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1.
Med Mycol J ; 61(3): 33-48, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32863327

RESUMO

Kawasaki disease (KD) is an inflammatory disease that was identified by Professor Tomisaku Kawasaki in 1961. Candida albicans-derived substances (CADS) such as the hot water extract of C. albicans and Candida water-soluble fractions (CAWS) induce coronary vasculitis similar to KD in mice. An increasing proportion of deep-seated candidiasis cases are caused by non-albicans Candida and are often resistant to antifungal drugs. We herein investigated whether the mannoprotein fractions (MN fractions) of clinically isolated Candida species induce vasculitis in mice. We prepared MN fractions from 26 strains of Candida species by conventional hot water extraction and compared vasculitis in DBA/2 mice. The results obtained revealed that the induction of vasculitis and resulting heart failure were significantly dependent on the species; namely, death rates on day 200 were as follows: Candida krusei (100%), Candida albicans (84%), Candida dubliniensis (47%), Candida parapsilosis (44%), Candida glabrata (32%), Candida guilliermondii (20%), and Candida tropicalis (20%). Even for C. albicans, some strains did not induce vasculitis. The present results suggest that MN-induced vasculitis is strongly dependent on the species and strains of Candida, and also that the MN fractions of some non-albicans Candida induce similar toxicity to those of C. albicans.


Assuntos
Candida albicans/química , Candida albicans/patogenicidade , Candidíase , Vasos Coronários/microbiologia , Proteínas Fúngicas/efeitos adversos , Vasculite/microbiologia , Animais , Candida albicans/classificação , Fracionamento Celular , Proteínas Fúngicas/isolamento & purificação , Camundongos Endogâmicos DBA , Especificidade da Espécie
2.
PLoS Genet ; 16(6): e1008881, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32525871

RESUMO

Iron is an essential nutrient required as a cofactor for many biological processes. As a fungal commensal-pathogen of humans, Candida albicans encounters a range of bioavailable iron levels in the human host and maintains homeostasis with a conserved regulatory circuit. How C. albicans senses and responds to iron availability is unknown. In model yeasts, regulation of the iron homeostasis circuit requires monothiol glutaredoxins (Grxs), but their functions beyond the regulatory circuit are unclear. Here, we show Grx3 is required for virulence and growth on low iron for C. albicans. To explore the global roles of Grx3, we applied a proteomic approach and performed in vivo cross-linked tandem affinity purification coupled with mass spectrometry. We identified a large number of Grx3 interacting proteins that function in diverse biological processes. This included Fra1 and Bol2/Fra2, which function with Grxs in intracellular iron trafficking in other organisms. Grx3 interacts with and regulates the activity of Sfu1 and Hap43, components of the C. albicans iron regulatory circuit. Unlike the regulatory circuit, which determines expression or repression of target genes in response to iron availability, Grx3 amplifies levels of gene expression or repression. Consistent with the proteomic data, the grx3 mutant is sensitive to heat shock, oxidative, nitrosative, and genotoxic stresses, and shows growth dependence on histidine, leucine, and tryptophan. We suggest Grx3 is a conserved global regulator of iron-dependent processes occurring within the cell.


Assuntos
Candida albicans/fisiologia , Candidíase Invasiva/microbiologia , Proteínas Fúngicas/metabolismo , Glutarredoxinas/metabolismo , Ferro/metabolismo , Animais , Candida albicans/patogenicidade , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Fatores de Transcrição GATA/metabolismo , Regulação Fúngica da Expressão Gênica , Glutarredoxinas/genética , Glutarredoxinas/isolamento & purificação , Homeostase , Humanos , Hifas , Masculino , Camundongos , Mutação , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/genética , Proteômica , Virulência/genética
3.
Nat Commun ; 11(1): 1026, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32094331

RESUMO

Structural and functional studies were conducted of the glucuronoyl esterase (GE) from Cerrena unicolor (CuGE), an enzyme catalyzing cleavage of lignin-carbohydrate ester bonds. CuGE is an α/ß-hydrolase belonging to carbohydrate esterase family 15 (CE15). The enzyme is modular, comprised of a catalytic and a carbohydrate-binding domain. SAXS data show CuGE as an elongated rigid molecule where the two domains are connected by a rigid linker. Detailed structural information of the catalytic domain in its apo- and inactivated form and complexes with aldouronic acids reveal well-defined binding of the 4-O-methyl-a-D-glucuronoyl moiety, not influenced by the nature of the attached xylo-oligosaccharide. Structural and sequence comparisons within CE15 enzymes reveal two distinct structural subgroups. CuGE belongs to the group of fungal CE15-B enzymes with an open and flat substrate-binding site. The interactions between CuGE and its natural substrates are explained and rationalized by the structural results, microscale thermophoresis and isothermal calorimetry.


Assuntos
Domínio Catalítico , Esterases/metabolismo , Proteínas Fúngicas/metabolismo , Ácido Glucurônico/metabolismo , Polyporales/enzimologia , Carboidratos , Parede Celular/metabolismo , Cristalografia por Raios X , Esterases/isolamento & purificação , Esterases/ultraestrutura , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/ultraestrutura , Hidrólise , Lignina/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Espalhamento a Baixo Ângulo , Relação Estrutura-Atividade , Especificidade por Substrato , Difração de Raios X
4.
Biochimie ; 168: 231-240, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31756400

RESUMO

A novel bgl1 gene, encoding GH3 family ß-glucosidase from Penicillium verruculosum (PvBGL), was cloned and heterologously expressed in P. canescens RN3-11-7 (niaD-) strain under the control of the strong xylA gene promoter. The recombinant rPvBGL was purified and their properties were studied in comparison with those of rAnBGL from Aspergillus niger expressed previously in the same fungal host. The rPvBGL had an observed molecular mass of 90 kDa (SDS-PAGE data) and displayed the enzyme maximum activity at pH 4.6 and 65 °C. The enzyme half-life time at 60 °C was found to be 87 min. Unlike the rAnBGL, the rPvBGL was not adsorbed on microcrystalline cellulose, which gives the latter enzyme an advantage in cellulose conversion with a longer time of hydrolysis.


Assuntos
Aspergillus niger/enzimologia , Proteínas Fúngicas , Penicillium/enzimologia , Proteínas Recombinantes , beta-Glucosidase , Celulose/química , Clonagem Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Especificidade por Substrato , beta-Glucosidase/química , beta-Glucosidase/isolamento & purificação
5.
Anal Bioanal Chem ; 412(2): 449-462, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31797019

RESUMO

In the last decades, microbial oils have been extensively investigated as a renewable platform for biofuel and oleochemical production. Offering a potent alternative to plant-based oils, oleaginous microorganisms have been the target of ongoing metabolic engineering aimed at increasing growth and lipid yields, in addition to specialty fatty acids. Discovery proteomics is an attractive tool for elucidating lipogenesis and identifying metabolic bottlenecks, feedback regulation, and competing biosynthetic pathways. One prominent microbial oil producer is Cutaneotrichosporon oleaginosus, due to its broad feedstock catabolism and high lipid yield. However, this yeast has a recalcitrant cell wall and high cell lipid content, which complicates efficient and unbiased protein extraction for downstream proteomic analysis. Optimization efforts of protein sample preparation from C. oleaginosus in the present study encompasses the comparison of 8 lysis methods, 13 extraction buffers, and 17 purification methods with respect to protein abundance, proteome coverage, applicability, and physiochemical properties (pI, MW, hydrophobicity in addition to COG, and GO analysis). The optimized protocol presented in this work entails a one-step extraction method utilizing an optimal lysis method (liquid homogenization), which is augmented with a superior extraction buffer (50 mM Tris, 8/2 M Urea/Thiourea, and 1% C7BzO), followed by either of 2 advantageous purification methods (hexane/ethanol or TCA/acetone), depending on subsequent applications and target studies. This work presents a significant step forward towards implementation of efficient C. oleaginosus proteome mining for the identification of potential targets for genetic optimization of this yeast to improve lipogenesis and production of specialty lipids. Graphical abstract.


Assuntos
Basidiomycota/metabolismo , Proteínas Fúngicas/metabolismo , Proteômica , Proteínas Fúngicas/isolamento & purificação , Lipídeos/isolamento & purificação , Proteólise , Solubilidade
6.
Nat Commun ; 10(1): 5807, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31862931

RESUMO

Transient interactions between the anaphase-promoting complex/cyclosome (APC/C) and its activator subunit Cdc20 or Cdh1 generate oscillations in ubiquitylation activity necessary to maintain the order of cell cycle events. Activator binds the APC/C with high affinity and exhibits negligible dissociation kinetics in vitro, and it is not clear how the rapid turnover of APC/C-activator complexes is achieved in vivo. Here, we describe a mechanism that controls APC/C-activator interactions based on the availability of substrates. We find that APC/C-activator dissociation is stimulated by abundant cellular polyanions such as nucleic acids and polyphosphate. Polyanions also interfere with substrate ubiquitylation. However, engagement with high-affinity substrate blocks the inhibitory effects of polyanions on activator binding and APC/C activity. We propose that this mechanism amplifies the effects of substrate affinity on APC/C function, stimulating processive ubiquitylation of high-affinity substrates and suppressing ubiquitylation of low-affinity substrates.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/imunologia , Proteínas Cdc20/metabolismo , Proteínas Cdh1/metabolismo , Proteínas Fúngicas/metabolismo , Polímeros/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/isolamento & purificação , Proteínas Cdc20/isolamento & purificação , Proteínas Cdh1/isolamento & purificação , Ciclo Celular , Proteínas Fúngicas/isolamento & purificação , Ácidos Nucleicos/metabolismo , Polifosfatos/metabolismo , Saccharomycetales/metabolismo , Especificidade por Substrato , Ubiquitinação
7.
J Vis Exp ; (154)2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31868175

RESUMO

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and lipids during endocytosis, sorting within the endosomal system, and autophagy. Central to SNX-BAR protein function is the ability to form homodimers or heterodimers that bind membranes using highly conserved phox-homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. In addition, oligomerization of SNX-BAR dimers on membranes can elicit the formation of membrane tubules and vesicles and this activity is thought to reflect their functions as coat proteins for endosome-derived transport carriers. Researchers have long utilized in vitro binding studies using recombinant SNX-BAR proteins on synthetic liposomes or giant unilamellar vesicles (GUVs) to reveal the precise makeup of lipids needed to drive membrane remodeling, thus revealing their mechanism of action. However, due to technical challenges with dual expression systems, toxicity of SNX-BAR protein expression in bacteria, and poor solubility of individual SNX-BAR proteins, most studies to date have examined SNX-BAR homodimers, including non-physiological dimers that form during expression in bacteria. Recently, we have optimized a protocol to overcome the major shortcomings of a typical bacterial expression system. Using this workflow, we demonstrate how to successfully express and purify large amounts of SNX-BAR heterodimers and how to reconstitute them on synthetic liposomes for binding and tubulation assays.


Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Lipossomos/metabolismo , Multimerização Proteica , Saccharomycetales/genética , Membrana Celular/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Expressão Gênica , Ligação Proteica , Estrutura Quaternária de Proteína , Transporte Proteico
8.
World J Microbiol Biotechnol ; 35(11): 171, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31673786

RESUMO

Fungal endo-ß-1,4-xylanases (endo-xylanases) can hydrolyze xylan into xylooligosaccharides (XOS), and have potential biotechnological applications for the exploitation of natural renewable polysaccharides. In the current study, we aimed to screen and characterize an efficient fungal endo-xylanase from 100 natural humus-rich soil samples collected in Guizhou Province, China, using extracted sugarcane bagasse xylan (SBX) as the sole carbon source. Initially, 182 fungal isolates producing xylanases were selected, among which Trichoderma sp. strain TP3-36 was identified as showing the highest xylanase activity of 295 U/mL with xylobiose (X2) as the main product when beechwood xylan was used as substrate. Subsequently, a glycoside hydrolase family 11 endo-xylanase, TXyn11A, was purified from strain TP3-36, and its optimal pH and temperature for activity against beechwood xylan were identified to be 5.0 and 55 °C, respectively. TXyn11A was stable across a broad pH range (3.0-10.0), and exhibited strict substrate specificity, including xylan from beechwood, wheat, rye, and sugarcane bagasse, with Km and Vmax values of 5 mg/mL and 1250 µmol/mg min, respectively, toward beechwood xylan. Intriguingly, the main product obtained from hydrolysis of beechwood xylan by TXyn11A was xylobiose, whereas SBX hydrolysis resulted in both X2 and xylotriose. Overall, these characteristics of the endo-xylanase TXyn11A indicate several potential industrial applications.


Assuntos
Dissacarídeos/metabolismo , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/isolamento & purificação , Trichoderma/enzimologia , Xilanos/metabolismo , Celulose , China , Estabilidade Enzimática , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Saccharum/metabolismo , Microbiologia do Solo , Especificidade por Substrato , Temperatura , Trichoderma/genética , Trichoderma/isolamento & purificação
9.
Nat Commun ; 10(1): 3795, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31439846

RESUMO

Histone H3 lysine 36 methylation (H3K36me) is a conserved histone modification deposited by the Set2 methyltransferases. Recent findings show that over-expression or mutation of Set2 enzymes promotes cancer progression, however, mechanisms of H3K36me are poorly understood. Set2 enzymes show spurious activity on histones and histone tails, and it is unknown how they obtain specificity to methylate H3K36 on the nucleosome. In this study, we present 3.8 Å cryo-EM structure of Set2 bound to the mimic of H2B ubiquitinated nucleosome. Our structure shows that Set2 makes extensive interactions with the H3 αN, the H3 tail, the H2A C-terminal tail and stabilizes DNA in the unwrapped conformation, which positions Set2 to specifically methylate H3K36. Moreover, we show that ubiquitin contributes to Set2 positioning on the nucleosome and stimulates the methyltransferase activity. Notably, our structure uncovers interfaces that can be targeted by small molecules for development of future cancer therapies.


Assuntos
Proteínas Fúngicas/metabolismo , Histonas/metabolismo , Metiltransferases/metabolismo , Nucleossomos/metabolismo , Ubiquitina/metabolismo , Chaetomium , Microscopia Crioeletrônica , Metilação de DNA , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/ultraestrutura , Código das Histonas , Histonas/isolamento & purificação , Histonas/ultraestrutura , Metiltransferases/isolamento & purificação , Metiltransferases/ultraestrutura , Modelos Moleculares , Nucleossomos/ultraestrutura , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Ubiquitina/ultraestrutura
10.
Int J Biol Macromol ; 139: 1028-1034, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31404600

RESUMO

We report cloning and expressing of recombinant human VEGF-A165, fused at the N-terminal with Hydrophobin II (HFBII) from Trichoderma reseei, in yeast Pichia pastoris. We validated the construct using SDS-PAGE and ELISA against VEGF-A165 and efficiently performed protein purification and enrichment based on HFBII counterpart and using an aqueous two-phase system (ATPS) with nonionic surfactant X-114. We studied the effects of various culture medium additives and interaction effects of positive factors to increase the recombinant HFBII-VEGF-A165 production. Supplementing the Pichia pastoris cell culture medium with Mg2+, Polysorbate 20 (PS 20), and 4-phenylbutyrate (PBA) improved the expression of the chimeric protein. Orthogonal experiments showed that the optimal condition to achieve maximal HFBII-VEGF-A165 production was with the addition of PBA, PS 20, and MgSO4. Under this condition, the production of the target protein was 4.5 times more than that in the medium without the additives. Overall, our approach to produce chimeric HFBII-VEGF-A165 and selectively capture it in ATPS is promising for large-scale protein production without laborious downstream processing.


Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Pichia/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Fator A de Crescimento do Endotélio Vascular/genética , Anticorpos Imobilizados/química , Anticorpos Imobilizados/metabolismo , Proliferação de Células , Proteínas Fúngicas/metabolismo , Expressão Gênica , Pichia/citologia , Ranibizumab/química , Ranibizumab/metabolismo , Trichoderma/genética
11.
Int J Biol Macromol ; 140: 761-770, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31434004

RESUMO

Lipase B from Candida antarctica (CalB) is the most widely used lipase, including in many industrial sectors, such as in biodiesel and pharmaceuticals production. CalB has been produced by heterologous expression using Pichia pastoris under PGK constitutive promoter (named LipB). Here, we have studied the structural features of commercial CalB and LipB enzymes using circular dichroism and fluorescence under different conditions. In the presence of denaturing agents CalB was more stable than LipB, in contrast, at increasing temperatures, LipB was more thermostable than CalB. Mass spectrometry data indicates that both enzymes have an insertion of amino acids related to α-factor yeast signal, however LipB enzyme showed the addition of nine residues at the N-terminal while CalB showed only four residues. Molecular modeling of LipB showed the formation of an amphipathic α-helix in N-terminal region that was not observed in CalB. This data suggests that this new α-helix possess could be involved in LipB thermostability. These results associated with new structural studies may provide information to the design of novel biocatalysts.


Assuntos
Candida/enzimologia , Proteínas Fúngicas/química , Lipase/química , Proteínas Recombinantes de Fusão , Sequência de Aminoácidos , Candida/genética , Ativação Enzimática , Estabilidade Enzimática , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Hidrólise , Lipase/genética , Lipase/isolamento & purificação , Lipase/metabolismo , Modelos Moleculares , Conformação Proteica , Relação Estrutura-Atividade , Temperatura , Termodinâmica
12.
Colloids Surf B Biointerfaces ; 183: 110418, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31404792

RESUMO

The design of interfaces that selectively react with molecules to transform them into compounds of industrial interest is an emerging area of research. An example of such reactions is the hydrolytic conversion of ester-based molecules to lipids and alcohols, which is of interest to the food, and pharmaceutical industries. In this study, a functional bio-interfaced layer was designed to hydrolyze 4-nitrophenyl acetate (pNPA) and Ricinus Communis (castor) oil rich in triglycerides using lipase b from Candida antarctica (CALB, EC 3.1.1.3). The attachment of CALB was performed via non-covalent immobilization over a polymer film of vertically aligned cylinders that resulted from the self-assembly of the di-block copolymer polystyrene-block-poly(4-vinyl pyridine) (PS-b-P4VP). This polymer-lipase model will serve as the groundwork for the design of further bioactive layers for separation applications requiring similar hydrolytic processes. Results from the fabricated functional bio-interfaced material include cylinders with featured pore size of 19 nm, d spacing of 34 nm, and ca. 40 nm of thickness. The polymer-enzyme layers were physically characterized using AFM, XPS, and FTIR. The immobilized enzyme was able to retain 91% of the initial enzymatic activity when using 4-nitrophenyl acetate (pNPA) and 78% when exposed to triglycerides from castor oil.


Assuntos
Poluentes Ambientais/química , Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Lipase/química , Nitrofenóis/química , Poliestirenos/química , Polivinil/química , Triglicerídeos/química , Candida/química , Candida/enzimologia , Óleo de Rícino/química , Enzimas Imobilizadas/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação , Humanos , Hidrólise , Lipase/isolamento & purificação , Porosidade , Ricinus/química
13.
J Basic Microbiol ; 59(9): 879-889, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31339587

RESUMO

Metallothionein (MT) is a low-molecular-weight protein with a high metal binding capacity and plays a key role in organism adaptation to heavy metals. In this study, a metallothionein gene was successfully cloned and sequenced from Antarctic sea-ice yeast Rhodotorula mucilaginosa AN5. Nucleotide sequencing and analysis revealed that the gene had four exons interrupted by three introns. MTs complementary DNA (named as RmMT) had an open reading frame of 321 bp encoding a 106 amino acid protein with a predicted molecular weight of 10.3 kDa and pI of 8.49. The number of amino acids and distribution of cysteine residues indicated that RmMT was a novel family of fungal MTs. Quantitative real-time polymerase chain reaction analysis showed that RmMT expression was elevated under copper-induced stress. The RmMT gene was transferred into E. coli and the RmMT expressing bacteria showed improved tolerance to copper ion and increased accumulation of heavy metals, such as Cu2+ , Pb2+ , Zn2+ , Cd2+ , and Ag+ . Moreover, in vitro studies, purified recombinant RmMT demonstrated that it could be used as a good scavenger of superoxide anion, hydroxyl, and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radicals. In summary, these results demonstrate that RmMT plays a key role in the tolerance and bioaccumulation of heavy metals.


Assuntos
Camada de Gelo/microbiologia , Metalotioneína/genética , Metalotioneína/metabolismo , Metais Pesados/metabolismo , Rhodotorula/genética , Adaptação Fisiológica/genética , Regiões Antárticas , Antioxidantes/isolamento & purificação , Antioxidantes/metabolismo , Sequência de Bases , Clonagem Molecular , Cobre/metabolismo , Escherichia coli/genética , Escherichia coli/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Expressão Gênica , Metalotioneína/isolamento & purificação , Fases de Leitura Aberta , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Rhodotorula/fisiologia
14.
Nat Commun ; 10(1): 3050, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296859

RESUMO

The Rea1 AAA+-ATPase dislodges assembly factors from pre-60S ribosomes upon ATP hydrolysis, thereby driving ribosome biogenesis. Here, we present crystal structures of Rea1-MIDAS, the conserved domain at the tip of the flexible Rea1 tail, alone and in complex with its substrate ligands, the UBL domains of Rsa4 or Ytm1. These complexes have structural similarity to integrin α-subunit domains when bound to extracellular matrix ligands, which for integrin biology is a key determinant for force-bearing cell-cell adhesion. However, the presence of additional motifs equips Rea1-MIDAS for its tasks in ribosome maturation. One loop insert cofunctions as an NLS and to activate the mechanochemical Rea1 cycle, whereas an additional ß-hairpin provides an anchor to hold the ligand UBL domains in place. Our data show the versatility of the MIDAS fold for mechanical force transmission in processes as varied as integrin-mediated cell adhesion and mechanochemical removal of assembly factors from pre-ribosomes.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/ultraestrutura , Proteínas Fúngicas/ultraestrutura , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , ATPases Associadas a Diversas Atividades Celulares/isolamento & purificação , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Adesão Celular/fisiologia , Chaetomium/fisiologia , Cristalografia por Raios X , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Integrinas/ultraestrutura , Ligantes , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
15.
Appl Biochem Biotechnol ; 189(4): 1327-1337, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31297753

RESUMO

A cellulase from the extreme obligate halophilic fungus, Aspergillus flavus, isolated from a man-made solar saltern in Phetchaburi, Thailand, was purified by ammonium sulfate precipitation and using Sephadex G-100 gel filtration column chromatography. The cellulase was found to be approximately 55 kDa by SDS-PAGE. Using CMC as a substrate, the specific activity of the cellulase was 62.9 units (U) mg-1 with Vmax and Km values of 37.87 mol min-1 mg-1 and 3.02 mg mL-1, respectively. Characterization of the enzyme revealed it to be an extremozyme, having an optimum activity at pH 10, 60 °C, and 200 g L-1 of NaCl. The enzyme activity was not significantly altered by the addition of divalent metal cations at 2 mM and neither did ß-mercaptoethanol, while EDTA was found strongly inhibiting the cellulase. Compared with commercial cellulase, the purified cellulase from A. flavus was more active in the extremity of conditions, especially at pH 10, 60 °C, and 150 g L-1 NaCl, whereas the commercial cellulase had a very low activity.


Assuntos
Aspergillus flavus/enzimologia , Biocombustíveis , Celulase , Etanol , Proteínas Fúngicas , Celulase/química , Celulase/isolamento & purificação , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação
16.
Rev Bras Parasitol Vet ; 28(3): 339-345, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31291435

RESUMO

Gastrointestinal nematode infection is an important cause of high economic losses in livestock production. Nematode control based on a synthetic chemical approach is considered unsustainable due to the increasing incidence of anthelmintic resistance. Control alternatives such as the use of natural products are therefore becoming relevant from an environmental and economic point of view. Proteins are macromolecules with various properties that can be obtained from a wide range of organisms, including plants and fungi. Proteins belonging to different classes have shown great potential for the control of nematodes. The action of proteins can occur at specific stages of the nematode life cycle, depending on the composition of the external layers of the nematode body and the active site of the protein. Advances in biotechnology have resulted in the emergence of numerous protein and peptide therapeutics; however, few have been discussed with a focus on the control of animal nematodes. Here, we discuss the use of exogenous proteins and peptides in the control of gastrointestinal.


Assuntos
Antinematódeos/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação , Gastroenteropatias/veterinária , Infecções por Nematoides/veterinária , Peptídeos/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Animais , Antinematódeos/administração & dosagem , Biotecnologia , Quitinases/administração & dosagem , Quitinases/isolamento & purificação , Proteínas Fúngicas/administração & dosagem , Gastroenteropatias/parasitologia , Infecções por Nematoides/tratamento farmacológico , Peptídeo Hidrolases/administração & dosagem , Peptídeo Hidrolases/isolamento & purificação , Peptídeos/administração & dosagem , Proteínas de Plantas/administração & dosagem
17.
Int J Mol Sci ; 20(13)2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269636

RESUMO

Marine microorganisms represent a reservoir of new promising secondary metabolites. Surface-active proteins with good emulsification activity can be isolated from fungal species that inhabit the marine environment and can be promising candidates for different biotechnological applications. In this study a novel surface-active protein, named Sap-Pc, was purified from a marine strain of Penicillium chrysogenum. The effect of salt concentration and temperature on protein production was analyzed, and a purification method was set up. The purified protein, identified as Pc13g06930, was annotated as a hypothetical protein. It was able to form emulsions, which were stable for at least one month, with an emulsification index comparable to that of other known surface-active proteins. The surface tension reduction was analyzed as function of protein concentration and a critical micellar concentration of 2 µM was determined. At neutral or alkaline pH, secondary structure changes were monitored over time, concurrently with the appearance of protein precipitation. Formation of amyloid-like fibrils of SAP-Pc was demonstrated by spectroscopic and microscopic analyses. Moreover, the effect of protein concentration, a parameter affecting kinetics of fibril formation, was investigated and an on-pathway involvement of micellar aggregates during the fibril formation process was suggested.


Assuntos
Proteínas Fúngicas/química , Penicillium chrysogenum/química , Tensoativos/química , Amiloide/química , Emulsificantes/química , Emulsificantes/isolamento & purificação , Emulsões/química , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Micelas , Tensão Superficial , Tensoativos/isolamento & purificação , Temperatura
18.
Can J Microbiol ; 65(11): 814-822, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31265796

RESUMO

Peptidases secreted by a clinical high-virulence Scedosporium aurantiacum isolate (strain WM 06.482; CBS 136046) under normoxic and hypoxic conditions were separated via size-exclusion chromatography, and peptidase activities present in each fraction were determined using class-specific substrates. The fractions demonstrating peptidase activity were assessed for their effects on the attachment and viability of A549 human lung epithelial cells in vitro. Of the peptidases detected in the size-exclusion chromatography fractions, the elastase-like peptidase reduced cell viability, the chymotrypsin-like peptidase was associated with cell detachment, and the cysteine peptidases were able to abolish both cell attachment and viability. The loss of cell viability and attachment became more prominent with an increase in the peptidase activity and could also be specifically prevented by addition of class-specific peptidase inhibitors. Our findings indicate that peptidases secreted by S. aurantiacum can breach the human alveolar epithelial cell barrier and, thus, may have a role in the pathobiology of the organism.


Assuntos
Células Epiteliais/microbiologia , Proteínas Fúngicas/metabolismo , Micoses/microbiologia , Peptídeo Hidrolases/metabolismo , Scedosporium/enzimologia , Transporte Biológico , Proteínas Fúngicas/isolamento & purificação , Humanos , Peptídeo Hidrolases/isolamento & purificação , Scedosporium/metabolismo , Scedosporium/patogenicidade , Virulência
19.
BMC Biotechnol ; 19(1): 43, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31262286

RESUMO

BACKGROUND: Proteases are hydrolytic enzymes that catalyze peptide linkage cleavage reactions at the level of proteins and peptides with different degrees of specificity. This group draws the attention of industry. More than one protease in three is a serine protease. Classically, they are active at neutral to alkaline pH. The serine proteases are researched for industrial uses, especially detergents. They are the most commercially available enzyme group in the world market. Overall, fungi produced extracellular proteases, easily separated from mycelium by filtration. RESULTS: A new basidiomycete fungus CTM10057, a hyperproducer of a novel protease (10,500 U/mL), was identified as Pleurotus sajor-caju (oyster mushroom). The enzyme, called SPPS, was purified to homogeneity by heat-treatment (80 °C for 20 min) followed by ammonium sulfate precipitation (35-55%)-dialysis, then UNO Q-6 FPLC ion-exchange chromatography and finally HPLC-ZORBAX PSM 300 HPSEC gel filtration chromatography, and submitted to biochemical characterization assays. The molecular mass was estimated to be 65 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion by HPLC. A high homology with mushroom proteases was displayed by the first 26 amino-acid residues of the NH2-terminal aminoacid sequence. Phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP) strongly inhibit SPPS, revealing that it is a member of the serine-proteases family. The pH and temperature optima were 9.5 and 70 °C, respectively. Interestingly, SPPS possesses the most elevated hydrolysis level and catalytic efficiency in comparison with SPTC, Flavourzyme® 500 L, and Thermolysin type X proteases. More remarkably, a high tolerance towards organic solvent tolerance was exhibited by SPPS, together with considerable detergent stability compared to the commercial proteases Thermolysin type X and Flavourzyme® 500 L, respectively. CONCLUSIONS: This proves the excellent proprieties characterizing SPPS, making it a potential candidate for industrial applications especially detergent formulations.


Assuntos
Proteínas Fúngicas/metabolismo , Temperatura Alta , Pleurotus/enzimologia , Serina Proteases/metabolismo , Detergentes/química , Estabilidade Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Concentração de Íons de Hidrogênio , Hidrólise , Microbiologia Industrial/métodos , Cinética , Peso Molecular , Serina Proteases/química , Serina Proteases/isolamento & purificação , Especificidade por Substrato
20.
Rev. bras. parasitol. vet ; 28(3): 339-345, July-Sept. 2019.
Artigo em Inglês | LILACS | ID: biblio-1042513

RESUMO

Abstract Gastrointestinal nematode infection is an important cause of high economic losses in livestock production. Nematode control based on a synthetic chemical approach is considered unsustainable due to the increasing incidence of anthelmintic resistance. Control alternatives such as the use of natural products are therefore becoming relevant from an environmental and economic point of view. Proteins are macromolecules with various properties that can be obtained from a wide range of organisms, including plants and fungi. Proteins belonging to different classes have shown great potential for the control of nematodes. The action of proteins can occur at specific stages of the nematode life cycle, depending on the composition of the external layers of the nematode body and the active site of the protein. Advances in biotechnology have resulted in the emergence of numerous protein and peptide therapeutics; however, few have been discussed with a focus on the control of animal nematodes. Here, we discuss the use of exogenous proteins and peptides in the control of gastrointestinal.


Resumo A infecção por nematoides gastrintestinais é uma importante causa de grandes perdas econômicas na pecuária. O controle de nematoides com compostos químicos sintéticos é considerado insustentável devido ao aumento da resistência anti-helmíntica. Alternativas de controle, como o uso de produtos naturais, estão se tornando relevantes do ponto de vista ambiental e econômico. As proteínas são macromoléculas com várias propriedades que podem ser obtidas de uma ampla gama de organismos, incluindo plantas e fungos. Proteínas pertencentes a diferentes classes têm mostrado grande potencial para o controle de nematoides. A ação das proteínas pode ocorrer em estágios específicos do ciclo de vida do nematoide, dependendo da composição das camadas externas do parasito e do sítio ativo da proteína. Avanços na biotecnologia resultaram no surgimento de numerosas terapias de proteínas e peptídeos; no entanto, pouco foi discutido com foco no controle de nematoides parasitos de animais. Na presente revisão foi discutido o uso de proteínas exógenas e peptídeos no controle de nematoides gastrintestinais, os mecanismos sugeridos de ação, e os desafios e perspectivas para o uso dessas biomoléculas como uma classe de anti-helmínticos.


Assuntos
Animais , Peptídeos/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação , Gastroenteropatias/veterinária , Infecções por Nematoides/veterinária , Antinematódeos/isolamento & purificação , Peptídeo Hidrolases/administração & dosagem , Peptídeo Hidrolases/isolamento & purificação , Peptídeos/administração & dosagem , Proteínas de Plantas/administração & dosagem , Biotecnologia , Proteínas Fúngicas/administração & dosagem , Quitinases/administração & dosagem , Quitinases/isolamento & purificação , Gastroenteropatias/parasitologia , Infecções por Nematoides/tratamento farmacológico , Antinematódeos/administração & dosagem
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