RESUMO
Herein, we fabricated nanoscale 2D CeO2 sheet structure to develop a stable resistive gas sensor for detection of low concentration (ppm) level formaldehyde vapors. The fabricated CeO2 nanosheets (NSs) showed an optical band gap of 3.53 eV and cubic fluorite crystal structure with enriched defect states. The formation of 2D NSs with well crystalline phases is clearly observed from HRTEM imag-es. The NSs have been shown tremendous blue-green emission related to large oxygen defects. A VOC sensing device based on fabricated two-dimensional NSs has been developed for the sensing of different VOCs. The device showed better sensing for formaldehyde compared with other VOCs (2-propanol, methanol, ethanol, and toluene). The response was found to be 4.35, with the response and recovery time of 71 s and 310 s, respectively. The device showed an increment of the recovery time (71 s to 100 s) with the decrement of the formaldehyde ppm (100 ppm to 20 ppm). Theoretical fit-tings provided the detection limit of formaldehyde ≈ 8.86 ± 0.45 ppm with sensitivity of 0.56 ± 0.05 ppm-1. The sensor device showed good reproducibility with excellent stability over the study period of 135 days, with a deviation of 1.8 % for 100 ppm formaldehyde. The average size of the NSs (≈ 24 nm) calculated from HRTEM observation showed lower value than the calculated Debye length (≈ 44 nm) of the charge accumulation during VOCs sensing. Different defect states, interstitial and surface states in the CeO2 NSs as observed from the Raman spectrum and emission spectrum are responsible for the formaldehyde sensing. This work offers an insight into 2D semiconductor-based oxide materi-al for highly sensitive and stable formaldehyde sensors. .
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Historically tools and technologies facilitated scientific discoveries. Steroid hormone research is not an exception. Unfortunately, the dramatic advancement of the field faded this research area and flagged it as a solved topic. However, it should have been the opposite. The area should glitter with its strong foundation and attract next-generation scientists. Over the past century, a myriad of new facts on biochemistry, molecular biology, cell biology, physiology and pathology of the steroid hormones was discovered. Several innovations were made and translated into life-saving treatment strategies such as synthetic steroids, and inhibitors of steroidogenesis and steroid signaling. Steroid molecules exhibit their diverse effects on cell metabolism, salt and water balance, development and function of the reproductive system, pregnancy, and immune-cell function. Despite vigorous research, the molecular basis of the immunomodulatory effect of steroids is still mysterious. The recent excitement on local extra-glandular steroidogenesis in regulating inflammation and immunity is revitalizing the topic with a new perspective. Therefore, here we review the role of steroidogenesis in regulating inflammation and immunity, discuss the unresolved questions, and how this area can bring another golden age of steroid hormone research with the development of new tools and technologies and advancement of the scientific methods.
Assuntos
Transdução de Sinais , EsteroidesRESUMO
The immune system is composed of a variety of cells that act in a coordinated fashion to protect the organism against a multitude of different pathogens. The great variability of existing pathogens corresponds to a similar high heterogeneity of the immune cells. The study of individual immune cells, the fundamental unit of immunity, has recently transformed from a qualitative microscopic imaging to a nearly complete quantitative transcriptomic analysis. This shift has been driven by the rapid development of multiple single-cell technologies. These new advances are expected to boost the detection of less frequent cell types and transient or intermediate cell states. They will highlight the individuality of each single cell and greatly expand the resolution of current available classifications and differentiation trajectories. In this review we discuss the recent advancement and application of single-cell technologies, their limitations and future applications to study the immune system.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Perfilação da Expressão Gênica , Sistema Imunitário/imunologia , Técnicas Imunológicas , Análise de Célula Única , Animais , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Linhagem da Célula , Difusão de Inovações , Previsões , Perfilação da Expressão Gênica/tendências , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/metabolismo , Técnicas Imunológicas/tendências , Imunofenotipagem , Fenótipo , RNA/genética , Análise de Sequência de RNA , Análise de Célula Única/tendências , TranscriptomaRESUMO
The work presents the identification of fish adulteration and quality assessment by incorporating a chemiresistive gas sensor and machine learning (ML) techniques. Highly sensitive SnO2 nanopetals were synthesized chemically and integrated with interdigitated electrodes to fabricate a sensor device. The sensor was calibrated with formaldehyde (37 %) with a theoretical detection limit of 75 ppb and further utilized to detect the vapors emitted from fresh and formalin-adulterated fish. An extensive sensing investigation was conducted with freshly caught Rohu fish samples. The sensing behavior was examined for all the samples at different time intervals to estimate the spoilage level. The classification between fresh and adulterated fish samples was obtained with 100 % accuracy by employing ML tools. Moreover, the storage duration and spoilage level of fish samples were quantified using regression models. This work emphasizes the potential of nanomaterials combined with machine learning for the accurate detection of adulteration in food systems.
Assuntos
Peixes , Gases , Animais , Aprendizado de Máquina , Alimentos Marinhos , EletrodosRESUMO
Osteoclasts play a central role in cancer-cell-induced osteolysis, but the molecular mechanisms of osteoclast activation during bone metastasis formation are incompletely understood. By performing RNA sequencing on a mouse breast carcinoma cell line with higher bone-metastatic potential, here we identify the enzyme CYP11A1 strongly upregulated in osteotropic tumor cells. Genetic deletion of Cyp11a1 in tumor cells leads to a decreased number of bone metastases but does not alter primary tumor growth and lung metastasis formation in mice. The product of CYP11A1 activity, pregnenolone, increases the number and function of mouse and human osteoclasts in vitro but does not alter osteoclast-specific gene expression. Instead, tumor-derived pregnenolone strongly enhances the fusion of pre-osteoclasts via prolyl 4-hydroxylase subunit beta (P4HB), identified as a potential interaction partner of pregnenolone. Taken together, our results demonstrate that Cyp11a1-expressing tumor cells produce pregnenolone, which is capable of promoting bone metastasis formation and osteoclast development via P4HB.
Assuntos
Neoplasias Ósseas , Neoplasias da Mama , Humanos , Feminino , Osteogênese , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Linhagem Celular Tumoral , Neoplasias Ósseas/metabolismo , Osteoclastos/metabolismo , Pregnenolona/metabolismo , Neoplasias da Mama/patologia , Diferenciação CelularRESUMO
Pregnenolone (P5) is synthesized as the first bioactive steroid in the mitochondria from cholesterol. Clusters of differentiation 4 (CD4+) and Clusters of differentiation 8 (CD8+) immune cells synthesize P5 de novo; P5, in turn, play important role in immune homeostasis and regulation. However, P5's biochemical mode of action in immune cells is still emerging. We envisage that revealing the complete spectrum of P5 target proteins in immune cells would have multifold applications, not only in basic understanding of steroids biochemistry in immune cells but also in developing new therapeutic applications. We employed a CLICK-enabled probe to capture P5-binding proteins in live T helper cell type 2 (Th2) cells. Subsequently, using high-throughput quantitative proteomics, we identified the P5 interactome in CD4+ Th2 cells. Our study revealed P5's mode of action in CD4+ immune cells. We identified novel proteins from mitochondrial and endoplasmic reticulum membranes to be the primary mediators of P5's biochemistry in CD4+ and to concur with our earlier finding in CD8+ immune cells. Applying advanced computational algorithms and molecular simulations, we were able to generate near-native maps of P5-protein key molecular interactions. We showed bonds and interactions between key amino acids and P5, which revealed the importance of ionic bond, hydrophobic interactions, and water channels. We point out that our results can lead to designing of novel molecular therapeutics strategies.
Assuntos
Pregnenolona , Células Th2 , Pregnenolona/metabolismo , Pregnenolona/farmacologia , Células Th2/metabolismo , Simulação de Dinâmica Molecular , Esteroides , Proteínas de Transporte/metabolismoRESUMO
Pregnenolone (P5) promotes prostate cancer cell growth, and de novo synthesis of intratumoural P5 is a potential cause of development of castration resistance. Immune cells can also synthesize P5 de novo. Despite its biological importance, little is known about P5's mode of actions, which appears to be context dependent and pleiotropic. A comprehensive proteome-wide spectrum of P5-binding proteins that are involved in its trafficking and functionality remains unknown. Here, we describe an approach that integrates chemical biology for probe synthesis with chemoproteomics to map P5-protein interactions in live prostate cancer cells and murine CD8+ T cells. We subsequently identified P5-binding proteins potentially involved in P5-trafficking and in P5's non-genomic action that may drive the promotion of castrate-resistance prostate cancer and regulate CD8+ T cell function. We envisage that this methodology could be employed for other steroids to map their interactomes directly in a broad range of living cells, tissues, and organisms.
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Mitochondrial dysfunction underlies a large number of acute or progressive diseases, as well as aging. However, proposed therapies for mitochondrial mutations suffer from poor transformation of mitochondria with exogenous DNA, or lack of functionality of the transferred nucleic acid within the organelle. We show that a transfer RNA import complex (RIC) from the parasitic protozoon Leishmania tropica rapidly and efficiently delivered signal-tagged antisense (STAS) RNA or DNA to mitochondria of cultured human cells. STAS-induced specific degradation of the targeted mitochondrial mRNA, with downstream effects on respiration. These results reveal the existence of a novel small RNA-mediated mRNA degradation pathway in mammalian mitochondria, and suggest that RIC-mediated delivery could be used to target therapeutic RNAs to the organelle within intact cells.
Assuntos
Marcação de Genes , Mitocôndrias/genética , Estabilidade de RNA , RNA Antissenso/genética , RNA de Transferência de Tirosina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Tumoral , Respiração Celular , Humanos , Leishmania tropica/genética , Mitocôndrias/fisiologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Transporte de RNA , RNA Antissenso/química , RNA Antissenso/fisiologia , RNA de Protozoário/genética , RNA de Protozoário/isolamento & purificação , RNA de Protozoário/fisiologia , RNA de Transferência de Tirosina/isolamento & purificação , RNA de Transferência de Tirosina/fisiologiaRESUMO
Here, using single-cell RNA sequencing, we examine the stromal compartment in murine melanoma and draining lymph nodes (LNs) at points across tumor development, providing data at http://www.teichlab.org/data/. Naive lymphocytes from LNs undergo activation and clonal expansion within the tumor, before PD1 and Lag3 expression, while tumor-associated myeloid cells promote the formation of a suppressive niche. We identify three temporally distinct stromal populations displaying unique functional signatures, conserved across mouse and human tumors. Whereas "immune" stromal cells are observed in early tumors, "contractile" cells become more prevalent at later time points. Complement component C3 is specifically expressed in the immune population. Its cleavage product C3a supports the recruitment of C3aR+ macrophages, and perturbation of C3a and C3aR disrupts immune infiltration, slowing tumor growth. Our results highlight the power of scRNA-seq to identify complex interplays and increase stromal diversity as a tumor develops, revealing that stromal cells acquire the capacity to modulate immune landscapes from early disease.
Assuntos
Melanoma/imunologia , Análise de Sequência de RNA/métodos , Células Estromais/metabolismo , Microambiente Tumoral/imunologia , Animais , Humanos , CamundongosRESUMO
Tumors subvert immune cell function to evade immune responses, yet the complex mechanisms driving immune evasion remain poorly understood. Here we show that tumors induce de novo steroidogenesis in T lymphocytes to evade anti-tumor immunity. Using a transgenic steroidogenesis-reporter mouse line we identify and characterize de novo steroidogenic immune cells, defining the global gene expression identity of these steroid-producing immune cells and gene regulatory networks by using single-cell transcriptomics. Genetic ablation of T cell steroidogenesis restricts primary tumor growth and metastatic dissemination in mouse models. Steroidogenic T cells dysregulate anti-tumor immunity, and inhibition of the steroidogenesis pathway is sufficient to restore anti-tumor immunity. This study demonstrates T cell de novo steroidogenesis as a mechanism of anti-tumor immunosuppression and a potential druggable target.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Melanoma/imunologia , Esteroides/imunologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular Tumoral , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/imunologia , Humanos , Evasão da Resposta Imune , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Knockout , Esteroides/biossínteseRESUMO
BACKGROUND: The IRE1a-XBP1 pathway is a conserved adaptive mediator of the unfolded protein response. The pathway is indispensable for the development of secretory cells by facilitating protein folding and enhancing secretory capacity. In the immune system, it is known to function in dendritic cells, plasma cells, and eosinophil development and differentiation, while its role in T helper cell is unexplored. Here, we investigated the role of the IRE1a-XBP1 pathway in regulating activation and differentiation of type-2 T helper cell (Th2), a major T helper cell type involved in allergy, asthma, helminth infection, pregnancy, and tumor immunosuppression. METHODS: We perturbed the IRE1a-XBP1 pathway and interrogated its role in Th2 cell differentiation. We performed genome-wide transcriptomic analysis of differential gene expression to reveal IRE1a-XBP1 pathway-regulated genes and predict their biological role. To identify direct target genes of XBP1 and define XBP1's regulatory network, we performed XBP1 ChIPmentation (ChIP-seq). We validated our predictions by flow cytometry, ELISA, and qPCR. We also used a fluorescent ubiquitin cell cycle indicator mouse to demonstrate the role of XBP1 in the cell cycle. RESULTS: We show that Th2 lymphocytes induce the IRE1a-XBP1 pathway during in vitro and in vivo activation. Genome-wide transcriptomic analysis of differential gene expression by perturbing the IRE1a-XBP1 pathway reveals XBP1-controlled genes and biological pathways. Performing XBP1 ChIPmentation (ChIP-seq) and integrating with transcriptomic data, we identify XBP1-controlled direct target genes and its transcriptional regulatory network. We observed that the IRE1a-XBP1 pathway controls cytokine secretion and the expression of two Th2 signature cytokines, IL13 and IL5. We also discovered that the IRE1a-XBP1 pathway facilitates activation-dependent Th2 cell proliferation by facilitating cell cycle progression through S and G2/M phase. CONCLUSIONS: We confirm and detail the critical role of the IRE1a-XBP1 pathway during Th2 lymphocyte activation in regulating cytokine expression, secretion, and cell proliferation. Our high-quality genome-wide XBP1 ChIP and gene expression data provide a rich resource for investigating XBP1-regulated genes. We provide a browsable online database available at http://data.teichlab.org .
Assuntos
Diferenciação Celular , Endorribonucleases/metabolismo , Estudo de Associação Genômica Ampla , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Estresse Fisiológico , Células Th2/citologia , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Sequência de Bases , Ciclo Celular , Proliferação de Células , Citocinas/metabolismo , Endorribonucleases/genética , Feminino , Regulação da Expressão Gênica , Ativação Linfocitária/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/genética , Regulação para Cima/genética , Proteína 1 de Ligação a X-Box/genéticaRESUMO
BACKGROUND: CD4(+) T cells are key regulators of the adaptive immune system and can be divided into T helper (Th) cells and regulatory T (Treg) cells. During an immune response Th cells mature from a naive state into one of several effector subtypes that exhibit distinct functions. The transcriptional mechanisms that underlie the specific functional identity of CD4(+) T cells are not fully understood. RESULTS: To assist investigations into the transcriptional identity and regulatory processes of these cells we performed mRNA-sequencing on three murine T helper subtypes (Th1, Th2 and Th17) as well as on splenic Treg cells and induced Treg (iTreg) cells. Our integrated analysis of this dataset revealed the gene expression changes associated with these related but distinct cellular identities. Each cell subtype differentially expresses a wealth of 'subtype upregulated' genes, some of which are well known whilst others promise new insights into signalling processes and transcriptional regulation. We show that hundreds of genes are regulated purely by alternative splicing to extend our knowledge of the role of post-transcriptional regulation in cell differentiation. CONCLUSIONS: This CD4(+) transcriptome atlas provides a valuable resource for the study of CD4(+) T cell populations. To facilitate its use by others, we have made the data available in an easily accessible online resource at www.th-express.org.
Assuntos
Regulação da Expressão Gênica , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Reguladores/citologia , Células Th17/citologia , Transcriptoma , Processamento Alternativo , Animais , Diferenciação Celular , Análise por Conglomerados , Citometria de Fluxo , Perfilação da Expressão Gênica , Sistema Imunitário , Camundongos , Camundongos Endogâmicos C57BL , Análise de Componente Principal , Isoformas de Proteínas/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Regulação para CimaRESUMO
T helper 2 (Th2) cells regulate helminth infections, allergic disorders, tumor immunity, and pregnancy by secreting various cytokines. It is likely that there are undiscovered Th2 signaling molecules. Although steroids are known to be immunoregulators, de novo steroid production from immune cells has not been previously characterized. Here, we demonstrate production of the steroid pregnenolone by Th2 cells in vitro and in vivo in a helminth infection model. Single-cell RNA sequencing and quantitative PCR analysis suggest that pregnenolone synthesis in Th2 cells is related to immunosuppression. In support of this, we show that pregnenolone inhibits Th cell proliferation and B cell immunoglobulin class switching. We also show that steroidogenic Th2 cells inhibit Th cell proliferation in a Cyp11a1 enzyme-dependent manner. We propose pregnenolone as a "lymphosteroid," a steroid produced by lymphocytes. We speculate that this de novo steroid production may be an intrinsic phenomenon of Th2-mediated immune responses to actively restore immune homeostasis.
Assuntos
Pregnenolona/biossíntese , RNA/metabolismo , Células Th1/imunologia , Células Th2/imunologia , Animais , Homeostase/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pregnenolona/genética , Pregnenolona/imunologia , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Células Th1/metabolismo , Células Th2/metabolismo , TranscriptomaRESUMO
Modification of proteins with ubiquitin and ubiquitin-like molecules is involved in the regulation of almost every biological process. Historically, each conjugation pathway has its unique set of E1, E2 and E3 enzymes that lead to activation and conjugation of their cognate molecules. Here, we present the unexpected finding that under stress conditions, the ubiquitin E1 enzyme Ube1 mediates conjugation of the ubiquitin-like molecule NEDD8. Inhibition of the 26S proteasome, heat shock and oxidative stress cause a global increase in NEDDylation. Surprisingly, this does not depend on the NEDD8 E1-activating enzyme, but rather on Ube1. A common event in the tested stress conditions is the depletion of "free" ubiquitin. A decrease in "free" ubiquitin levels in the absence of additional stress is sufficient to stimulate NEDDylation through Ube1. Further analysis on the NEDD8 proteome shows that the modified NEDDylated proteins are simultaneously ubiquitinated. Mass spectrometry on the complex proteome under stress reveals the existence of mixed chains between NEDD8 and ubiquitin. We further show that NEDDylation of the p53 tumor suppressor upon stress is mediated mainly through Ube1. Our studies reveal an unprecedented interplay between NEDD8 and ubiquitin pathways operating in diverse cellular stress conditions.
Assuntos
Estresse Oxidativo , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Western Blotting , Linhagem Celular Tumoral , Ciclopentanos/farmacologia , Ativação Enzimática , Técnicas de Silenciamento de Genes , Humanos , Leupeptinas/farmacologia , Espectrometria de Massas , Dados de Sequência Molecular , Proteína NEDD8 , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirimidinas/farmacologia , Fatores de Tempo , Transfecção , Proteína Supressora de Tumor p53/metabolismo , Enzimas Ativadoras de Ubiquitina/genética , Ubiquitinas/genéticaRESUMO
Many maternally inherited and incurable neuromyopathies are caused by mutations in mitochondrial (mt) transfer RNA (tRNA) genes. Kinetoplastid protozoa, including Leishmania, have evolved specialized systems for importing nucleus-encoded tRNAs into mitochondria. We found that the Leishmania RNA import complex (RIC) could enter human cells by a caveolin-1-dependent pathway, where it induced import of endogenous cytosolic tRNAs, including tRNA(Lys), and restored mitochondrial function in a cybrid harboring a mutant mt tRNA(Lys) (MT-TK) gene. The use of protein complexes to modulate mitochondrial function may help in the management of such genetic disorders.
Assuntos
Citosol/metabolismo , Leishmania tropica , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Protozoários/metabolismo , RNA de Transferência de Lisina/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Caveolina 1/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Respiração Celular , Células Cultivadas , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Endocitose , Humanos , Síndrome de Kearns-Sayre/metabolismo , Síndrome MERRF/metabolismo , Mitocôndrias/genética , Mutação , Consumo de Oxigênio , Biossíntese de Proteínas , RNA de Transferência de Lisina/genética , TransfecçãoRESUMO
In kinetoplastid protozoa, import of cytosolic tRNAs into mitochondria occurs through tRNAs interacting with membrane-bound proteins, the identities of which are unknown. The inner membrane RNA import complex of Leishmania tropica contains multiple proteins and is active for import in vitro. RIC1, the largest subunit of this complex, is structurally homologous to the conserved alpha subunit of F1 ATP synthase. The RIC1 gene complemented an atpA mutation in Escherichia coli. Antisense-mediated knockdown of RIC1/F1alpha in Leishmania resulted in depletion of several mitochondrial tRNAs belonging to distinct subsets (types I and II) that interact cooperatively or antagonistically within the import complex. The knockdown-induced defect in import of type I tRNAs was rectified in a reconstituted system by purified RIC1/F1alpha alone, but recovery of type II tRNA import additionally required a type I tRNA. RIC1/F1alpha formed stable complexes with type I, but not type II, tRNAs through the cooperation of its nucleotide binding and C-terminal domains. Thus, RIC1/F1alpha is a type I tRNA import receptor. As expected of a bifunctional protein, RIC1/F1alpha is shared by both the import complex and by respiratory complex V. Alternative use of ancient respiratory proteins may have been an important step in the evolution of tRNA import.
Assuntos
Leishmania tropica/citologia , Leishmania tropica/metabolismo , Mitocôndrias/metabolismo , Proteínas de Protozoários/metabolismo , RNA de Transferência/metabolismo , Animais , Transporte Biológico , Leishmania tropica/genética , Mitocôndrias/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Homologia Estrutural de ProteínaRESUMO
Transport of tRNAs across the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania requires interactions with specific binding proteins (receptors) in a multi-subunit complex. The allosteric model of import regulation proposes cooperative and antagonistic interactions between two or more receptors with binding specificities for distinct tRNA families (types I and II, respectively). To identify the type II receptor, the gene encoding RIC8A, a subunit of the complex, was cloned. The C-terminal region of RIC8A is homologous to subunit 6b of ubiquinol cytochrome c reductase (respiratory complex III), while the N-terminal region has intrinsic affinity for type II, but not for type I, tRNAs. RIC8A is shared by the import complex and complex III, indicating its bi-functionality, but is assembled differently in the two complexes. Knockdown of RIC8A in Leishmania lowered the mitochondrial content of type II tRNAs but raised that of type I tRNAs, with downstream effects on mitochondrial translation and respiration, and cell death. In RIC8A knockdown cells, a subcomplex was formed that interacted with type I tRNA, but the negative regulation by type II tRNA was lost. Mitochondrial extracts from these cells were defective for type II, but not type I, import; import and regulation were restored by purified RIC8A. These results provide evidence for the relevance of allosteric regulation in vivo and indicate that acquisition of new tRNA-binding domains by ancient respiratory components have played a key role in the evolution of mitochondrial tRNA import.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/química , Leishmania tropica/metabolismo , Proteínas de Membrana Transportadoras/química , Mitocôndrias/metabolismo , RNA de Transferência/química , RNA/metabolismo , Sítio Alostérico , Animais , Transporte Biológico , Clonagem Molecular , Reagentes de Ligações Cruzadas/farmacologia , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Proteínas de Membrana Transportadoras/fisiologia , Modelos Moleculares , Estrutura Terciária de Proteína , Proteínas Recombinantes/químicaRESUMO
A variety of clinical disorders result from mutations in mitochondrial tRNA genes, leading to translational defects. We show here that a protein complex from the kinetoplastid protozoon Leishmania induces specific, ATP-dependent import of human cytoplasmic tRNA(1)(Lys) into human mitochondria in vitro. The imported tRNA undergoes efficient aminoacylation within the organelle and supports organellar protein synthesis. Moreover, translation in mitochondria from patients with myclonic epilepsy with ragged red fibers (MERRF) and Kearns-Sayre syndrome (KSS), containing mutant tRNA(Lys) genes, is stimulated to near-wild-type levels and the formation of aberrant polypeptides suppressed by complex-mediated import. These results suggest a novel way to introduce RNAs for the modulation of mitochondrial gene expression.