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1.
Nat Commun ; 11(1): 4025, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788667

RESUMO

Droplet-based high throughput single cell sequencing techniques tremendously advanced our insight into cell-to-cell heterogeneity. However, those approaches only allow analysis of one extremity of the transcript after short read sequencing. In consequence, information on splicing and sequence heterogeneity is lost. To overcome this limitation, several approaches that use long-read sequencing were introduced recently. Yet, those techniques are limited by low sequencing depth and/or lacking or inaccurate assignment of unique molecular identifiers (UMIs), which are critical for elimination of PCR bias and artifacts. We introduce ScNaUmi-seq, an approach that combines the high throughput of Oxford Nanopore sequencing with an accurate cell barcode and UMI assignment strategy. UMI guided error correction allows to generate high accuracy full length sequence information with the 10x Genomics single cell isolation system at high sequencing depths. We analyzed transcript isoform diversity in embryonic mouse brain and show that ScNaUmi-seq allows defining splicing and SNVs (RNA editing) at a single cell level.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento por Nanoporos , Nanoporos , Transcriptoma , Animais , Encéfalo , Expressão Gênica , Perfilação da Expressão Gênica , Genômica , Camundongos , Camundongos Endogâmicos C57BL , Isoformas de Proteínas , Receptores de AMPA/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos
2.
Nat Commun ; 11(1): 4067, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792493

RESUMO

The brain is organized morphologically and functionally into a columnar structure. According to the radial unit hypothesis, neurons from the same lineage form a radial unit that contributes to column formation. However, the molecular mechanisms that link neuronal lineage and column formation remain elusive. Here, we show that neurons from the same lineage project to different columns under control of Down syndrome cell adhesion molecule (Dscam) in the fly brain. Dscam1 is temporally expressed in newly born neuroblasts and is inherited by their daughter neurons. The transient transcription of Dscam1 in neuroblasts enables the expression of the same Dscam1 splice isoform within cells of the same lineage, causing lineage-dependent repulsion. In the absence of Dscam1 function, neurons from the same lineage project to the same column. When the splice diversity of Dscam1 is reduced, column formation is significantly compromised. Thus, Dscam1 controls column formation through lineage-dependent repulsion.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas de Drosophila/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Axônios/metabolismo , Moléculas de Adesão Celular/genética , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurogênese/genética , Neurogênese/fisiologia , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
PLoS Genet ; 16(7): e1008923, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32735630

RESUMO

Mitochondrial translation defects can be due to mutations affecting mitochondrial- or nuclear-encoded components. The number of known nuclear genes involved in mitochondrial translation has significantly increased in the past years. RCC1L (WBSCR16), a putative GDP/GTP exchange factor, has recently been described to interact with the mitochondrial large ribosomal subunit. In humans, three different RCC1L isoforms have been identified that originate from alternative splicing but share the same N-terminus, RCC1LV1, RCC1LV2 and RCC1LV3. All three isoforms were exclusively localized to mitochondria, interacted with its inner membrane and could associate with homopolymeric oligos to different extent. Mitochondrial immunoprecipitation experiments showed that RCC1LV1 and RCC1LV3 associated with the mitochondrial large and small ribosomal subunit, respectively, while no significant association was observed for RCC1LV2. Overexpression and silencing of RCC1LV1 or RCC1LV3 led to mitoribosome biogenesis defects that resulted in decreased translation. Indeed, significant changes in steady-state levels and distribution on isokinetic sucrose gradients were detected not only for mitoribosome proteins but also for GTPases, (GTPBP10, ERAL1 and C4orf14), and pseudouridylation proteins, (TRUB2, RPUSD3 and RPUSD4). All in all, our data suggest that RCC1L is essential for mitochondrial function and that the coordination of at least two isoforms is essential for proper ribosomal assembly.


Assuntos
GTP Fosfo-Hidrolases/genética , Proteínas Mitocondriais/genética , Isoformas de Proteínas/genética , Proteínas Ribossômicas/genética , Proteínas de Ligação ao GTP/genética , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Mitocôndrias/genética , Ribossomos Mitocondriais/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Biossíntese de Proteínas/genética , RNA/genética , Proteínas de Ligação a RNA/genética
4.
Proc Natl Acad Sci U S A ; 117(29): 17409-17417, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32616567

RESUMO

Proteolytic cascades regulate immunity and development in animals, but these cascades in plants have not yet been reported. Here we report that the extracellular immune protease Rcr3 of tomato is activated by P69B and other subtilases (SBTs), revealing a proteolytic cascade regulating extracellular immunity in solanaceous plants. Rcr3 is a secreted papain-like Cys protease (PLCP) of tomato that acts both in basal resistance against late blight disease (Phytophthora infestans) and in gene-for-gene resistance against the fungal pathogen Cladosporium fulvum (syn. Passalora fulva) Despite the prevalent model that Rcr3-like proteases can activate themselves at low pH, we found that catalytically inactive proRcr3 mutant precursors are still processed into mature mRcr3 isoforms. ProRcr3 is processed by secreted P69B and other Asp-selective SBTs in solanaceous plants, providing robust immunity through SBT redundancy. The apoplastic effector EPI1 of P. infestans can block Rcr3 activation by inhibiting SBTs, suggesting that this effector promotes virulence indirectly by preventing the activation of Rcr3(-like) immune proteases. Rcr3 activation in Nicotiana benthamiana requires a SBT from a different subfamily, indicating that extracellular proteolytic cascades have evolved convergently in solanaceous plants or are very ancient in the plant kingdom. The frequent incidence of Asp residues in the cleavage region of Rcr3-like proteases in solanaceous plants indicates that activation of immune proteases by SBTs is a general mechanism, illuminating a proteolytic cascade that provides robust apoplastic immunity.


Assuntos
Lycopersicon esculentum/metabolismo , Peptídeo Hidrolases/metabolismo , Doenças das Plantas/imunologia , Imunidade Vegetal , Proteólise , Cladosporium , Lycopersicon esculentum/genética , Peptídeo Hidrolases/genética , Phytophthora infestans , Doenças das Plantas/parasitologia , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/metabolismo , Isoformas de Proteínas , Virulência
5.
Anticancer Res ; 40(7): 3781-3792, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32620617

RESUMO

BACKGROUND/AIM: Canine B-cell lymphoma represents a useful in vivo model for human diffuse large B-cell lymphoma (DLBCL). Pan-Bromodomain and extra-terminal (BET) inhibition targeting BRD2/3/4 and selective inhibition of BRD4, as well as spleen tyrosine kinase (SYK) inhibition, are currently evaluated as haematologic cancer therapy. Herein, we characterized the differences in the biologic response of isoform-specific or pan-BET inhibition alone or in combination with SYK inhibition. MATERIALS AND METHODS: I-BET151 (pan-inhibitor) and AZD5153 (BRD4 inhibitor) were combined with Entospletinib (SYK inhibitor) and comparatively analysed in the canine DLBCL cell line CLBL-1. Dose- and time-dependent cellular responses were analysed by cell number, metabolic activity, apoptosis/necrosis, and cell morphology. The synergistic potential was evaluated through the Bliss independence model. RESULTS: I-BET151 and AZD5153 showed significant dose- and time-dependent inhibitory effects. Adding Entospletinib to I-BET151 or AZD5153 had no additional synergistic effects. CONCLUSION: Entospletinib did not enhance the inhibitory effects of the pan- or isoform-specific BET.


Assuntos
Indazóis/farmacologia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Isoformas de Proteínas/antagonistas & inibidores , Pirazinas/farmacologia , Animais , Linhagem Celular Tumoral , Cães , Compostos Heterocíclicos com 2 Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Linfoma Difuso de Grandes Células B/metabolismo , Piperazinas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Quinase Syk/metabolismo
6.
Mol Cell ; 79(3): 425-442.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32615088

RESUMO

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.


Assuntos
Adenosina/análogos & derivados , Neoplasias de Cabeça e Pescoço/genética , Metiltransferases/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adenosina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Bleomicina/farmacologia , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Células HEK293 , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Hibridização de Ácido Nucleico , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Nat Struct Mol Biol ; 27(8): 763-767, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32647346

RESUMO

SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. Here, we investigate the relationship of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related bat virus, RaTG13. We determined cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; we compared these with recently reported structures for uncleaved SARS-CoV-2 S. We also biochemically characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and about 1,000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor.


Assuntos
Betacoronavirus/genética , Interações Hospedeiro-Patógeno/genética , Peptidil Dipeptidase A/química , Receptores Virais/química , Glicoproteína da Espícula de Coronavírus/química , Animais , Betacoronavirus/metabolismo , Betacoronavirus/ultraestrutura , Sítios de Ligação , Quirópteros/virologia , Infecções por Coronavirus/virologia , Microscopia Crioeletrônica , Evolução Molecular , Furina/química , Expressão Gênica , Células HEK293 , Humanos , Modelos Moleculares , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Proteólise , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Homologia Estrutural de Proteína
8.
PLoS Comput Biol ; 16(7): e1007801, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32628657

RESUMO

Recent experiments with super-resolution live cell microscopy revealed that nonmuscle myosin II minifilaments are much more dynamic than formerly appreciated, often showing plastic processes such as splitting, concatenation and stacking. Here we combine sequence information, electrostatics and elasticity theory to demonstrate that the parallel staggers at 14.3, 43.2 and 72 nm have a strong tendency to splay their heads away from the minifilament, thus potentially initiating the diverse processes seen in live cells. In contrast, the straight antiparallel stagger with an overlap of 43 nm is very stable and likely initiates minifilament nucleation. Using stochastic dynamics in a newly defined energy landscape, we predict that the optimal parallel staggers between the myosin rods are obtained by a trial-and-error process in which two rods attach and re-attach at different staggers by rolling and zipping motion. The experimentally observed staggers emerge as the configurations with the largest contact times. We find that contact times increase from isoforms C to B to A, that A-B-heterodimers are surprisingly stable and that myosin 18A should incorporate into mixed filaments with a small stagger. Our findings suggest that nonmuscle myosin II minifilaments in the cell are first formed by isoform A and then convert to mixed A-B-filaments, as observed experimentally.


Assuntos
Miosina Tipo II , Eletricidade Estática , Biologia Computacional , Humanos , Modelos Moleculares , Miosina Tipo II/química , Miosina Tipo II/metabolismo , Miosina Tipo II/ultraestrutura , Conformação Proteica , Isoformas de Proteínas
9.
Tumour Biol ; 42(7): 1010428320937863, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32686600

RESUMO

Maintaining intracellular pH is crucial for preserving healthy cellular behavior and, when dysregulated, results in increased proliferation, migration, and invasion. The Na+/H+ exchanger isoform 1 is a highly regulated transmembrane antiporter that maintains pH homeostasis by exporting protons in response to intra- and extracellular signals. Activation of Na+/H+ exchanger isoform 1 is exquisitely regulated by the extracellular environment and protein cofactors, including calcineurin B homologous proteins 1 and 2. While Na+/H+ exchanger isoform 1 and calcineurin B homologous protein 1 are ubiquitously expressed, calcineurin B homologous protein 2 shows tissue-specific expression and upregulation in a variety of cancer cells. In addition, calcineurin B homologous protein 2 expression is modulated by tumorigenic extracellular conditions like low nutrients. To understand the role of calcineurin B homologous protein 2 in tumorigenesis and survival in lung cancer, we surveyed existing databases and formed a comprehensive report of Na+/H+ exchanger isoform 1, calcineurin B homologous protein 1, and calcineurin B homologous protein 2 expression in diseased and non-diseased tissues. We show that calcineurin B homologous protein 2 is upregulated during oncogenesis in many adeno and squamous carcinomas. To understand the functional role of calcineurin B homologous protein 2 upregulation, we evaluated the effect of Na+/H+ exchanger isoform 1 and calcineurin B homologous protein 2 depletion on cellular function during cancer progression in situ. Here, we show that calcineurin B homologous protein 2 functions through Na+/H+ exchanger isoform 1 to effect cell proliferation, cell migration, steady-state pHi, and anchorage-independent tumor growth. Finally, we present evidence that calcineurin B homologous protein 2 depletion in vivo has potential to reduce tumor burden in a xenograft model. Together, these data support the tumor-promoting potential of aberrant calcineurin B homologous protein 2 expression and position calcineurin B homologous protein 2 as a potential therapeutic target for the treatment of non-small cell lung cancer.


Assuntos
Calcineurina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Calcineurina/genética , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Nus , Transplante de Neoplasias , Isoformas de Proteínas/metabolismo , Transplante Heterólogo
10.
PLoS One ; 15(6): e0232946, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32484809

RESUMO

High throughput sequencing of RNA (RNA-Seq) has become a staple in modern molecular biology, with applications not only in quantifying gene expression but also in isoform-level analysis of the RNA transcripts. To enable such an isoform-level analysis, a transcriptome assembly algorithm is utilized to stitch together the observed short reads into the corresponding transcripts. This task is complicated due to the complexity of alternative splicing - a mechanism by which the same gene may generate multiple distinct RNA transcripts. We develop a novel genome-guided transcriptome assembler, RefShannon, that exploits the varying abundances of the different transcripts, in enabling an accurate reconstruction of the transcripts. Our evaluation shows RefShannon is able to improve sensitivity effectively (up to 22%) at a given specificity in comparison with other state-of-the-art assemblers. RefShannon is written in Python and is available from Github (https://github.com/shunfumao/RefShannon).


Assuntos
Biologia Computacional/métodos , Transcriptoma , Processamento Alternativo , Simulação por Computador , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Rim/metabolismo , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Análise de Sequência de RNA , Software
11.
Mol Cell ; 78(6): 993-995, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32559427

RESUMO

In this issue of Molecular Cell, Wu et al. (2020) describe studies that establish oncogenic versus tumor-suppressive functions of two BRD4 isoforms in the regulation of gene expression and breast cancer development.


Assuntos
Proteínas Nucleares/genética , Fatores de Transcrição/genética , Proteínas de Ciclo Celular , Oncogenes , Isoformas de Proteínas
12.
N Engl J Med ; 382(24): 2337-2343, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32521134

RESUMO

We describe a case of life-threatening disseminated coccidioidomycosis in a previously healthy child. Like most patients with disseminated coccidioidomycosis, this child had no genomic evidence of any known, rare immune disease. However, comprehensive immunologic testing showed exaggerated production of interleukin-4 and reduced production of interferon-γ. Supplementation of antifungal agents with interferon-γ treatment slowed disease progression, and the addition of interleukin-4 and interleukin-13 blockade with dupilumab resulted in rapid resolution of the patient's clinical symptoms. This report shows that blocking of type 2 immune responses can treat infection. This immunomodulatory approach could be used to enhance immune clearance of refractory fungal, mycobacterial, and viral infections. (Supported by the Jeffrey Modell Foundation and the National Institutes of Health.).


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Antifúngicos/uso terapêutico , Coccidioidomicose/tratamento farmacológico , Interferon gama/uso terapêutico , Encéfalo/diagnóstico por imagem , Pré-Escolar , Coccidioidomicose/imunologia , Progressão da Doença , Quimioterapia Combinada , Humanos , Interferon gama/metabolismo , Interleucina-12/metabolismo , Interleucina-13/antagonistas & inibidores , Interleucina-4/antagonistas & inibidores , Interleucina-4/metabolismo , Imagem por Ressonância Magnética , Masculino , Isoformas de Proteínas , Receptores de Interleucina-12/química , Receptores de Interleucina-12/genética , Coluna Vertebral/diagnóstico por imagem , Células Th1/imunologia
14.
Proc Natl Acad Sci U S A ; 117(27): 15702-15711, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32576691

RESUMO

Mammalian cells contain two isoforms of RNA polymerase III (Pol III) that differ in only a single subunit, with POLR3G in one form (Pol IIIα) and the related POLR3GL in the other form (Pol IIIß). Previous research indicates that POLR3G and POLR3GL are differentially expressed, with POLR3G expression being highly enriched in embryonic stem cells (ESCs) and tumor cells relative to the ubiquitously expressed POLR3GL. To date, the functional differences between these two subunits remain largely unexplored, especially in vivo. Here, we show that POLR3G and POLR3GL containing Pol III complexes bind the same target genes and assume the same functions both in vitro and in vivo and, to a significant degree, can compensate for each other in vivo. Notably, an observed defect in the differentiation ability of POLR3G knockout ESCs can be rescued by exogenous expression of POLR3GL. Moreover, whereas POLR3G knockout mice die at a very early embryonic stage, POLR3GL knockout mice complete embryonic development without noticeable defects but die at about 3 wk after birth with signs of both general growth defects and potential cerebellum-related neuronal defects. The different phenotypes of the knockout mice likely reflect differential expression levels of POLR3G and POLR3GL across developmental stages and between tissues and insufficient amounts of total Pol III in vivo.


Assuntos
Cerebelo/crescimento & desenvolvimento , Desenvolvimento Embrionário/genética , Neurônios/metabolismo , RNA Polimerase III/genética , Animais , Sítios de Ligação/genética , Diferenciação Celular/genética , Cerebelo/patologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Camundongos , Camundongos Knockout , Neurônios/patologia , Ligação Proteica/genética , Isoformas de Proteínas/genética , Subunidades Proteicas/genética
15.
Nat Commun ; 11(1): 3182, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576858

RESUMO

Most eukaryotic genes produce alternative polyadenylation (APA) isoforms. Here we report that, unlike previously characterized cell lineages, differentiation of syncytiotrophoblast (SCT), a cell type critical for hormone production and secretion during pregnancy, elicits widespread transcript shortening through APA in 3'UTRs and in introns. This global APA change is observed in multiple in vitro trophoblast differentiation models, and in single cells from placentas at different stages of pregnancy. Strikingly, the transcript shortening is unrelated to cell proliferation, a feature previously associated with APA control, but instead accompanies increased secretory functions. We show that 3'UTR shortening leads to transcripts with higher mRNA stability, which augments transcriptional activation, especially for genes involved in secretion. Moreover, this mechanism, named secretion-coupled APA (SCAP), is also executed in B cell differentiation to plasma cells. Together, our data indicate that SCAP tailors the transcriptome during formation of secretory cells, boosting their protein production and secretion capacity.


Assuntos
Diferenciação Celular/fisiologia , Poliadenilação/fisiologia , Transporte Proteico/fisiologia , Transcriptoma , Regiões 3' não Traduzidas , Diferenciação Celular/genética , Linhagem da Célula , Proliferação de Células , Células-Tronco Embrionárias , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Isoformas de Proteínas , Transporte Proteico/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo
16.
PLoS Genet ; 16(6): e1008829, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32502151

RESUMO

Ion channels are present at specific levels within subcellular compartments of excitable cells. The regulation of ion channel trafficking and targeting is an effective way to control cell excitability. The BK channel is a calcium-activated potassium channel that serves as a negative feedback mechanism at presynaptic axon terminals and sites of muscle excitation. The C. elegans BK channel ortholog, SLO-1, requires an endoplasmic reticulum (ER) membrane protein for efficient anterograde transport to these locations. Here, we found that, in the absence of this ER membrane protein, SLO-1 channels that are seemingly normally folded and expressed at physiological levels undergo SEL-11/HRD1-mediated ER-associated degradation (ERAD). This SLO-1 degradation is also indirectly regulated by a SKN-1A/NRF1-mediated transcriptional mechanism that controls proteasome levels. Therefore, our data indicate that SLO-1 channel density is regulated by the competitive balance between the efficiency of ER trafficking machinery and the capacity of ERAD.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Ligação a DNA/metabolismo , Degradação Associada com o Retículo Endoplasmático/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Terminações Pré-Sinápticas/metabolismo , Fatores de Transcrição/metabolismo , Aldicarb/farmacologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Acoplamento Excitação-Contração/efeitos dos fármacos , Acoplamento Excitação-Contração/genética , Retroalimentação Fisiológica/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Músculos/inervação , Terminações Pré-Sinápticas/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma , Isoformas de Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo
17.
Life Sci ; 256: 117912, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32504755

RESUMO

Histone deacetylase enzymes were prominent chromatin remodeling drug that targets in the pathophysiology of Alzheimer's disease associated with transcriptional dysregulation. In vitro and in vivo models of AD have demonstrated overexpression of HDAC activity. Non-specificity and non-selectivity of HDAC are the major problems of existing HDAC inhibitors. Hence, we aim to set up a methodology describing the rational development of isoform-selective HDAC inhibitor targeting class, I and class IIb. A convenient multistage virtual screening followed by machine learning and IC50 screenings were used to classify the 5064 compounds into inhibitors and non-inhibitors classes retrieved from the ChEMBL database. ADMET analysis identified the pharmacokinetics and pharmacodynamics properties of selected compounds. Molecular docking, along with mutational analysis of eleven compounds, characterized the inhibiting potency. Herein, for the first time, we reported ChEMBL1834473 (2-[[5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl]amino]-N-hydroxypyrimidine-5-carboxamide) as the isoform-selective HDAC inhibitor, which interact central Zn2+ atom. The negative energy and interacting residue of the ChEMBL1834473 with six HDAC isoform has also been tabulated and mapped. Moreover, our findings concluded histidine, glycine, phenylalanine, and aspartic acid as key residues in protein-ligand interaction and classify 2347 compounds as HDAC inhibitors. Later, a protein-protein interaction network of six HDAC with the key proteins involved in the progression of an AD and signaling pathway, which describes the relationship between ChEMBL1834473 and AD, has been demonstrated using PPI network where the chosen inhibitor will work. Altogether, we conclude that the compound ChEMBL1834473 may be capable of inhibiting all isoforms of class I and class IIb HDAC based on computational analysis for AD therapeutics.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/uso terapêutico , Pirimidinas/química , Pirimidinas/uso terapêutico , Ácido Aspártico/metabolismo , Bases de Dados de Compostos Químicos , Avaliação Pré-Clínica de Medicamentos , Glicina/metabolismo , Histona Desacetilases/metabolismo , Humanos , Aprendizado de Máquina , Simulação de Acoplamento Molecular , Estrutura Molecular , Terapia de Alvo Molecular , Fenilalanina/metabolismo , Isoformas de Proteínas/química , Termodinâmica , Zinco/metabolismo
18.
Proc Natl Acad Sci U S A ; 117(27): 15554-15564, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32561649

RESUMO

The serum haptoglobin protein (Hp) scavenges toxic hemoglobin (Hb) leaked into the bloodstream from erythrocytes. In humans, there are two frequently occurring allelic forms of Hp, resulting in three genotypes: Homozygous Hp 1-1 and Hp 2-2, and heterozygous Hp 2-1. The Hp genetic polymorphism has an intriguing effect on the quaternary structure of Hp. The simplest form, Hp 1-1, forms dimers consisting of two α1ß units, connected by disulfide bridges. Hp 2-1 forms mixtures of linear (α1)2(α2)n-2(ß)n oligomers (n > 1) while Hp 2-2 occurs in cyclic (α2)n(ß)n oligomers (n > 2). Different Hp genotypes bind Hb with different affinities, with Hp 2-2 being the weakest binder. This behavior has a significant influence on Hp's antioxidant capacity, with potentially distinctive personalized clinical consequences. Although Hp has been studied extensively in the past, the finest molecular details of the observed differences in interactions between Hp and Hb are not yet fully understood. Here, we determined the full proteoform profiles and proteoform assemblies of all three most common genetic Hp variants. We combined several state-of-the-art analytical methods, including various forms of chromatography, mass photometry, and different tiers of mass spectrometry, to reveal how the tens to hundreds distinct proteoforms and their assemblies influence Hp's capacity for Hb binding. We extend the current knowledge by showing that Hb binding does not just depend on the donor's genotype, but is also affected by variations in Hp oligomerization, glycosylation, and proteolytic processing of the Hp α-chain.


Assuntos
Haptoglobinas/genética , Hemoglobinas/metabolismo , Alelos , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/metabolismo , Glicosilação , Haptoglobinas/química , Haptoglobinas/isolamento & purificação , Haptoglobinas/metabolismo , Hemoglobinas/toxicidade , Humanos , Espectrometria de Massas , Modelos Moleculares , Estrutura Molecular , Polimorfismo Genético , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Relação Estrutura-Atividade
19.
Proc Natl Acad Sci U S A ; 117(27): 15895-15901, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571932

RESUMO

In eukaryotic cells, the N-terminal amino moiety of many proteins is modified by N-acetyltransferases (NATs). This protein modification can alter the folding of the target protein; can affect binding interactions of the target protein with substrates, allosteric effectors, or other proteins; or can trigger protein degradation. In prokaryotes, only ribosomal proteins are known to be N-terminally acetylated, and the acetyltransferases responsible for this modification belong to the Rim family of proteins. Here, we report that, in Salmonella enterica, the sirtuin deacylase CobB long isoform (CobBL) is N-terminally acetylated by the YiaC protein of this bacterium. Results of in vitro acetylation assays showed that CobBL was acetylated by YiaC; liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to confirm these results. Results of in vitro and in vivo experiments showed that CobBL deacetylase activity was negatively affected when YiaC acetylated its N terminus. We report 1) modulation of a bacterial sirtuin deacylase activity by acetylation, 2) that the Gcn5-related YiaC protein is the acetyltransferase that modifies CobBL, and 3) that YiaC is an NAT. Based on our data, we propose the name of NatA (N-acyltransferase A) in lieu of YiaC to reflect the function of the enzyme.


Assuntos
Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Salmonella enterica/metabolismo , Sirtuínas/metabolismo , Acetilação , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida , Isoformas de Proteínas , Salmonella enterica/enzimologia , Espectrometria de Massas em Tandem
20.
PLoS Negl Trop Dis ; 14(6): e0008299, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32511239

RESUMO

Snake venoms are complex mixtures of proteins with toxic activities, with many distinct isoforms, affecting different physiological targets, comprised in a few protein families. It is currently accepted that this diversity in venom composition is an adaptive advantage for venom efficacy on a wide range of prey. However, on the other side, variability on isoforms expression has implications in the clinics of human victims of snakebites and in the efficacy of antivenoms. B. atrox snakes are responsible for most of the human accidents in Brazilian Amazon and the type and abundance of protein families on their venoms present individual variability. Thus, in this study we attempted to correlate the individual venom proteome of the snake brought to the hospital by the patient seeking for medical assistance with the clinical signs observed in the same patient. Individual variability was confirmed in venoms of the 14 snakes selected for the study. The abundance of each protein family was quite similar among the venom samples, while the isoforms composition was highly variable. Considering the protein families, the SVMP group presented the best correlation with bleeding disorders and edema. Considering individual isoforms, some isoforms of venom metalloproteinase (SVMP), C-type lectin-like toxins (CTL) and snake venom serine proteinases (SVSP) presented expression levels that with statistically significant positive correlation to signs and symptoms presented by the patients as bleeding disorders, edema, ecchymosis and blister formation. However, some unexpected data were also observed as the correlation between a CTL, CRISP or LAAO isoforms with blister formation, still to be confirmed with a larger number of samples. Although this is still a small number of patient samples, we were able to indicate that venom composition modulates clinical manifestations of snakebites, to confirm at the bedside the prominent role of SVMPs and to include new possible toxin candidates for the development of toxin inhibitors or to improve antivenom selectiveness, important actions for the next generation treatments of snakebites.


Assuntos
Bothrops , Venenos de Crotalídeos/análise , Proteoma/análise , Serina Proteases/análise , Animais , Antivenenos , Brasil , Metaloproteases/análise , Isoformas de Proteínas/análise , Proteômica , Mordeduras de Serpentes/terapia
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