacologia , Mycobacterium tuberculosis/fisiologia , Animais , Linhagem Celular , Ácidos Graxos Insaturados/metabolismo , Feminino , Humanos , Imunidade Inata , Fatores Imunológicos/metabolismo , Masculino , Camundongos , Mycobacterium tuberculosis/metabolismo , Nutrientes/metabolismo
4.
J Virol ; 83(6): 2770-7, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19109377

RESUMO

Nonpathogenic simian immunodeficiency virus SIVagm infection of African green monkeys (AGMs) is characterized by the absence of a robust antibody response against Gag p27. To determine if this is accompanied by a selective loss of T-cell responses to Gag p27, we studied CD4(+) and CD8(+) T-cell responses against Gag p27 and other SIVagm antigens in the peripheral blood and lymph nodes of acutely and chronically infected AGMs. Our data show that AGMs can mount a T-cell response against Gag p27, indicating that the absence of anti-p27 antibodies is not due to the absence of Gag p27-specific T cells.


Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Produtos do Gene gag/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Sangue/imunologia , Células Cultivadas , Chlorocebus aethiops , Anticorpos Anti-HIV/sangue , Linfonodos/imunologia , Subpopulações de Linfócitos/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia
5.
JCI Insight ; 5(20)2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32970636

RESUMO

Hidradenitis suppurativa (HS) is a chronic skin disorder of unknown etiology that manifests as recurrent, painful lesions. Cutaneous dysbiosis and unresolved inflammation are hallmarks of active HS, but their origin and interplay remain unclear. Our metabolomic profiling of HS skin revealed an abnormal induction of the kynurenine pathway of tryptophan catabolism in dermal fibroblasts, correlating with the release of kynurenine pathway-inducing cytokines by inflammatory cell infiltrates. Notably, overactivation of the kynurenine pathway in lesional skin was associated with local and systemic depletion in tryptophan. Yet the skin microbiota normally degrades host tryptophan into indoles regulating tissue inflammation via engagement of the aryl hydrocarbon receptor (AHR). In HS skin lesions, we detected contextual defects in AHR activation coinciding with impaired production of bacteria-derived AHR agonists and decreased incidence of AHR ligand-producing bacteria in the resident flora. Dysregulation of tryptophan catabolism at the skin-microbiota interface thus provides a mechanism linking the immunological and microbiological features of HS lesions. In addition to revealing metabolic alterations in patients with HS, our study suggests that correcting AHR signaling would help restore immune homeostasis in HS skin.


Assuntos
Hidradenite Supurativa/genética , Inflamação/genética , Receptores de Hidrocarboneto Arílico/genética , Pele/metabolismo , Triptofano/metabolismo , Adulto , Axila/microbiologia , Axila/patologia , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Hidradenite Supurativa/microbiologia , Hidradenite Supurativa/patologia , Interações entre Hospedeiro e Microrganismos/genética , Humanos , Inflamação/microbiologia , Inflamação/patologia , Cinurenina/genética , Masculino , Metabolismo/genética , Pessoa de Meia-Idade , Pele/microbiologia , Pele/patologia
6.
Front Immunol ; 10: 2913, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31921172

RESUMO

Mycobacterium leprae, the causative agent of leprosy, is unique amongst human pathogens in its capacity to produce the virulence factor phenolic glycolipid (PGL)-I. In addition to mediating bacterial tropism for neurons, PGL-I interacts with Complement Receptor (CR)3 on macrophages (MPs) to promote infection. We demonstrate here that PGL-I binding to CR3 also enhances bacterial invasion of both polymorphonuclear neutrophils (PMNs) and dendritic cells (DCs). Moreover, in all cell types CR3 engagement by PGL-I activates the Syk tyrosine kinase, inducing calcineurin-dependent nuclear translocation of the transcription factor NFATc. This selectively augments the production of IL-2 by DCs, IL-10 by PMNs and IL-1ß by MPs. In intranasally-infected mice PGL-I binding to CR3 heightens mycobacterial phagocytosis by lung PMNs and MPs, and stimulates NFATc-controlled production of Syk-dependent cytokines. Our study thus identifies the CR3-Syk-NFATc axis as a novel signaling pathway activated by PGL-I in innate immune cells, rewiring host cytokine responses to M. leprae.


Assuntos
Antígenos de Bactérias/imunologia , Calcineurina/imunologia , Glicolipídeos/imunologia , Imunidade Inata , Hanseníase/imunologia , Antígeno de Macrófago 1/imunologia , Mycobacterium leprae/imunologia , Fatores de Transcrição NFATC/imunologia , Transdução de Sinais/imunologia , Quinase Syk/imunologia , Animais , Calcineurina/genética , Citocinas/genética , Citocinas/imunologia , Células Dendríticas/imunologia , Hanseníase/genética , Antígeno de Macrófago 1/genética , Masculino , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Neutrófilos/imunologia , Fagocitose , Transdução de Sinais/genética , Quinase Syk/genética
7.
FASEB J ; 21(12): 3262-71, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17507667

RESUMO

Nonhuman primates, including African green monkey (AGM), are important models for biomedical research. The information on monkey genomes is still limited and no versatile gene expression screening tool is available. We tested human whole genome microarrays for cross-species reactivity with AGM transcripts using both long oligonucleotide arrays (60-mer probes) and short oligonucleotide arrays (25-mer). Using the long oligonucleotide arrays, we detected 4-fold more AGM transcripts than with the short oligonucleotide technology. The number of detected transcripts was comparable to that detected using human RNA, with 87% of the detected genes being shared between both species. The specificity of the signals obtained with the long oligonucleotide arrays was determined by analyzing the transcriptome of concanavalin A-activated CD4+ T cells vs. nonactivated T cells of two monkey species AGM and macaque. For both species, the genes showing the most significant changes in expression, such as IL-2R, were those known to be regulated in human CD4+ T cell activation. Finally, tissue specificity of the signals was established by comparing the transcription profiles of AGM brain and tonsil cells. In conclusion, the ABI human microarray platform provides a highly valuable tool for the assessment of AGM gene expression profiles.


Assuntos
Chlorocebus aethiops/genética , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Animais , Sequência de Bases , Linfócitos T CD4-Positivos/fisiologia , Expressão Gênica , Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/métodos , Genoma Humano , Humanos , Sistema Imunitário/fisiologia , Macaca mulatta , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Sensibilidade e Especificidade , Análise de Sequência de DNA
8.
Front Immunol ; 9: 2, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29403489

RESUMO

Phenolic glycolipids (PGLs) are cell wall components of a subset of pathogenic mycobacteria, with immunomodulatory properties. Here, we show that in addition, PGLs exert antibactericidal activity by limiting the production of nitric oxide synthase (iNOS) in mycobacteria-infected macrophages. PGL-mediated downregulation of iNOS was complement receptor 3-dependent and comparably induced by bacterial and purified PGLs. Using Mycobacterium leprae PGL-1 as a model, we found that PGLs dampen the toll-like receptor (TLR)4 signaling pathway, with macrophage exposure to PGLs leading to significant reduction in TIR-domain-containing adapter-inducing interferon-ß (TRIF) protein level. PGL-driven decrease in TRIF operated posttranscriptionally and independently of Src-family tyrosine kinases, lysosomal and proteasomal degradation. It resulted in the defective production of TRIF-dependent IFN-ß and CXCL10 in TLR4-stimulated macrophages, in addition to iNOS. Our results unravel a mechanism by which PGLs hijack both the bactericidal and inflammatory responses of host macrophages. Moreover, they identify TRIF as a critical node in the crosstalk between CR3 and TLR4.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antígenos de Bactérias/metabolismo , Glicolipídeos/metabolismo , Macrófagos/imunologia , Mycobacterium leprae/imunologia , Óxido Nítrico Sintase Tipo II/biossíntese , Receptor 4 Toll-Like/metabolismo , Animais , Parede Celular/metabolismo , Células Cultivadas , Quimiocina CXCL10/biossíntese , Interferon beta/biossíntese , Hanseníase/imunologia , Hanseníase/microbiologia , Hanseníase/patologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais
9.
J Med Chem ; 57(17): 7382-95, 2014 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-25158122

RESUMO

Mycolactone is a complex macrolide toxin produced by Mycobacterium ulcerans, the causative agent of skin lesions called Buruli ulcers. Mycolactone-mediated activation of neural (N) Wiskott-Aldrich syndrome proteins (WASP) induces defects in cell adhesion underpinning cytotoxicity and disease pathogenesis. We describe the chemical synthesis of 23 novel mycolactone analogues that differ in structure and modular assembly of the lactone core with its northern and southern polyketide side chains. The lactone core linked to southern chain was the minimal structure binding N-WASP and hematopoietic homolog WASP, where the number and configuration of hydroxyl groups on the acyl side chain impacted the degree of binding. A fluorescent derivative of this compound showed time-dependent accumulation in target cells. Furthermore, a simplified version of mycolactone mimicked the natural toxin for activation of WASP in vitro and induced comparable alterations of epithelial cell adhesion. Therefore, it constitutes a structural and functional surrogate of mycolactone for WASP/N-WASP-dependent effects.


Assuntos
Toxinas Bacterianas/química , Macrolídeos/química , Proteína da Síndrome de Wiskott-Aldrich/química , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacologia , Adesão Celular/efeitos dos fármacos , Células HeLa , Humanos , Cinética , Macrolídeos/metabolismo , Macrolídeos/farmacologia , Modelos Químicos , Estrutura Molecular , Mycobacterium ulcerans/química , Ligação Proteica , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
10.
Genetics ; 182(4): 1101-8, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19528324

RESUMO

Lateral inhibition mediated by Notch receptor signaling regulates the determination of sensory organ precursor cells (SOPs) in Drosophila. The selection of SOPs from proneural cluster cells appears to rely on a negative feedback loop linking activation of the Notch receptor to downregulation of its ligand Delta within each cell. The molecular basis of this regulatory feedback mechanism is not known. Here, we have tested the role of the Bearded (Brd) family genes in this process. The Drosophila genome encodes eight Brd family members that interact with the E3 ubiquitin ligase Neuralized (Neur) and act as inhibitors of Neur-mediated Delta signaling. Genome engineering technologies were used to create specific deletions of all eight Brd family genes. We find that the Brd family genes malpha, m4, and m6 encoded by the Enhancer of split Complex (E(spl)-C) are dispensable for Drosophila development and that deletion of the five Brd family genes encoded by the Brd Complex only reduces viability. However, deletion of all Brd family genes results in embryonic lethality. Additionally, the malpha, m4, and m6 genes act redundantly with the other five Brd family genes to spatially restrict Notch activation in stage 5 embryos. These data reveal that the Brd family genes have an essential but redundant activity. While the activity of all eight Brd genes appears to be dispensable for SOP determination, clone border studies indicate that both the relative activity levels of Neur and Brd family members influence competition for the SOP fate during lateral inhibition. We propose that inhibition of Neur-Delta interaction by Brd family members is part of the feedback loop that underlies lateral inhibition in Drosophila.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila/embriologia , Retroalimentação Fisiológica , Receptores Notch/genética , Animais , Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Engenharia Genética/métodos , Genoma de Inseto , Órgãos dos Sentidos/citologia , Órgãos dos Sentidos/embriologia , Deleção de Sequência , Células-Tronco , Ubiquitina-Proteína Ligases/metabolismo
11.
J Clin Invest ; 119(12): 3544-55, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19959873

RESUMO

African green monkeys (AGMs) infected with the AGM type of SIV (SIVagm) do not develop chronic immune activation and AIDS, despite viral loads similar to those detected in humans infected with HIV-1 and rhesus macaques (RMs) infected with the RM type of SIV (SIVmac). Because chronic immune activation drives progressive CD4+ T cell depletion and immune cell dysfunctions, factors that characterize disease progression, we sought to understand the molecular basis of this AGM phenotype. To this end, we longitudinally assessed the gene expression profiles of blood- and lymph node-derived CD4+ cells from AGMs and RMs in response to SIVagm and SIVmac infection, respectively, using a genomic microarray platform. The molecular signature of acute infection was characterized, in both species, by strong upregulation of type I IFN-stimulated genes (ISGs). ISG expression returned to basal levels after postinfection day 28 in AGMs but was sustained in RMs, especially in the lymph node-derived cells. We also found that SIVagm induced IFN-alpha production by AGM cells in vitro and that low IFN-alpha levels were sufficient to induce strong ISG responses. In conclusion, SIV infection triggered a rapid and strong IFN-alpha response in vivo in both AGMs and RMs, with this response being efficiently controlled only in AGMs, possibly as a result of active regulatory mechanisms.


Assuntos
Interferon Tipo I/biossíntese , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/patogenicidade , Animais , Linfócitos T CD4-Positivos/imunologia , Chlorocebus aethiops , Perfilação da Expressão Gênica , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1 , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interferon Tipo I/genética , Interferon-alfa/biossíntese , Interferon-alfa/genética , Macaca mulatta , Análise de Sequência com Séries de Oligonucleotídeos , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/fisiologia , Especificidade da Espécie , Virulência/imunologia , Replicação Viral
12.
Hum Mol Genet ; 11(23): 2887-94, 2002 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-12393800

RESUMO

Gene amplification plays a critical role in tumor progression. Hence, understanding the factors triggering this process in human cancers is an important concern. Unfortunately, the structures formed at early stages are usually unavailable for study, hampering the identification of the initiating events in tumors. Here, we show that the region containing the PIP gene, which is overexpressed in 80% of primary and metastatic breast cancers, is duplicated in the breast carcinoma cell line T47D. The two copies are organized as a large palindrome, lying 'in loco' on one chromosome 7. Such features constitute the landmark of the breakage-fusion-bridge (BFB) cycle mechanism. In hamster cells selected in vitro to resist cytotoxic drugs, common fragile site (CFS) activation has been shown to trigger this mechanism. Here, we characterize FRA7I at the molecular level and demonstrate that it lies 2 Mb telomeric to the PIP gene and sets the distal end of the repeated sequence. Moreover, our results suggest that the BFB process was frozen within the first cycle by healing of the broken chromosome. T47D cells thus offer a unique opportunity to observe the earliest products of the BFB cycle mechanism. Our findings constitute the first evidence that this amplification mechanism can be initiated in vivo by fragile site activation.


Assuntos
Apolipoproteínas , Fusão Gênica Artificial , Neoplasias da Mama/genética , Proteínas de Transporte/genética , Quebra Cromossômica/genética , Fragilidade Cromossômica/genética , Duplicação Gênica , Glicoproteínas , Proteínas de Membrana Transportadoras , Animais , Apolipoproteínas D , Células CHO , Sítios Frágeis do Cromossomo , Cromossomos Humanos Par 7/genética , Cricetinae , Sondas de DNA , Biblioteca Gênica , Humanos , Hibridização in Situ Fluorescente , Técnicas In Vitro , Cariotipagem , Sequências Repetitivas de Ácido Nucleico , Telômero/genética , Células Tumorais Cultivadas
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