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Neutrophils are sentinel immune cells with essential roles for antimicrobial defense. Most of our knowledge on neutrophil tissue navigation derived from wounding and infection models, whereas allergic conditions remained largely neglected. Here, we analyzed allergen-challenged mouse tissues and discovered that degranulating mast cells (MCs) trap living neutrophils inside them. MCs release the attractant leukotriene B4 to re-route neutrophils toward them, thus exploiting a chemotactic system that neutrophils normally use for intercellular communication. After MC intracellular trap (MIT) formation, neutrophils die, but their undigested material remains inside MC vacuoles over days. MCs benefit from MIT formation, increasing their functional and metabolic fitness. Additionally, they are more pro-inflammatory and can exocytose active neutrophilic compounds with a time delay (nexocytosis), eliciting a type 1 interferon response in surrounding macrophages. Together, our study highlights neutrophil trapping and nexocytosis as MC-mediated processes, which may relay neutrophilic features over the course of chronic allergic inflammation.
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Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here, we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone heat shock protein 70 (HSP70) binds to non-coding RNA transcribed by RNA polymerase III (RNA Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by RNA Pol III. Detailed biochemical analyses showed that HSP70 alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription. Thus, our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in cancer therapeutics.
Assuntos
Proteínas de Choque Térmico HSP70 , Neoplasias , Humanos , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , RNA , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , RNA de Transferência/genética , RNA não Traduzido/genéticaRESUMO
In response to stress, human cells coordinately downregulate transcription and translation of housekeeping genes. To downregulate transcription, the negative elongation factor (NELF) is recruited to gene promoters impairing RNA polymerase II elongation. Here we report that NELF rapidly forms nuclear condensates upon stress in human cells. Condensate formation requires NELF dephosphorylation and SUMOylation induced by stress. The intrinsically disordered region (IDR) in NELFA is necessary for nuclear NELF condensation and can be functionally replaced by the IDR of FUS or EWSR1 protein. We find that biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation. Importantly, NELF condensation is required for cellular viability under stressful conditions. We propose that stress-induced NELF condensates reported here are nuclear counterparts of cytosolic stress granules. These two stress-inducible condensates may drive the coordinated downregulation of transcription and translation, likely forming a critical node of the stress survival strategy.
Assuntos
Resposta ao Choque Térmico/genética , Proteínas Intrinsicamente Desordenadas/genética , Processamento de Proteína Pós-Traducional , RNA Polimerase II/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Cromatina/química , Cromatina/metabolismo , Células Clonais , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Genes Reporter , Células HEK293 , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fosforilação , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Transdução de Sinais , Estresse Fisiológico , Sumoilação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Proteína Vermelha FluorescenteRESUMO
The production of alternative RNA variants contributes to the tissue-specific regulation of gene expression. In the animal nervous system, a systematic shift toward distal sites of transcription termination produces transcript signatures that are crucial for neuron development and function. Here, we report that, in Drosophila, the highly conserved protein ELAV globally regulates all sites of neuronal 3' end processing and directly binds to proximal polyadenylation sites of target mRNAs in vivo. We uncover an endogenous strategy of functional gene rescue that safeguards neuronal RNA signatures in an ELAV loss-of-function context. When not directly repressed by ELAV, the transcript encoding the ELAV paralog FNE acquires a mini-exon, generating a new protein able to translocate to the nucleus and rescue ELAV-mediated alternative polyadenylation and alternative splicing. We propose that exon-activated functional rescue is a more widespread mechanism that ensures robustness of processes regulated by a hierarchy, rather than redundancy, of effectors.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas ELAV/metabolismo , Éxons/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Masculino , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genéticaRESUMO
Pluripotent stem cells are invaluable resources to study development and disease, holding a great promise for regenerative medicine. Here we use human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) from patients with Huntington's disease (HD-iPSCs) to shed light into the normal function of huntingtin (HTT) and its demise in disease. We find that HTT binds ATF7IP, a regulator of the histone H3 methyltransferase SETDB1. HTT inhibits the interaction of the ATF7IP-SETDB1 complex with other heterochromatin regulators and transcriptional repressors, maintaining low levels of H3K9 trimethylation (H3K9me3) in hESCs. Loss of HTT promotes global increased H3K9me3 levels and enrichment of H3K9me3 marks at distinct genes, including transcriptional regulators of neuronal differentiation. Although these genes are normally expressed at low amounts in hESCs, HTT knockdown (KD) reduces their induction during neural differentiation. Notably, mutant expanded polyglutamine repeats in HTT diminish its interaction with ATF7IP-SETDB1 complex and trigger H3K9me3 in HD-iPSCs. Conversely, KD of ATF7IP in HD-iPSCs reduces H3K9me3 alterations and ameliorates gene expression changes in their neural counterparts. Taken together, our results indicate ATF7IP as a potential target to correct aberrant H3K9me3 levels induced by mutant HTT.
Assuntos
Proteína Huntingtina/genética , Doença de Huntington/genética , Proteínas Metiltransferases/genética , Fatores de Transcrição/genética , Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/patologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Heterocromatina/genética , Histona Metiltransferases/genética , Histona-Lisina N-Metiltransferase , Humanos , Doença de Huntington/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Lentivirus/genética , Neurônios/metabolismo , Neurônios/patologia , Peptídeos/genética , Proteínas RepressorasRESUMO
BACKGROUND: As we emerge into the genomic medicine era, the epidemiology of diseases is taken for granted. Accurate prevalence figures, especially of rare diseases (RDs, ≤50/100,000), will become even more important for purposes of health care and societal planning. We noticed that the numbers of affected individuals in regionally established registries for mainly hereditary RDs do not align with published estimated and expected prevalence figures. We therefore hypothesized that such non-population-based means overestimate RDs and sought to address this by recalculating prevalence for an important 'common' hereditary disease, autosomal-dominant polycystic kidney disease (ADPKD) whereby presumed-prevalence is 100-250/100,000 METHODS: The Else-Kroener-Fresenius-ADPKD-Study in south-west Germany with a population of 2,727,351 inhabitants was established with the cooperation of all nephrology centres. Furthermore, general practitioners, internists, urologists, human geneticists and neurosurgery centres were contacted with questionnaires for demographic, family and kidney function data. Germline-mutation screening of susceptibility genes PKD1 and PKD2 was offered. Official population data for 2010 were used for overall and kidney function-adjusted prevalence estimations. RESULTS: A total of 891 subjects, 658 index-cases and 233 relatives, aged 10-89 (mean 52), were registered, with >90% response rate, 398 by nephrologists and 493 by non-nephrologists. Molecular-genetic analyses contributed to confirmation of the diagnosis in 57%. The overall prevalence of ADPKD was 32.7/100,000 reaching a maximum of 57.3/100,000 in the 6th decade of life. CONCLUSIONS: Prevalence of ADPKD is overestimated by 2- to 5-fold and close to the limit of RDs which may be of broad clinical, logistic and policy implications.
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Mutação em Linhagem Germinativa/genética , Rim Policístico Autossômico Dominante/epidemiologia , Canais de Cátion TRPP/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Ligação Genética , Alemanha/epidemiologia , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Prevalência , Prognóstico , Sistema de Registros , Adulto JovemRESUMO
Upon progesterone stimulation, Endometrial Stromal Cells (EnSCs) undergo a differentiation program into secretory cells (decidualization) to release in abundance factors crucial for embryo implantation. We previously demonstrated that decidualization requires massive reshaping of the secretory pathway and, in particular, of the Golgi complex. To decipher the underlying mechanisms, we performed a time-course transcriptomic analysis of in vitro decidualizing EnSC. Pathway analysis shows that Gene Ontology terms associated with vesicular trafficking and early secretory pathway compartments are the most represented among those enriched for upregulated genes. Among these, we identified a cluster of co-regulated genes that share CREB3L1 and CREB3L2 binding elements in their promoter regions. Indeed, both CREB3L1 and CREB3L2 transcription factors are up-regulated during decidualization. Simultaneous downregulation of CREB3L1 and CREB3L2 impairs Golgi enlargement, and causes dramatic changes in decidualizing EnSC, including Golgi fragmentation, collagen accumulation in dilated Endoplasmic Reticulum cisternae, and overall decreased protein secretion. Thus, both CREB3L1 and CREB3L2 are required for Golgi reshaping and efficient protein secretion, and, as such, for successful decidualization.
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RNA binding proteins and messenger RNAs (mRNAs) assemble into ribonucleoprotein granules that regulate mRNA trafficking, local translation, and turnover. The dysregulation of RNA-protein condensation disturbs synaptic plasticity and neuron survival and has been widely associated with human neurological disease. Neuronal granules are thought to condense around particular proteins that dictate the identity and composition of each granule type. Here, we show in Drosophila that a previously uncharacterized long noncoding RNA, mimi, is required to scaffold large neuronal granules in the adult nervous system. Neuronal ELAV-like proteins directly bind mimi and mediate granule assembly, while Staufen maintains condensate integrity. mimi granules contain mRNAs and proteins involved in synaptic processes; granule loss in mimi mutant flies impairs nervous system maturity and neuropeptide-mediated signaling and causes phenotypes of neurodegeneration. Our work reports an architectural RNA for a neuronal granule and provides a handle to interrogate functions of a condensate independently of those of its constituent proteins.
Assuntos
Neuropeptídeos , RNA Longo não Codificante , Grânulos de Ribonucleoproteínas Citoplasmáticas , Humanos , Neurônios/fisiologia , Neuropeptídeos/metabolismo , RNA/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
The precise regulation of RNA Polymerase II (Pol II) transcription after genotoxic stress is crucial for proper execution of the DNA damage-induced stress response. While stalling of Pol II on transcription-blocking lesions (TBLs) blocks transcript elongation and initiates DNA repair in cis, TBLs additionally elicit a response in trans that regulates transcription genome-wide. Here we uncover that, after an initial elongation block in cis, TBLs trigger the genome-wide VCP-mediated proteasomal degradation of promoter-bound, P-Ser5-modified Pol II in trans. This degradation is mechanistically distinct from processing of TBL-stalled Pol II, is signaled via GSK3, and contributes to the TBL-induced transcription block, even in transcription-coupled repair-deficient cells. Thus, our data reveal the targeted degradation of promoter-bound Pol II as a critical pathway that allows cells to cope with DNA damage-induced transcription stress and enables the genome-wide adaptation of transcription to genotoxic stress.
Assuntos
Quinase 3 da Glicogênio Sintase , Transcrição Gênica , Dano ao DNA/genética , Reparo do DNA/genética , Quinase 3 da Glicogênio Sintase/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismoRESUMO
One of the critical events that regulates muscle cell differentiation is the replacement of the lamin B receptor (LBR)-tether with the lamin A/C (LMNA)-tether to remodel transcription and induce differentiation-specific genes. Here, we report that localization and activity of the LBR-tether are crucially dependent on the muscle-specific chaperone HSPB3 and that depletion of HSPB3 prevents muscle cell differentiation. We further show that HSPB3 binds to LBR in the nucleoplasm and maintains it in a dynamic state, thus promoting the transcription of myogenic genes, including the genes to remodel the extracellular matrix. Remarkably, HSPB3 overexpression alone is sufficient to induce the differentiation of two human muscle cell lines, LHCNM2 cells, and rhabdomyosarcoma cells. We also show that mutant R116P-HSPB3 from a myopathy patient with chromatin alterations and muscle fiber disorganization, forms nuclear aggregates that immobilize LBR. We find that R116P-HSPB3 is unable to induce myoblast differentiation and instead activates the unfolded protein response. We propose that HSPB3 is a specialized chaperone engaged in muscle cell differentiation and that dysfunctional HSPB3 causes neuromuscular disease by deregulating LBR.
Assuntos
Proteínas de Choque Térmico Pequenas/genética , Proteínas de Choque Térmico/metabolismo , Desenvolvimento Muscular/imunologia , Músculo Esquelético/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Linhagem Celular , Células HeLa , Humanos , Músculo Esquelético/citologia , Transfecção , Receptor de Lamina BRESUMO
Haematopoietic stem cells (HSCs) are normally quiescent, but have evolved mechanisms to respond to stress. Here, we evaluate haematopoietic regeneration induced by chemotherapy. We detect robust chromatin reorganization followed by increased transcription of transposable elements (TEs) during early recovery. TE transcripts bind to and activate the innate immune receptor melanoma differentiation-associated protein 5 (MDA5) that generates an inflammatory response that is necessary for HSCs to exit quiescence. HSCs that lack MDA5 exhibit an impaired inflammatory response after chemotherapy and retain their quiescence, with consequent better long-term repopulation capacity. We show that the overexpression of ERV and LINE superfamily TE copies in wild-type HSCs, but not in Mda5-/- HSCs, results in their cycling. By contrast, after knockdown of LINE1 family copies, HSCs retain their quiescence. Our results show that TE transcripts act as ligands that activate MDA5 during haematopoietic regeneration, thereby enabling HSCs to mount an inflammatory response necessary for their exit from quiescence.
Assuntos
Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Elementos de DNA Transponíveis , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Helicase IFIH1 Induzida por Interferon/metabolismo , Agonistas Mieloablativos/farmacologia , Animais , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Retrovirus Endógenos/genética , Ativação Enzimática , Células HEK293 , Células-Tronco Hematopoéticas/enzimologia , Humanos , Helicase IFIH1 Induzida por Interferon/genética , Ligantes , Elementos Nucleotídeos Longos e Dispersos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
Genome-wide association studies identify genomic variants associated with human traits and diseases. Most trait-associated variants are located within cell-type-specific enhancers, but the molecular mechanisms governing phenotypic variation are less well understood. Here, we show that many enhancer variants associated with red blood cell (RBC) traits map to enhancers that are co-bound by lineage-specific master transcription factors (MTFs) and signaling transcription factors (STFs) responsive to extracellular signals. The majority of enhancer variants reside on STF and not MTF motifs, perturbing DNA binding by various STFs (BMP/TGF-ß-directed SMADs or WNT-induced TCFs) and affecting target gene expression. Analyses of engineered human blood cells and expression quantitative trait loci verify that disrupted STF binding leads to altered gene expression. Our results propose that the majority of the RBC-trait-associated variants that reside on transcription-factor-binding sequences fall in STF target sequences, suggesting that the phenotypic variation of RBC traits could stem from altered responsiveness to extracellular stimuli.
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Eritrócitos/fisiologia , Regulação da Expressão Gênica/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Eritrócitos/citologia , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Humanos , Locos de Características Quantitativas/genética , Proteína Smad1/genética , Proteína Smad1/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica/genéticaRESUMO
Proteotoxic stress such as heat shock causes heat-shock factor (HSF)-dependent transcriptional upregulation of chaperones. Heat shock also leads to a rapid and reversible downregulation of many genes, a process we term stress-induced transcriptional attenuation (SITA). The mechanism underlying this conserved phenomenon is unknown. Here we report that enhanced recruitment of negative transcription elongation factors to gene promoters in human cell lines induces SITA. A chemical inhibitor screen showed that active translation and protein ubiquitination are required for the response. We further find that proteins translated during heat shock are subjected to ubiquitination and that p38 kinase signaling connects cytosolic translation with gene downregulation. Notably, brain samples of subjects with Huntington's disease also show transcriptional attenuation, which is recapitulated in cellular models of protein aggregation similar to heat shock. Thus our work identifies an HSF-independent mechanism that links nascent-protein ubiquitination to transcriptional downregulation during heat shock, with potential ramifications in neurodegenerative diseases.
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Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/fisiologia , Ubiquitinação/fisiologia , Citosol/metabolismo , Regulação para Baixo , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico/genética , Humanos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Ubiquitinação/genéticaRESUMO
Molecular chaperones such as heat-shock proteins (HSPs) help in protein folding. Their function in the cytosol has been well studied. Notably, chaperones are also present in the nucleus, a compartment where proteins enter after completing de novo folding in the cytosol, and this raises an important question about chaperone function in the nucleus. We performed a systematic analysis of the nuclear pool of heat-shock protein 90. Three orthogonal and independent analyses led us to the core functional interactome of HSP90. Computational and biochemical analyses identify host cell factor C1 (HCFC1) as a transcriptional regulator that depends on HSP90 for its stability. HSP90 was required to maintain the expression of HCFC1-targeted cell-cycle genes. The regulatory nexus between HSP90 and the HCFC1 module identified in this study sheds light on the relevance of chaperones in the transcription of cell-cycle genes. Our study also suggests a therapeutic avenue of combining chaperone and transcription inhibitors for cancer treatment.
Assuntos
Cromatina/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Genes cdc , Proteínas de Choque Térmico HSP90/metabolismo , Fator C1 de Célula Hospedeira/metabolismo , Animais , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células/genética , Sequenciamento de Cromatina por Imunoprecipitação , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Citosol/metabolismo , Bases de Dados Genéticas , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Fator C1 de Célula Hospedeira/genética , Humanos , Camundongos , Ligação Proteica , Mapas de Interação de Proteínas , RNA-SeqRESUMO
Interrogation and control of cellular fate and function using optogenetics is providing revolutionary insights into biology. Optogenetic control of cells is achieved by coupling genetically encoded photoreceptors to cellular effectors and enables unprecedented spatiotemporal control of signaling processes. Here, a fast and reversibly switchable photoreceptor is used to tune the mechanical properties of polymer materials in a fully reversible, wavelength-specific, and dose- and space-controlled manner. By integrating engineered cyanobacterial phytochrome 1 into a poly(ethylene glycol) matrix, hydrogel materials responsive to light in the cell-compatible red/far-red spectrum are synthesized. These materials are applied to study in human mesenchymal stem cells how different mechanosignaling pathways respond to changing mechanical environments and to control the migration of primary immune cells in 3D. This optogenetics-inspired matrix allows fundamental questions of how cells react to dynamic mechanical environments to be addressed. Further, remote control of such matrices can create new opportunities for tissue engineering or provide a basis for optically stimulated drug depots.
RESUMO
Understanding how genotypes are linked to phenotypes is important in biomedical and evolutionary studies. The chaperone heat-shock protein 90 (HSP90) buffers genetic variation by stabilizing proteins with variant sequences, thereby uncoupling phenotypes from genotypes. Here we report an unexpected role of HSP90 in buffering cis-regulatory variation affecting gene expression. By using the tripartite-motif-containing 28 (TRIM28; also known as KAP1)-mediated epigenetic pathway, HSP90 represses the regulatory influence of endogenous retroviruses (ERVs) on neighboring genes that are critical for mouse development. Our data based on natural variations in the mouse genome show that genes respond to HSP90 inhibition in a manner dependent on their genomic location with regard to strain-specific ERV-insertion sites. The evolutionary-capacitor function of HSP90 may thus have facilitated the exaptation of ERVs as key modifiers of gene expression and morphological diversification. Our findings add a new regulatory layer through which HSP90 uncouples phenotypic outcomes from individual genotypes.
Assuntos
Retrovirus Endógenos/metabolismo , Evolução Molecular , Proteínas de Choque Térmico HSP90/metabolismo , Mamíferos/genética , Animais , Pareamento de Bases/genética , Sequência de Bases , Elementos de DNA Transponíveis/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Anotação de Sequência Molecular , Mutagênese Insercional/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína 28 com Motivo Tripartido , Regulação para Cima/genéticaRESUMO
BACKGROUND: Mood stabilizers appear to be more potent in treating mania than depression. The anticonvulsant lamotrigine has been shown to be effective for bipolar depression. This study examines putative antidepressive properties of lamotrigine in a mainly unipolar routine clinical patient population. METHOD: Forty patients with a depressive episode (DSM-IV criteria) requiring psychiatric intervention received lamotrigine or placebo using a fixed dose escalation scheme with a target dose of 200 mg/day for 9 weeks. Additionally, all patients were treated with paroxetine. Hamilton Rating Scale for Depression (HAM-D) and Clinical Global Impressions scale (CGI) ratings were used to monitor therapeutic efficacy. RESULTS: Adjunctive treatment with lamotrigine did not result in a significant difference in HAM-D total score at the endpoint of the study when compared with paroxetine alone. However, lamotrigine demonstrated significant efficacy on core depressive symptoms as reflected by HAM-D items 1 (depressed mood; p = .0019), 2 (guilt feelings; p = .0011), and 7 (work and interest; p = .049) and the CGI-Severity of Illness scale (p < .0001). Patients receiving lamotrigine had fewer days on treatment with benzodiazepines and fewer withdrawals for treatment failure. Lamotrigine appeared to accelerate the onset of action of the antidepressant. Two patients on lamotrigine treatment developed neutropenia, and 1 developed a benign rash. There was no detectable pharmacokinetic interaction between lamotrigine and paroxetine. CONCLUSION: Lamotrigine might have antidepressive properties in unipolar patients and may accelerate onset of action when given in combination with typical antidepressants.
Assuntos
Anticonvulsivantes/uso terapêutico , Transtorno Depressivo/tratamento farmacológico , Paroxetina/uso terapêutico , Inibidores Seletivos de Recaptação de Serotonina/uso terapêutico , Triazinas/uso terapêutico , Doença Aguda , Adolescente , Adulto , Idoso , Transtorno Depressivo/diagnóstico , Método Duplo-Cego , Quimioterapia Combinada , Feminino , Humanos , Lamotrigina , Masculino , Pessoa de Meia-Idade , Placebos , Escalas de Graduação Psiquiátrica , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
The major threat in prostate cancer is the occurrence of metastases in androgen-independent tumor stage, for which no causative cure is available. Here we show that metastatic behavior of androgen-independent prostate tumor cells requires the protein-kinase-C-related kinase (PRK1/PKN1) in vitro and in vivo. PRK1 regulates cell migration and gene expression through its kinase activity, but does not affect cell proliferation. Transcriptome and interactome analyses uncover that PRK1 regulates expression of migration-relevant genes by interacting with the scaffold protein sperm-associated antigen 9 (SPAG9/JIP4). SPAG9 and PRK1 colocalize in human cancer tissue and are required for p38-phosphorylation and cell migration. Accordingly, depletion of either ETS domain-containing protein Elk-1 (ELK1), an effector of p38-signalling or p38 depletion hinders cell migration and changes expression of migration-relevant genes as observed upon PRK1-depletion. Importantly, a PRK1 inhibitor prevents metastases in mice, showing that the PRK1-pathway is a promising target to hamper prostate cancer metastases in vivo. Here we describe a novel mechanism controlling the metastatic behavior of PCa cells and identify PRK1 as a promising therapeutic target to treat androgen-independent metastatic prostate cancer.
Assuntos
Androgênios/metabolismo , Movimento Celular/fisiologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteína Quinase C/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Proliferação de Células/fisiologia , Humanos , Masculino , Camundongos , Metástase Neoplásica , Fosforilação , Neoplasias da Próstata/genética , Proteína Quinase C/genética , Transcriptoma , TransfecçãoRESUMO
Despite significant methodological advances in protein structure determination high-resolution structures of membrane proteins are still rare, leaving sequence-based predictions as the only option for exploring the structural variability of membrane proteins at large scale. Here, a new structural classification approach for α-helical membrane proteins is introduced based on the similarity of predicted helix interaction patterns. Its application to proteins with known 3D structure showed that it is able to reliably detect structurally similar proteins even in the absence of any sequence similarity, reproducing the SCOP and CATH classifications with a sensitivity of 65% at a specificity of 90%. We applied the new approach to enhance our comprehensive structural classification of α-helical membrane proteins (CAMPS), which is primarily based on sequence and topology similarity, in order to find protein clusters that describe the same fold in the absence of sequence similarity. The total of 151 helix architectures were delineated for proteins with more than four transmembrane segments. Interestingly, we observed that proteins with 8 and more transmembrane helices correspond to fewer different architectures than proteins with up to 7 helices, suggesting that in large membrane proteins the evolutionary tendency to re-use already available folds is more pronounced.