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1.
Chem Commun (Camb) ; 55(87): 13093-13095, 2019 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-31612161

RESUMO

Ubiquitin monomers functionalized with an azide or multiple alkynes were utilized for the assembly of branched ubiquitin oligomers (K6/K11, K11/K48, K11/K63, K6/K11/K48) by click chemistry. The oligomers resist deubiquitylase-catalysed hydrolysis and exhibit stability in eukaryotic cell lysates.


Assuntos
Ubiquitina/biossíntese , Alquinos/química , Azidas/química , Biocatálise , Química Click , Enzimas Desubiquitinantes/metabolismo , Células Eucarióticas/metabolismo , Humanos , Hidrólise , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinação
2.
BMC Bioinformatics ; 20(1): 340, 2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31208316

RESUMO

BACKGROUND: As a highly efficient and specific gene regulation technology, RNAi has broad application fields and good prospects. The effect of RNAi enhances as the dosage of siRNA increases, while an exorbitant siRNA dosage will inhibit the RNAi effect. So it is crucial to formulate a dose-effect model to describe the degradation effects of the target mRNA at different siRNA dosages. RESULTS: In this work, a simple RNA interference model with hill kinetic function (Giulia Cuccato et al. (2011)) is extended. Firstly, by introducing both the degradation time delay τ1 of mRNA caused by siRNA and the transportation time delay τ2 of mRNA from the nucleus to the cytoplasm during protein translation, one acquires a novel delay differential equations (DDEs) model with physiology lags. Secondly, qualitative analyses are executed to identify regions of stability of the positive equilibrium and to determine the corresponding parameter scales. Next, the approximate period of the limit cycle at Hopf bifurcation points is computed. Furthermore we analyze the parameter sensitivity of the limit cycle. Finally, we propose an optimal strategy to select siRNA dosage which arouses significant silencing efficiency. CONCLUSIONS: Our researches indicate that when the dosage of siRNA is large, oscillating periods are identical for disparate number of siRNA target sites even if it greatly impacts the critical siRNA dosage which is the switch of oscillating behavior. Furthermore, parametric sensitivity analyses of limit cycle disclose that both of degradation lag and maximum degradation rate of mRNA due to RNAi are principal elements on determining periodic oscillation. Our explorations will provide evidence for gene regulation and RNAi.


Assuntos
Células Eucarióticas/metabolismo , Periodicidade , Interferência de RNA , Animais , Simulação por Computador , Humanos , Modelos Genéticos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética
3.
Nat Commun ; 10(1): 1977, 2019 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-31036831

RESUMO

Protein phosphorylation is the best characterized post-translational modification that regulates almost all cellular processes through diverse mechanisms such as changing protein conformations, interactions, and localization. While the inventory for phosphorylation sites across different species has rapidly expanded, their functional role remains poorly investigated. Here, we combine 537,321 phosphosites from 40 eukaryotic species to identify highly conserved phosphorylation hotspot regions within domain families. Mapping these regions onto structural data reveals that they are often found at interfaces, near catalytic residues and tend to harbor functionally important phosphosites. Notably, functional studies of a phospho-deficient mutant in the C-terminal hotspot region within the ribosomal S11 domain in the yeast ribosomal protein uS11 shows impaired growth and defective cytoplasmic 20S pre-rRNA processing at 16 °C and 20 °C. Altogether, our study identifies phosphorylation hotspots for 162 protein domains suggestive of an ancient role for the control of diverse eukaryotic domain families.


Assuntos
Células Eucarióticas/metabolismo , Proteínas Fúngicas/metabolismo , Fosforilação , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo
4.
Mol Cell ; 74(4): 640-650, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31100245

RESUMO

Cellular RNAs are naturally decorated with a variety of chemical modifications. The structural diversity of the modified nucleosides provides regulatory potential to sort groups of RNAs for organized metabolism and functions, thus affecting gene expression. Recent years have witnessed a burst of interest in and understanding of RNA modification biology, thanks to the emerging transcriptome-wide sequencing methods for mapping modified sites, highly sensitive mass spectrometry for precise modification detection and quantification, and extensive characterization of the modification "effectors," including enzymes ("writers" and "erasers") that alter the modification level and binding proteins ("readers") that recognize the chemical marks. However, challenges remain due to the vast heterogeneity in expression abundance of different RNA species, further complicated by divergent cell-type-specific and tissue-specific expression and localization of the effectors as well as modifications. In this review, we highlight recent progress in understanding the function of N6-methyladenosine (m6A), the most abundant internal mark on eukaryotic mRNA, in light of the specific biological contexts of m6A effectors. We emphasize the importance of context for RNA modification regulation and function.


Assuntos
Adenosina/análogos & derivados , Metilação , RNA Mensageiro/genética , RNA/genética , Adenosina/genética , Células Eucarióticas/metabolismo , Regulação da Expressão Gênica/genética , Especificidade de Órgãos/genética , Processamento Pós-Transcricional do RNA/genética , Transcriptoma
5.
J Trace Elem Med Biol ; 54: 142-149, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31109604

RESUMO

BACKGROUND: Metal ions are essential for numerous life processes. This study aims to investigate the relationship between seminal quality and ion levels in seminal plasma. BASIC PROCEDURES: A total of 205 semen samples were collected and seminal plasma ion levels were examined with inductively-coupled plasma-mass spectrometry. The nickel function was demonstrated by in vitro assay and cell growth. MAIN FINDINGS: The low sperm motility group showed distinctively reduced nickel concentration in seminal plasma compared with the normal sperm motility group. However, arsenic, sulfur, selenium, magnesium and zinc were negatively associated with sperm quality. No significant relationship between other examined cations and semen quality was observed. In vitro assay suggested low concentration of nickel significantly increased sperm total motility and progressive motility. Cell growth assay further confirmed nickel promoted eukaryotic yeast cell growth. Nickel level in seminal plasma may play important functions to determine sperm quality. PRINCIPAL CONCLUSIONS: Our study reveals a strong correlation between S, Mg, Se, Zn, As, Ni and seminal quality as well as discovers a novel functional role of nickel in sperm motility and eukaryotic cell growth. These findings may provide a potential avenue for assessment of sperm quality and treatment of reproduction disorders.


Assuntos
Níquel/farmacologia , Motilidade Espermática/efeitos dos fármacos , Células Cultivadas , Células Eucarióticas/efeitos dos fármacos , Células Eucarióticas/metabolismo , Humanos , Masculino , Níquel/metabolismo , Estresse Oxidativo/fisiologia , Selênio/metabolismo , Sêmen/química , Motilidade Espermática/fisiologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Oligoelementos/metabolismo , Zinco/metabolismo
6.
Phys Rev Lett ; 122(12): 128101, 2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30978101

RESUMO

The more we learn about the cytoplasm of cells, the more we realize that the cytoplasm is not uniform but instead is highly inhomogeneous. In any inhomogeneous solution, there are concentration gradients, and particles move either up or down these gradients due to a mechanism called diffusiophoresis. I estimate that inside metabolically active cells, the dynamics of particles can be strongly accelerated by diffusiophoresis, provided that they are at least tens of nanometers across. The dynamics of smaller objects, such as single proteins, are largely unaffected.


Assuntos
Bactérias/metabolismo , Citoplasma/metabolismo , Células Eucarióticas/metabolismo , Modelos Biológicos , Complexos de ATP Sintetase/metabolismo , Adenosina Trifosfatases , Trifosfato de Adenosina/metabolismo , Bactérias/citologia , Citoplasma/química , Difusão , Eletroforese , Células Eucarióticas/citologia
7.
Nat Microbiol ; 4(7): 1138-1148, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30936488

RESUMO

The origin of eukaryotes represents an unresolved puzzle in evolutionary biology. Current research suggests that eukaryotes evolved from a merger between a host of archaeal descent and an alphaproteobacterial endosymbiont. The discovery of the Asgard archaea, a proposed archaeal superphylum that includes Lokiarchaeota, Thorarchaeota, Odinarchaeota and Heimdallarchaeota suggested to comprise the closest archaeal relatives of eukaryotes, has helped to elucidate the identity of the putative archaeal host. Whereas Lokiarchaeota are assumed to employ a hydrogen-dependent metabolism, little is known about the metabolic potential of other members of the Asgard superphylum. We infer the central metabolic pathways of Asgard archaea using comparative genomics and phylogenetics to be able to refine current models for the origin of eukaryotes. Our analyses indicate that Thorarchaeota and Lokiarchaeota encode proteins necessary for carbon fixation via the Wood-Ljungdahl pathway and for obtaining reducing equivalents from organic substrates. By contrast, Heimdallarchaeum LC2 and LC3 genomes encode enzymes potentially enabling the oxidation of organic substrates using nitrate or oxygen as electron acceptors. The gene repertoire of Heimdallarchaeum AB125 and Odinarchaeum indicates that these organisms can ferment organic substrates and conserve energy by coupling ferredoxin reoxidation to respiratory proton reduction. Altogether, our genome analyses suggest that Asgard representatives are primarily organoheterotrophs with variable capacity for hydrogen consumption and production. On this basis, we propose the 'reverse flow model', an updated symbiogenetic model for the origin of eukaryotes that involves electron or hydrogen flow from an organoheterotrophic archaeal host to a bacterial symbiont.


Assuntos
Archaea/genética , Archaea/metabolismo , Evolução Biológica , Células Eucarióticas/fisiologia , Modelos Biológicos , Filogenia , Archaea/classificação , Proteínas Arqueais/genética , Células Eucarióticas/metabolismo , Genoma Arqueal/genética , Processos Heterotróficos , Hidrogênio/metabolismo , Redes e Vias Metabólicas , Oxirredução , Simbiose
8.
RNA ; 25(7): 869-880, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31019095

RESUMO

Bacterial RNA has emerged as an important activator of innate immune responses by stimulating Toll-like receptors TLR7 and TLR8 in humans. Guanosine 2'-O-methylation at position 18 (Gm18) in bacterial tRNA was shown to antagonize tRNA-induced TLR7/8 activation, suggesting a potential role of Gm18 as an immune escape mechanism. This modification also occurs in eukaryotic tRNA, yet a physiological immune function remained to be tested. We therefore set out to investigate the immune modulatory role of Gm18 in both prokaryotic and eukaryotic microorganisms, Escherichia coli and Saccharomyces cerevisiae, and in human cells. Using RiboMethSeq analysis we show that mutation of trmH in E. coli, trm3 in S. cereviase, and CRISPR/Cas9-induced knockout of TARBP1 in H. sapiens results in loss of Gm18 within tRNA. Lack of Gm18 across the kingdoms resulted in increased immunostimulation of peripheral blood mononuclear cells when activated by tRNA preparations. In E. coli, lack of 2'-O-methyltransferase trmH also enhanced immune stimulatory properties by whole cellular RNA. In contrast, lack of Gm18 in yeasts and human cells did not affect immunostimulation by whole RNA preparations. When using live E. coli bacteria, lack of trmH did not affect overall immune stimulation although we detected a defined TLR8/RNA-dependent gene expression signature upon E. coli infection. Together, these results demonstrate that Gm18 is a global immune inhibitory tRNA modification across the kingdoms and contributes to tRNA recognition by innate immune cells, but as an individual modification has insufficient potency to modulate recognition of the investigated microorganisms.


Assuntos
Endossomos/metabolismo , Células Eucarióticas/imunologia , Guanosina/química , Imunidade Inata/imunologia , Células Procarióticas/imunologia , RNA de Transferência/metabolismo , Receptores Toll-Like/metabolismo , Células Eucarióticas/metabolismo , Humanos , Metilação , Células Procarióticas/metabolismo , RNA de Transferência/genética , Receptores Toll-Like/genética , tRNA Metiltransferases/genética , tRNA Metiltransferases/metabolismo
9.
Nucleic Acids Res ; 47(10): 4986-5000, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30976803

RESUMO

Transcription factors (TF) are central to transcriptional regulation, but they are often studied in relative isolation and without close control of the metabolic state of the cell. Here, we describe genome-wide binding (by ChIP-exo) of 15 yeast TFs in four chemostat conditions that cover a range of metabolic states. We integrate this data with transcriptomics and six additional recently mapped TFs to identify predictive models describing how TFs control gene expression in different metabolic conditions. Contributions by TFs to gene regulation are predicted to be mostly activating, additive and well approximated by assuming linear effects from TF binding signal. Notably, using TF binding peaks from peak finding algorithms gave distinctly worse predictions than simply summing the low-noise and high-resolution TF ChIP-exo reads on promoters. Finally, we discover indications of a novel functional role for three TFs; Gcn4, Ert1 and Sut1 during nitrogen limited aerobic fermentation. In only this condition, the three TFs have correlated binding to a large number of genes (enriched for glycolytic and translation processes) and a negative correlation to target gene transcript levels.


Assuntos
Células Eucarióticas/metabolismo , Perfilação da Expressão Gênica , Regiões Promotoras Genéticas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Análise por Conglomerados , Ontologia Genética , Modelos Genéticos , Nitrogênio/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
10.
Nucleic Acids Res ; 47(10): 4970-4973, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-30997511

RESUMO

The alignment between the boundaries of protein domains and the boundaries of exons could provide evidence for the evolution of proteins via domain shuffling, but literature in the field has so far struggled to conclusively show this. Here, on larger data sets than previously possible, we do finally show that this phenomenon is indisputably found widely across the eukaryotic tree. In contrast, the alignment between exons and the boundaries of intrinsically disordered regions of proteins is not a general property of eukaryotes. Most interesting of all is the discovery that domain-exon alignment is much more common in recently evolved protein sequences than older ones.


Assuntos
Células Eucarióticas/metabolismo , Éxons/genética , Íntrons/genética , Proteínas/genética , Animais , Evolução Molecular , Genoma/genética , Humanos
11.
Nat Commun ; 10(1): 1287, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894536

RESUMO

Close proximities between organelles have been described for decades. However, only recently a specific field dealing with organelle communication at membrane contact sites has gained wide acceptance, attracting scientists from multiple areas of cell biology. The diversity of approaches warrants a unified vocabulary for the field. Such definitions would facilitate laying the foundations of this field, streamlining communication and resolving semantic controversies. This opinion, written by a panel of experts in the field, aims to provide this burgeoning area with guidelines for the experimental definition and analysis of contact sites. It also includes suggestions on how to operationally and tractably measure and analyze them with the hope of ultimately facilitating knowledge production and dissemination within and outside the field of contact-site research.


Assuntos
Membrana Celular/metabolismo , Células Eucarióticas/metabolismo , Membranas Intracelulares/metabolismo , Organelas/metabolismo , Terminologia como Assunto , Animais , Fracionamento Celular/métodos , Membrana Celular/ultraestrutura , Células Eucarióticas/ultraestrutura , Humanos , Membranas Intracelulares/ultraestrutura , Microscopia/instrumentação , Microscopia/métodos , Organelas/ultraestrutura , Proteínas/genética , Proteínas/metabolismo , Coloração e Rotulagem/métodos
12.
BMB Rep ; 52(3): 163-164, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30885288

RESUMO

The ribosomal synthesis of proteins in the eukaryotic cytosol has always been thought to start from the unformylated N-terminal (Nt) methionine (Met). In contrast, in virtually all nascent proteins in bacteria and eukaryotic organelles, such as mitochondria and chloroplasts, Nt-formyl-methionine (fMet) is the first building block of ribosomal synthesis. Through extensive approaches, including mass spectrometric analyses of the N-termini of proteins and molecular genetic techniques with an affinity-purified antibody for Nt-formylation, we investigated whether Nt-formylated proteins could also be produced and have their own metabolic fate in the cytosol of a eukaryote, such as yeast Saccharomyces cerevisiae. We discovered that Nt-formylated proteins could be generated in the cytosol by yeast mitochondrial formyltransferase (Fmt1). These Nt-formylated proteins were massively upregulated in the stationary phase or upon starvation for specific amino acids and were crucial for the adaptation to specific stresses. The stress-activated kinase Gcn2 was strictly required for the upregulation of Nt-formylated proteins by regulating the activity of Fmt1 and its retention in the cytosol. We also found that the Nt-fMet residues of Nt-formylated proteins could be distinct N-terminal degradation signals, termed fMet/N-degrons, and that Psh1 E3 ubiquitin ligase mediated the selective destruction of Nt-formylated proteins as the recognition component of a novel eukaryotic fMet/N-end rule pathway, termed fMet/N-recognin. [BMB Reports 2019; 52(3): 163-164].


Assuntos
Hidroximetil e Formil Transferases/fisiologia , Biossíntese de Proteínas/fisiologia , Aminoácidos/metabolismo , Citosol/metabolismo , Células Eucarióticas/metabolismo , Hidroximetil e Formil Transferases/metabolismo , Metionina/metabolismo , Mitocôndrias/metabolismo , Proteínas/metabolismo , Proteólise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Regulação para Cima
13.
Int J Mol Sci ; 20(6)2019 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-30893886

RESUMO

For flawless translation of mRNA sequence into protein, tRNAs must undergo a series of essential maturation steps to be properly recognized and aminoacylated by aminoacyl-tRNA synthetase, and subsequently utilized by the ribosome. While all tRNAs carry a 3'-terminal CCA sequence that includes the site of aminoacylation, the additional 5'-G-1 position is a unique feature of most histidine tRNA species, serving as an identity element for the corresponding synthetase. In eukaryotes including yeast, both 3'-CCA and 5'-G-1 are added post-transcriptionally by tRNA nucleotidyltransferase and tRNAHis guanylyltransferase, respectively. Hence, it is possible that these two cytosolic enzymes compete for the same tRNA. Here, we investigate substrate preferences associated with CCA and G-1-addition to yeast cytosolic tRNAHis, which might result in a temporal order to these important processing events. We show that tRNA nucleotidyltransferase accepts tRNAHis transcripts independent of the presence of G-1; however, tRNAHis guanylyltransferase clearly prefers a substrate carrying a CCA terminus. Although many tRNA maturation steps can occur in a rather random order, our data demonstrate a likely pathway where CCA-addition precedes G-1 incorporation in S. cerevisiae. Evidently, the 3'-CCA triplet and a discriminator position A73 act as positive elements for G-1 incorporation, ensuring the fidelity of G-1 addition.


Assuntos
Células Eucarióticas/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência de Histidina/genética , Saccharomyces cerevisiae/genética , Citosol/metabolismo , Cinética , Nucleotídeos/metabolismo , Fatores de Tempo
14.
Nat Rev Genet ; 20(5): 283-297, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30886348

RESUMO

Sophisticated gene-regulatory mechanisms probably evolved in prokaryotes billions of years before the emergence of modern eukaryotes, which inherited the same basic enzymatic machineries. However, the epigenomic landscapes of eukaryotes are dominated by nucleosomes, which have acquired roles in genome packaging, mitotic condensation and silencing parasitic genomic elements. Although the molecular mechanisms by which nucleosomes are displaced and modified have been described, just how transcription factors, histone variants and modifications and chromatin regulators act on nucleosomes to regulate transcription is the subject of considerable ongoing study. We explore the extent to which these transcriptional regulatory components function in the context of the evolutionarily ancient role of chromatin as a barrier to processes acting on DNA and how chromatin proteins have diversified to carry out evolutionarily recent functions that accompanied the emergence of differentiation and development in multicellular eukaryotes.


Assuntos
Montagem e Desmontagem da Cromatina , DNA/genética , Genoma , Nucleossomos/genética , Transcrição Genética , Animais , Evolução Biológica , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/história , Proteínas Cromossômicas não Histona/metabolismo , DNA/história , DNA/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Genômica/métodos , Histonas/genética , Histonas/história , Histonas/metabolismo , História do Século XXI , História Antiga , Humanos , Nucleossomos/química , Nucleossomos/metabolismo , Células Procarióticas/citologia , Células Procarióticas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/história , Fatores de Transcrição/metabolismo
15.
Keio J Med ; 68(1): 26, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30905885

RESUMO

Many powerful molecular biology tools have their origin in nature. From restriction enzymes to CRISPR-Cas9, microbes utilize a diverse array of systems to get ahead evolutionarily. We are exploring this natural diversity through bioinformatics, biochemical, and molecular work to better understand the fundamental ways in which microbes and other living organisms sense and respond to their environment and as possible to develop these natural systems as molecular tools and to improve human health. Building on our demonstration that Cas9 can be repurposed for precision genome editing in mammalian cells, we look for novel CRISPR-Cas systems that are different and may have other useful properties. This led to the discovery of several new CRISPR systems, including the CRISPR-Cas13 family that target RNA, rather than DNA. We have developed a toolbox for RNA modulation based on Cas13, including methods for precision base editing, adding to our robust toolbox for DNA based on Cas9 and Cas12. We are expanding our biodiscovery efforts to search for new microbial proteins that may be adapted for applications beyond genome and transcriptome modulation, capitalizing on the growing volume of microbial genomic sequences. We are particularly interested in identifying new therapeutic modalities and vehicles for delivering them into patients. We hope that additional robust tools and delivery options will further accelerate research into human disease and open up new therapeutic possibilities.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes/métodos , Genoma , Terapia de Alvo Molecular , RNA Guia/genética , Animais , Archaea/química , Archaea/classificação , Archaea/genética , Archaea/imunologia , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Bactérias/química , Bactérias/classificação , Bactérias/genética , Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Biologia Computacional , Elementos de DNA Transponíveis , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , RNA Guia/metabolismo
16.
Cell Host Microbe ; 25(3): 454-462.e6, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30827827

RESUMO

Legionella pneumophila (L.p.), the microbe responsible for Legionnaires' disease, secretes ∼300 bacterial proteins into the host cell cytosol. A subset of these proteins affects a wide range of post-translational modifications (PTMs) to disrupt host cellular pathways. L.p. has 5 conserved eukaryotic-like Ser/Thr effector kinases, LegK1-4 and LegK7, which are translocated during infection. Using a chemical genetic screen, we identified the Hsp70 chaperone family as a direct host target of LegK4. Phosphorylation of Hsp70s at T495 in the substrate-binding domain disrupted Hsp70's ATPase activity and greatly inhibited its protein folding capacity. Phosphorylation of cytosolic Hsp70 by LegK4 resulted in global translation inhibition and an increase in the amount of Hsp70 on highly translating polysomes. LegK4's ability to inhibit host translation via a single PTM uncovers a role for Hsp70 in protein synthesis and directly links it to the cellular translational machinery.


Assuntos
Células Eucarióticas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Interações Hospedeiro-Patógeno , Legionella pneumophila/enzimologia , Fosfotransferases/metabolismo , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Células Eucarióticas/microbiologia , Doença dos Legionários/microbiologia , Fosforilação , Fatores de Virulência/metabolismo
17.
BMC Evol Biol ; 19(1): 40, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30704394

RESUMO

BACKGROUND: Cilia and flagella are complex cellular structures thought to have first evolved in a last ciliated eukaryotic ancestor due to the conserved 9 + 2 microtubule doublet structure of the axoneme and associated proteins. The Tektin family of coiled-coil domain containing proteins was previously identified in cilia of organisms as diverse as green algae and sea urchin. While studies have shown that some Tektins are necessary for ciliary function, there has been no comprehensive phylogenetic survey of tektin genes. To fill this gap, we sampled tektin sequences broadly among metazoan and unicellular lineages in order to determine how the tektin gene complements evolved in over 100 different extant species. RESULTS: Using Bayesian and Maximum Likelihood analyses, we have ascertained with high confidence that all metazoan tektins arose from a single ancestral tektin gene in the last common ancestor of metazoans and choanoflagellates. Gene duplications gave rise to two tektin genes in the metazoan ancestor, and a subsequent expansion to three and four tektin genes in early bilaterian ancestors. While all four tektin genes remained highly conserved in most deuterostome and spiralian species surveyed, most tektin genes in ecdysozoans are highly derived with extensive gene loss in several lineages including nematodes and some crustaceans. In addition, while tektin-1, - 2, and - 4 have remained as single copy genes in most lineages, tektin-3/5 has been duplicated independently several times, notably at the base of the spiralian, vertebrate and hymenopteran (Ecdysozoa) clades. CONCLUSIONS: We provide a solid description of tektin evolution supporting one, two, three, and four ancestral tektin genes in a holozoan, metazoan, bilaterian, and nephrozoan ancestor, respectively. The isolated presence of tektin in a cryptophyte and a chlorophyte branch invokes events of horizontal gene transfer, and that the last common ciliated eukaryotic ancestor lacked a tektin gene. Reconstructing the evolutionary history of the tektin complement in each extant metazoan species enabled us to pinpoint lineage specific expansions and losses. Our analysis will help to direct future studies on Tektin function, and how gain and loss of tektin genes might have contributed to the evolution of various types of cilia and flagella.


Assuntos
Biodiversidade , Cílios/metabolismo , Proteínas dos Microtúbulos/classificação , Filogenia , Sequência de Aminoácidos , Animais , Células Eucarióticas/metabolismo , Evolução Molecular , Genoma , Proteínas dos Microtúbulos/química , Proteínas dos Microtúbulos/genética
18.
Proc Natl Acad Sci U S A ; 116(11): 4973-4982, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30718387

RESUMO

Each genomic locus in a eukaryotic cell has a distinct average time of replication during S phase that depends on the spatial and temporal pattern of replication initiation events. Replication timing can affect genomic integrity because late replication is associated with an increased mutation rate. For most eukaryotes, the features of the genome that specify the location and timing of initiation events are unknown. To investigate these features for the fission yeast, Schizosaccharomyces pombe, we developed an integrative model to analyze large single-molecule and global genomic datasets. The model provides an accurate description of the complex dynamics of S. pombe DNA replication at high resolution. We present evidence that there are many more potential initiation sites in the S. pombe genome than previously identified and that the distribution of these sites is primarily determined by two factors: the sequence preferences of the origin recognition complex (ORC), and the interference of transcription with the assembly or stability of prereplication complexes (pre-RCs). We suggest that in addition to directly interfering with initiation, transcription has driven the evolution of the binding properties of ORC in S. pombe and other eukaryotic species to target pre-RC assembly to regions of the genome that are less likely to be transcribed.


Assuntos
Replicação do DNA , Células Eucarióticas/metabolismo , Schizosaccharomyces/metabolismo , Sequência Rica em At , Cromossomos Fúngicos/genética , Simulação por Computador , Período de Replicação do DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Células Eucarióticas/citologia , Genoma Fúngico , Modelos Biológicos , Complexo de Reconhecimento de Origem/genética , Probabilidade , Schizosaccharomyces/citologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Transcrição Genética
19.
Mol Genet Genomics ; 294(3): 637-647, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30758669

RESUMO

Genomes can be considered a combination of 16 dinucleotides. Analysing the relative abundance of different dinucleotides may reveal important features of genome evolution. In present study, we conducted extensive surveys on the relative abundances of dinucleotides in various genomic components of 28 bacterial, 20 archaean, 19 fungal, 24 plant and 29 animal species. We found that TA, GT and AC are significantly under-represented in open reading frames of all organisms and in intergenic regions and introns of most organisms. Specific dinucleotides are of greatly varied usage at different codon positions. The significantly low representations of TA, GT and AC are considered the evolutionary consequences of preventing formation of pre-mature stop codons and of reducing intron-splicing options in candidate primary mRNA sequences. These data suggest that a reduction of TA and GT occurred on both strands of the DNA sequence at an early stage of de novo gene birth. Interestingly, GT and AC are also significantly under-represented in current prokaryotic genomes, suggesting that ancient prokaryotic protein-coding genes might have contained introns. The greatly varied usages of specific dinucleotides at different codon positions are considered evolutionary accommodations to compensate the unavailability of specific codons and to avoid formation of pre-mature stop codons. This is the first report presenting data of dinucleotide relative abundance to indicate the possible existence of spliceosomal introns in ancient prokaryotic genes and to hypothesize early steps of de novo gene birth.


Assuntos
Archaea/genética , Composição de Bases/genética , Células Eucarióticas/metabolismo , Fases de Leitura Aberta/genética , Células Procarióticas/metabolismo , Animais , Proteínas Arqueais/genética , Proteínas de Bactérias/genética , Sequência de Bases , Códon/genética , DNA Intergênico/genética , Genoma/genética , Íntrons/genética , Especificidade da Espécie
20.
Nucleic Acids Res ; 47(5): 2681-2698, 2019 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-30726994

RESUMO

Most eukaryotic expression systems make use of host-cell nuclear transcriptional and post-transcriptional machineries. Here, we present the first generation of the chimeric cytoplasmic capping-prone phage polymerase (C3P3-G1) expression system developed by biological engineering, which generates capped and polyadenylated transcripts in host-cell cytoplasm by means of two components. First, an artificial single-unit chimeric enzyme made by fusing an mRNA capping enzyme and a DNA-dependent RNA polymerase. Second, specific DNA templates designed to operate with the C3P3-G1 enzyme, which encode for the transcripts and their artificial polyadenylation. This system, which can potentially be adapted to any in cellulo or in vivo eukaryotic expression applications, was optimized for transient expression in mammalian cells. C3P3-G1 shows promising results for protein production in Chinese Hamster Ovary (CHO-K1) cells. This work also provides avenues for enhancing the performances for next generation C3P3 systems.


Assuntos
Núcleo Celular/genética , Citoplasma/genética , RNA Polimerases Dirigidas por DNA/genética , Transcrição Genética , Animais , Células CHO , Cricetulus , Citoplasma/química , RNA Polimerases Dirigidas por DNA/química , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Humanos , Poli A/genética , Poliadenilação/genética
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