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1.
PLoS Genet ; 17(10): e1009817, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34624015

RESUMO

The yeast-to-hypha transition is tightly associated with pathogenicity in many human pathogenic fungi, such as the model fungal pathogen Cryptococcus neoformans, which is responsible for approximately 180,000 deaths annually. In this pathogen, the yeast-to-hypha transition can be initiated by distinct stimuli: mating stimulation or glucosamine (GlcN), the monomer of cell wall chitosan. However, it remains poorly understood how the signal specificity for Cryptococcus morphological transition by disparate stimuli is ensured. Here, by integrating temporal expression signature analysis and phenome-based clustering evaluation, we demonstrate that GlcN specifically triggers a unique cellular response, which acts as a critical determinant underlying the activation of GlcN-induced filamentation (GIF). This cellular response is defined by an unusually hyperactive cell wall synthesis that is highly ATP-consuming. A novel cell surface protein Gis1 was identified as the indicator molecule for the GlcN-induced cell wall response. The Mpk1-directed cell wall pathway critically bridges global cell wall gene induction and intracellular ATP supply, ensuring the Gis1-dependent cell wall response and the stimulus specificity of GIF. We further reveal that the ability of Mpk1 to coordinate the cell wall response and GIF activation is conserved in different Cryptococcus pathogens. Phosphoproteomics-based profiling together with genetic and phenotypic analysis revealed that the Mpk1 kinase mediates the regulatory specificity of GIF through a coordinated downstream regulatory network centered on Skn7 and Crz1. Overall, our findings discover an unprecedented and conserved cell wall biosynthesis-dependent fungal differentiation commitment mechanism, which enables the signal specificity of pathogenicity-related dimorphism induced by GlcN in Cryptococcus pathogens.


Assuntos
Parede Celular/genética , Cryptococcus neoformans/genética , Glucosamina/genética , Virulência/genética , Proteínas Fúngicas/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica/genética
2.
Water Sci Technol ; 83(9): 2218-2231, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33989188

RESUMO

To improve the efficiency of the Fe(II)/Fe(III) cycle and continuous reactivity of pyrite, a pyrite/H2O2/hydroxylamine (HA) system was proposed to treat rhodamine B (RhB). The results showed that near-complete decolorization and 52.8% mineralization 50 mg L-1 RhB were achieved under its optimum conditions: HA 0.8 mM, H2O2 1.6 mM, pyrite 0.4 g L-1, and initial pH 4.0. The degradation reaction was dominated by an •OH radical produced by the reaction of Fe2+ with H2O2 in solution. HA primarily had two roles: in solution, HA could accelerate the Fe(II)/Fe(III) cycle through its strong reducibility to enhance RhB decolorization; on the pyrite surface, HA could improve the continuous reactivity of pyrite by inhibiting the oxidation of pyrite. In addition, the dosing manner of HA had a significant effect on RhB decolorization. In addition, the high decolorization and mineralization efficiency of other dye pollutants suggested that the pyrite/H2O2/HA system might be widely used in textile wastewater treatment.


Assuntos
Peróxido de Hidrogênio , Poluentes Químicos da Água , Compostos Férricos , Hidroxilamina , Hidroxilaminas , Ferro , Oxirredução , Rodaminas , Sulfetos , Poluentes Químicos da Água/análise
3.
Nucleic Acids Res ; 43(9): e58, 2015 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-25722369

RESUMO

In this study, a universal protein expression enhancement RNA tool, termed RNAe, was developed by modifying a recently discovered natural long non-coding RNA. At the moment, RNAe is the only technology for gene expression enhancement, as opposed to silencing, at the post-transcriptional level. With this technology, an expression enhancement of 50-1000% is achievable, with more than 200% enhancement achieved in most cases. This work identified the sufficient and necessary element for RNAe function, which was found to be merely 300 nucleotides long and was named minRNAe. It contains a 72-nt 5' pairing sequence which determines the specificity, a 167-nt short non-pairing interspersed nuclear element (SINE) B2 sequence which enhances ribosome recruitment to the target mRNA, and a poly(A) tail, provided together on a plasmid bearing the appropriate sequences. Cellular delivery of RNAe was achieved using routine transfection. The RNAe platform was validated in several widely-used mammalian cell lines. It was proven to be efficient and flexible in specifically enhancing the expression of various endogenous and exogenous proteins of diverse functions in a dose-dependent manner. Compared to the expression-inhibitory tool RNAi, the RNAe tool has a comparable effect size, with an enhancing as opposed to inhibitory effect. One may predict that this brand new technology for enhancing the production of proteins will find wide applications in both research and biopharmaceutical production.


Assuntos
Regulação da Expressão Gênica , Biossíntese de Proteínas , Engenharia de Proteínas/métodos , RNA Longo não Codificante/química , Formação de Anticorpos , Linhagem Celular , Vetores Genéticos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Proteômica , RNA Antissenso/química , Sequências Repetitivas de Ácido Nucleico , Ribossomos/metabolismo
4.
Biochim Biophys Acta ; 1844(12): 2077-85, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25239613

RESUMO

Poly(A)-specific ribonuclease (PARN) is a deadenylase with three RNA-binding domains (the nuclease, R3H and RRM domains) and a C-terminal domain. PARN participates in diverse physiological processes by regulating mRNA fates through deadenylation. PARN mainly exists as a dimer in dilute solutions. In this research, we found that PARN could self-associate into tetramer and high-order oligomers both in vitro and in living cells. Mutational and spectroscopic analysis indicated that PARN oligomerization was triggered by the R3H domain, which led to the solvent-exposed Trp219 fluorophore to become buried in a solvent-inaccessible microenvironment. The RRM and C-terminal domains also played a role in modulating the dissociation rate of the tetrameric PARN. Enzymatic analysis indicated that tetramerization did not affect the catalytic behavior of the full-length PARN and truncated enzymes containing the RRM domain, which might be caused by the high propensity of the dimeric proteins to self-associate into oligomers. Tetramerization significantly enhanced the catalytic activity and processivity of the truncated form with the removal of the RRM and C-terminal domains. The results herein suggested that self-association might be one of the regulation methods for PARN to achieve a highly regulated deadenylase activity. We propose that self-association may facilitate PARN to concentrate around the target mRNAs by restricted diffusion.

5.
Biochim Biophys Acta ; 1834(6): 1089-98, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23388391

RESUMO

Deadenylases specifically catalyze the degradation of eukaryotic mRNA poly(A) tail in the 3'- to 5'-end direction with the release of 5'-AMP as the product. Among the deadenylase family, poly(A)-specific ribonuclease (PARN) is unique in its domain composition, which contains three potential RNA-binding domains: the catalytic nuclease domain, the R3H domain and the RRM domain. In this research, we investigated the roles of these RNA-binding domains by comparing the structural features and enzymatic properties of mutants lacking either one or two of the three RNA-binding domains. The results showed that the R3H domain had the ability to bind various oligonucleotides at the micromolar level with no oligo(A) specificity. The removal of the R3H domain dissociated PARN into monomers, which still possessed the RNA-binding ability and catalytic functions. Unlike the critical role of the RRM domain in PARN processivity, the removal of the R3H domain did not affect the catalytic pattern of PARN. Our results suggested that both R3H and RRM domains were essential for the high affinity of long poly(A) substrate, but the R3H domain did not contribute to the substrate recognition of PARN. Compared to the RRM domain, the R3H domain played a more important role in the structural integrity of the dimeric PARN. The multiple RNA-binding domain architecture endows PARN the property of highly efficient catalysis in a highly processive mode.


Assuntos
Exorribonucleases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação , Catálise , Exorribonucleases/genética , Humanos , Mutação , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
Nat Microbiol ; 9(2): 346-358, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38225460

RESUMO

Antibiotic tolerance is the ability of a susceptible population to survive high doses of cidal drugs and has been shown to compromise therapeutic outcomes in bacterial infections. In comparison, whether fungicide tolerance can be induced by host-derived factors during fungal diseases remains largely unknown. Here, through a systematic evaluation of metabolite-drug-fungal interactions in the leading fungal meningitis pathogen, Cryptococcus neoformans, we found that brain glucose induces fungal tolerance to amphotericin B (AmB) in mouse brain tissue and patient cerebrospinal fluid via the fungal glucose repression activator Mig1. Mig1-mediated tolerance limits treatment efficacy for cryptococcal meningitis in mice via inhibiting the synthesis of ergosterol, the target of AmB, and promoting the production of inositolphosphorylceramide, which competes with AmB for ergosterol. Furthermore, AmB combined with an inhibitor of fungal-specific inositolphosphorylceramide synthase, aureobasidin A, shows better efficacy against cryptococcal meningitis in mice than do clinically recommended therapies.


Assuntos
Cryptococcus neoformans , Meningite Criptocócica , Humanos , Animais , Camundongos , Anfotericina B/farmacologia , Anfotericina B/uso terapêutico , Meningite Criptocócica/tratamento farmacológico , Meningite Criptocócica/microbiologia , Antifúngicos/farmacologia , Encéfalo , Ergosterol/uso terapêutico
7.
Microbiol Spectr ; 10(3): e0265321, 2022 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-35471092

RESUMO

Sexual reproduction facilitates infection by the production of both a lineage advantage and infectious sexual spores in the ubiquitous human fungal pathogen Cryptococcus deneoformans. However, the regulatory determinants specific for initiating mating remain poorly understood. Here, we identified a velvet family regulator, Cva1, that strongly promotes sexual reproduction in C. deneoformans. This regulation was determined to be specific, based on a comprehensive phenotypic analysis of cva1Δ under 26 distinct in vitro and in vivo growth conditions. We further revealed that Cva1 plays a critical role in the initiation of early mating events, including sexual cell-cell fusion, but is not important for the late sexual development stages or meiosis. Thus, Cva1 specifically contributes to mating activation. Importantly, a novel mating-responsive protein, Cfs1, serves as the key target of Cva1 during mating, since its absence nearly blocks cell-cell fusion in C. deneoformans and its sister species C. neoformans. Together, our findings provide insight into how C. deneoformans ensures the regulatory specificity of mating. IMPORTANCE The human fungal pathogen C. deneoformans is a model organism for studying fungal sexual reproduction, which is considered to be important to infection. However, the specific regulatory determinants for activation of sexual reproduction remain poorly understood. In this study, by combining transcriptomic and comprehensive phenotypic analysis, we identified a velvet family regulator Cva1 that specifically and critically elicits early mating events, including sexual cell-cell fusion. Significantly, Cva1 induces mating through the novel mating-responsive protein Cfs1, which is essential for cell-cell fusion in C. deneoformans and its sister species C. neoformans. Considering that Cva1 and Cfs1 are highly conserved in species belonging to Cryptococcaeceae, they may play conserved and specific roles in the initiation of sexual reproduction in this important fungal clade, which includes multiple human fungal pathogens.


Assuntos
Cryptococcus neoformans , Proteínas Fúngicas , Genes Fúngicos Tipo Acasalamento , Fatores de Transcrição , Cryptococcus neoformans/genética , Proteínas Fúngicas/genética , Meiose , Fatores de Transcrição/genética
8.
Nat Commun ; 13(1): 7938, 2022 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-36566249

RESUMO

Pathogenic fungi of the genus Cryptococcus can undergo two sexual cycles, involving either bisexual diploidization (after fusion of haploid cells of different mating type) or unisexual diploidization (by autodiploidization of a single cell). Here, we construct a gene-deletion library for 111 transcription factor genes in Cryptococcus deneoformans, and explore the roles of these regulatory networks in the two reproductive modes. We show that transcription factors crucial for bisexual syngamy induce the expression of known mating determinants as well as other conserved genes of unknown function. Deletion of one of these genes, which we term FMP1, leads to defects in bisexual reproduction in C. deneoformans, its sister species Cryptococcus neoformans, and the ascomycete Neurospora crassa. Furthermore, we show that a recently evolved regulatory cascade mediates pre-meiotic unisexual autodiploidization, supporting that this reproductive process is a recent evolutionary innovation. Our findings indicate that genetic circuits with different evolutionary ages govern hallmark events distinguishing unisexual and bisexual reproduction in Cryptococcus.


Assuntos
Cryptococcus neoformans , Proteínas Fúngicas , Meningite Criptocócica , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/patogenicidade , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos Tipo Acasalamento/genética , Reprodução Assexuada/genética , Meningite Criptocócica/parasitologia
9.
Int J Mol Sci ; 12(5): 2901-16, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21686157

RESUMO

Divalent metal ions are essential for the efficient catalysis and structural stability of many nucleotidyl-transfer enzymes. Poly(A)-specific ribonuclease (PARN) belongs to the DEDD superfamily of 3'-exonucleases, and the active site of PARN contains four conserved acidic amino acid residues that coordinate two Mg(2+) ions. In this research, we studied the roles of these four acidic residues in PARN thermal stability by mutational analysis. It was found that Mg(2+) significantly decreased the rate but increased the aggregate size of the 54 kDa wild-type PARN in a concentration-dependent manner. All of the four mutants decreased PARN thermal aggregation, while the aggregation kinetics of the mutants exhibited dissimilar Mg(2+)-dependent behavior. A comparison of the kinetic parameters indicated that Asp28 was the most crucial one to the binding of the two Mg(2+) ions, while metal B might be more important in PARN structural stability. The spectroscopic and aggregation results also suggested that the alterations in the active site structure by metal binding or mutations might lead to a global conformational change of the PARN molecule.


Assuntos
Sequência Conservada , Exorribonucleases/química , Estabilidade Proteica , Domínio Catalítico , Cinética , Magnésio/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação
10.
Cells ; 9(1)2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31936572

RESUMO

Translation is spatiotemporally regulated and endoplasmic reticulum (ER)-associated mRNAs are generally in efficient translation. It is unclear whether the ER-associated mRNAs are deadenylated or degraded on the ER surface in situ or in the cytosol. Here, we showed that ER possessed active deadenylases, particularly the poly(A)-specific ribonuclease (PARN), in common cell lines and mouse tissues. Consistently, purified recombinant PARN exhibited a strong ability to insert into the Langmuir monolayer and liposome. ER-anchored PARN was found to be able to reshape the poly(A) length profile of the ER-associated RNAs by suppressing long poly(A) tails without significantly influencing the cytosolic RNAs. The shortening of long poly(A) tails did not affect global translation efficiency, which suggests that the non-specific action of PARN towards long poly(A) tails was beyond the scope of translation regulation on the ER surface. Transcriptome sequencing analysis indicated that the ER-anchored PARN trigged the degradation of a small subset of ER-enriched transcripts. The ER-anchored PARN modulated the translation of its targets by redistributing ribosomes to heavy polysomes, which suggests that PARN might play a role in dynamic ribosome reallocation. During DNA damage response, MK2 phosphorylated PARN-Ser557 to modulate PARN translocation from the ER to cytosol. The ER-anchored PARN modulated DNA damage response and thereby cell viability by promoting the decay of ER-associated MDM2 transcripts with low ribosome occupancy. These findings revealed that highly regulated communication between mRNA degradation rate and translation efficiency is present on the ER surface in situ and PARN might contribute to this communication by modulating the dynamic ribosome reallocation between transcripts with low and high ribosome occupancies.


Assuntos
Retículo Endoplasmático/metabolismo , Exorribonucleases/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA , Membrana Celular/metabolismo , Dano ao DNA , Exorribonucleases/química , Células HEK293 , Células HeLa , Humanos , Modelos Biológicos , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
11.
Biochem Biophys Rep ; 18: 100626, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30949591

RESUMO

Poly(A)-specific ribonuclease (PARN) catalyzes the degradation of mRNA poly(A) tail to regulate translation efficiency and mRNA decay in higher eukaryotic cells. The full-length PARN is a multi-domain protein containing the catalytic nuclease domain, the R3H domain, the RRM domain and the C-terminal intrinsically unstructured domain (CTD). The roles of the three well-structured RNA-binding domains have been extensively studied, while little is known about CTD. In this research, the impact of CTD on PARN stability and aggregatory potency was studied by comparing the thermal inactivation and denaturation behaviors of full-length PARN with two N-terminal fragments lacking CTD. Our results showed that K+ induced additional regular secondary structures and enhanced PARN stability against heat-induced inactivation, unfolding and aggregation. CTD prevented PARN from thermal inactivation but promoted thermal aggregation to initiate at a temperature much lower than that required for inactivation and unfolding. Blue-shift of Trp fluorescence during thermal transitions suggested that heat treatment induced rearrangements of domain organizations. CTD amplified the stabilizing effect of K+, implying the roles of CTD was mainly achieved by electrostatic interactions. These results suggested that CTD might dynamically interact with the main body of the molecule and release of CTD promoted self-association via electrostatic interactions.

12.
Cells ; 8(8)2019 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-31387300

RESUMO

Poly(A)-specific ribonuclease (PARN), a multifunctional multi-domain deadenylase, is crucial to the regulation of mRNA turnover and the maturation of various non-coding RNAs. Despite extensive studies of the well-folding domains responsible for PARN catalysis, the structure and function of the C-terminal domain (CTD) remains elusive. PARN is a cytoplasm-nucleus shuttle protein with concentrated nucleolar distribution. Here, we identify the nuclear and nucleolar localization signals in the CTD of PARN. Spectroscopic studies indicated that PARN-CTD is intrinsically disordered with loosely packed local structures/tertiary structure. Phosphorylation-mimic mutation S557D disrupted the local structure and facilitated the binding of the CTD with the well-folded domains, with no impact on PARN deadenylase activity. Under normal conditions, the nucleolus-residing PARN recruited CBP80 into the nucleoli to repress its deadenylase activity, while DNA damage-induced phosphorylation of PARN-S557 expelled CBP80 from the nucleoli to discharge activity inhibition and attracted nucleoplasm-located CstF-50 into the nucleoli to activate deadenylation. The structure switch-induced function switch of PARN reshaped the profile of small nuclear non-coding RNAs to respond to DNA damage. Our findings highlight that the structure switch of the CTD induced by posttranslational modifications redefines the subset of binding partners, and thereby the RNA targets in the nucleoli.


Assuntos
Núcleo Celular/metabolismo , Dano ao DNA , Exorribonucleases/metabolismo , Sinais de Localização Nuclear , Dobramento de Proteína , Transporte Ativo do Núcleo Celular , Animais , Células CHO , Cricetinae , Cricetulus , Exorribonucleases/química , Células HEK293 , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/química
13.
Elife ; 72018 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-30281018

RESUMO

In the human fungal pathogen Cryptococcus neoformans, sex can benefit its pathogenicity through production of meiospores, which are believed to offer both physical and meiosis-created lineage advantages for its infections. Cryptococcus sporulation occurs following two parallel events, meiosis and differentiation of the basidium, the characteristic sexual structure of the basidiomycetes. However, the circuit integrating these events to ensure subsequent sporulation is unclear. Here, we show the spatiotemporal coordination of meiosis and basidial maturation by visualizing event-specific molecules in developing basidia defined by a quantitative approach. Monitoring of gene induction timing together with genetic analysis reveals co-regulation of the coordinated events by a shared regulatory program. Two RRM family regulators, Csa1 and Csa2, are crucial components that bridge meiosis and basidial maturation, further determining sporulation. We propose that the regulatory coordination of meiosis and basidial development serves as a determinant underlying the production of infectious meiospores in C. neoformans.


Assuntos
Cryptococcus neoformans/citologia , Cryptococcus neoformans/genética , Meiose/genética , Cryptococcus neoformans/crescimento & desenvolvimento , Cryptococcus neoformans/ultraestrutura , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Genes Fúngicos Tipo Acasalamento , Mutação/genética , Fenótipo , Filogenia , Esporos Fúngicos/metabolismo
14.
Nat Microbiol ; 3(6): 698-707, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29784977

RESUMO

Bacterial quorum sensing is a well-characterized communication system that governs a large variety of collective behaviours. By comparison, quorum sensing regulation in eukaryotic microbes remains poorly understood, especially its functional role in eukaryote-specific behaviours, such as sexual reproduction. Cryptococcus neoformans is a prevalent fungal pathogen that has two defined sexual cycles (bisexual and unisexual) and is a model organism for studying sexual reproduction in fungi. Here, we show that the quorum sensing peptide Qsp1 serves as an important signalling molecule for both forms of sexual reproduction. Qsp1 orchestrates various differentiation and molecular processes, including meiosis, the hallmark of sexual reproduction. It activates bisexual mating, at least in part through the control of pheromone, a signal necessary for bisexual activation. Notably, Qsp1 also plays a major role in the intercellular regulation of unisexual initiation and coordination, in which pheromone is not strictly required. Through a multi-layered genetic screening approach, we identified the atypical zinc finger regulator Cqs2 as an important component of the Qsp1 signalling cascade during both bisexual and unisexual reproduction. The absence of Cqs2 eliminates the Qsp1-stimulated mating response. Together, these findings extend the range of behaviours governed by quorum sensing to sexual development and meiosis.


Assuntos
Cryptococcus neoformans/fisiologia , Peptídeos/genética , Peptídeos/metabolismo , Percepção de Quorum , Cryptococcus neoformans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos Tipo Acasalamento , Testes Genéticos , Hifas/crescimento & desenvolvimento , Meiose , Feromônios/metabolismo , Transdução de Sinais
15.
PLoS One ; 8(7): e69582, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23936053

RESUMO

The modulation of mRNA turnover has been increasingly recognized as a hotpoint for gene expression regulation at the post-transcriptional level. In eukaryotic cells, most mRNAs are degraded via the deadenylation-dependent pathway, in which the removal of the poly(A) tail is the initial and rate-limiting step. Caf1, a deadenylase specifically degrades poly(A) from the 3'-end, is highly conserved from yeast to mammalians. Caf1s in higher plants have been shown to be involved in plant development and stress response. However, little is known about the biochemical and biophysical properties of Caf1s in plants. In this research, we cloned the crcaf1 gene from Chlamydomonas reinhardtii and studied the properties of the recombinant proteins. The results showed that CrCaf1 was a deadenylase with conserved sequence motifs, structural features, and catalytic properties of the Caf1 family. CrCaf1 degraded poly(A) in a distributive mode with the optimal reacting conditions at pH 7 and 35°C. CrCaf1 had similar activity when coordinated with Mg(2+) and Mn(2+), while the enzyme bound to Ca(2+) or Zn(2+) was almost inactivated. Zn(2+) could induce CrCaf1 aggregation with the disruption of the native structure, while Mg(2+), Mn(2+) and Ca(2+) could stabilize CrCaf1 against thermal denaturation by reducing protein aggregation. Among the various metal ions, Mn(2+) showed the strongest protective effect on CrCaf1 stability, implying that Mn(2+) might play a role in regulating CrCaf1 stability in the C. reinhardtii cells under some stressed conditions. These findings provide a starting point for further investigation of the physiological functions of CrCaf1 in C. reinhardtii.


Assuntos
Fenômenos Biofísicos , Chlamydomonas reinhardtii/enzimologia , Proteínas de Plantas/metabolismo , Ribonucleases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Fenômenos Biofísicos/efeitos dos fármacos , Cátions Bivalentes/farmacologia , Chlamydomonas reinhardtii/efeitos dos fármacos , Cristalografia por Raios X , Estabilidade Enzimática/efeitos dos fármacos , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas Recombinantes/química , Ribonucleases/química , Alinhamento de Sequência , Temperatura
16.
PLoS One ; 7(3): e33700, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22442711

RESUMO

The shortening of the 3'-end poly(A) tail, also called deadenylation, is crucial to the regulation of mRNA processing, transportation, translation and degradation. The deadenylation process is achieved by deadenylases, which specifically catalyze the removal of the poly(A) tail at the 3'-end of eukaryotic mRNAs and release 5'-AMP as the product. To achieve their physiological functions, all deadenylases have numerous binding partners that may regulate their catalytic properties or recruit them into various protein complexes. To study the effects of various partners, it is important to develop new deadenylase assay that can be applied either in vivo or in vitro. In this research, we developed the deadenylase assay by the size-exclusion chromatography (SEC) method. The SEC analysis indicated that the poly(A) or oligo(A) substrate and the product AMP could be successfully separated and quantified. The enzymatic parameters of deadenylase could be obtained by quantifying the AMP generation. When using the commercial poly(A) as the substrate, a biphasic catalytic process was observed, which might correlate to the two distinct states of poly(A) in the commercial samples. Different lots of commercial poly(A) had dissimilar size distributions and were dissimilar in response to the degradation of deadenylase. The deadenylation pattern, processive or distributive, could also be investigated using the SEC assay by monitoring the status of the substrate and the generation kinetics of AMP and A2. The SEC assay was applicable to both simple samples using the purified enzyme and complex enzyme reaction conditions such as using protein mixtures or crude cell extracts as samples. The influence of solutes with absorption at 254 nm could be successfully eliminated by constructing the different SEC profiles.


Assuntos
Monofosfato de Adenosina/química , Bioensaio/métodos , Cromatografia em Gel/métodos , RNA Mensageiro/química , Ribonucleases/análise , Células HEK293 , Humanos , Ribonucleases/química
17.
FEBS J ; 276(10): 2849-60, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19459940

RESUMO

Poly(A)-specific ribonuclease (PARN) specifically catalyzes the degradation of the poly(A) tails of single-stranded mRNAs in a highly processive mode. PARN participates in diverse and important intracellular processes by acting as a regulator of mRNA stability and translational efficiency. In this article, the equilibrium unfolding of PARN was studied using both guanidine hydrochloride and urea as chemical denaturants. The unfolding of PARN was characterized as a multistate process, but involving dissimilar equilibrium intermediates when denatured by the two denaturants. A comparison of the spectral characteristics of these intermediates indicated that the conformational changes at low concentrations of the chemical denaturants were more likely to be rearrangements of the tertiary and quaternary structures. In particular, an inactive molten globule-like intermediate was identified to exist as soluble non-native oligomers, and the formation of the oligomers was modulated by electrostatic interactions. An active dimeric intermediate unique to urea-induced unfolding was characterized to have increased regular secondary structures and modified tertiary structures, implying that additional regular structures could be induced by environmental stresses. The dissimilarity in the unfolding pathways induced by guanidine hydrochloride and urea suggest that electrostatic interactions play an important role in PARN stability and regulation. The appearance of multiple intermediates with distinct properties provides the structural basis for the multilevel regulation of PARN by conformational changes.


Assuntos
Poli A/metabolismo , Ribonucleases/química , Guanidina/química , Conformação Proteica , Desnaturação Proteica , Ribonucleases/metabolismo , Eletricidade Estática , Ureia/química
18.
Biochem Biophys Res Commun ; 360(4): 846-51, 2007 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-17624302

RESUMO

Poly(A)-specific ribonuclease (PARN), a key enzyme involved in eukaryotic mRNA decay, contains one catalytic domain and two RNA-binding domains. Here we found that at least one RNA-binding domain is required for the substrate binding, but not for the catalysis of PARN. The removal of the R3H domain led to a dramatic decrease in PARN stability and a change in the aggregation kinetic regime, while only minor effects were observed for the removal of the RRM domain or both RNA-binding domains. Thus the R3H domain might stabilize PARN by acting as a protector or intermolecular chaperone of the RRM domain.


Assuntos
Exorribonucleases/metabolismo , Sequência de Bases , Primers do DNA , Estabilidade Enzimática , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/química , Mutação , Espectrofotometria Ultravioleta
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