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1.
PLoS Comput Biol ; 16(9): e1007922, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32946455

RESUMO

Prions are self-replicative protein particles lacking nucleic acids. Originally discovered for causing infectious neurodegenerative disorders, they have also been found to play several physiological roles in a variety of species. Functional and pathogenic prions share a common mechanism of replication, characterized by the ability of an amyloid conformer to propagate by inducing the conversion of its physiological, soluble counterpart. Since time-resolved biophysical experiments are currently unable to provide full reconstruction of the physico-chemical mechanisms responsible for prion replication, one must rely on computer simulations. In this work, we show that a recently developed algorithm called Self-Consistent Path Sampling (SCPS) overcomes the computational limitations of plain MD and provides a viable tool to investigate prion replication processes using state-of-the-art all-atom force fields in explicit solvent. First, we validate the reliability of SCPS simulations by characterizing the folding of a class of small proteins and comparing against the results of plain MD simulations. Next, we use SCPS to investigate the replication of the prion forming domain of HET-s, a physiological fungal prion for which high-resolution structural data are available. Our atomistic reconstruction shows remarkable similarities with a previously reported mechanism of mammalian PrPSc propagation obtained using a simpler and more approximate path sampling algorithm. Together, these results suggest that the propagation of prions generated by evolutionary distant proteins may share common features. In particular, in both these cases, prions propagate their conformation through a very similar templating mechanism.


Assuntos
Proteínas Fúngicas , Simulação de Dinâmica Molecular , Príons , Algoritmos , Biologia Computacional , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Príons/química , Príons/metabolismo , Conformação Proteica , Dobramento de Proteína
2.
Nat Commun ; 11(1): 4667, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938938

RESUMO

The pyruvate dehydrogenase complex (PDC) is a multienzyme complex central to aerobic respiration, connecting glycolysis to mitochondrial oxidation of pyruvate. Similar to the E3-binding protein (E3BP) of mammalian PDC, PX selectively recruits E3 to the fungal PDC, but its divergent sequence suggests a distinct structural mechanism. Here, we report reconstructions of PDC from the filamentous fungus Neurospora crassa by cryo-electron microscopy, where we find protein X (PX) interior to the PDC core as opposed to substituting E2 core subunits as in mammals. Steric occlusion limits PX binding, resulting in predominantly tetrahedral symmetry, explaining previous observations in Saccharomyces cerevisiae. The PX-binding site is conserved in (and specific to) fungi, and complements possible C-terminal binding motifs in PX that are absent in mammalian E3BP. Consideration of multiple symmetries thus reveals a differential structural basis for E3BP-like function in fungal PDC.


Assuntos
Proteínas Fúngicas/química , Neurospora crassa/química , Complexo Piruvato Desidrogenase/química , Sítios de Ligação , Microscopia Crioeletrônica , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Complexo Piruvato Desidrogenase/genética , Complexo Piruvato Desidrogenase/metabolismo
3.
Nat Commun ; 11(1): 4031, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788582

RESUMO

Calcium (Ca2+) influx into mitochondria occurs through a Ca2+-selective uniporter channel, which regulates essential cellular processes in eukaryotic organisms. Previous evolutionary analyses of its pore-forming subunits MCU and EMRE, and gatekeeper MICU1, pinpointed an evolutionary paradox: the presence of MCU homologs in fungal species devoid of any other uniporter components and of mt-Ca2+ uptake. Here, we trace the mt-Ca2+ uniporter evolution across 1,156 fully-sequenced eukaryotes and show that animal and fungal MCUs represent two distinct paralogous subfamilies originating from an ancestral duplication. Accordingly, we find EMRE orthologs outside Holoza and uncover the existence of an animal-like uniporter within chytrid fungi, which enables mt-Ca2+ uptake when reconstituted in vivo in the yeast Saccharomyces cerevisiae. Our study represents the most comprehensive phylogenomic analysis of the mt-Ca2+ uptake system and demonstrates that MCU, EMRE, and MICU formed the core of the ancestral opisthokont uniporter, with major implications for comparative structural and functional studies.


Assuntos
Canais de Cálcio/genética , Evolução Molecular , Proteínas Fúngicas/genética , Sequência de Aminoácidos , Cálcio/metabolismo , Canais de Cálcio/química , Quitridiomicetos/genética , Proteínas Fúngicas/química , Células HeLa , Humanos , Funções Verossimilhança , Filogenia , Especificidade da Espécie
4.
Proc Natl Acad Sci U S A ; 117(31): 18600-18607, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32703806

RESUMO

Programmed cell death (PCD) in filamentous fungi prevents cytoplasmic mixing following fusion between conspecific genetically distinct individuals (allorecognition) and serves as a defense mechanism against mycoparasitism, genome exploitation, and deleterious cytoplasmic elements (i.e., senescence plasmids). Recently, we identified regulator of cell death-1 (rcd-1), a gene controlling PCD in germinated asexual spores in the filamentous fungus Neurospora crassa rcd-1 alleles are highly polymorphic and fall into two haplogroups in N. crassa populations. Coexpression of alleles from the two haplogroups, rcd-1-1 and rcd-1-2, is necessary and sufficient to trigger a cell death reaction. Here, we investigated the molecular bases of rcd-1-dependent cell death. Based on in silico analyses, we found that RCD-1 is a remote homolog of the N-terminal pore-forming domain of gasdermin, the executioner protein of a highly inflammatory cell death reaction termed pyroptosis, which plays a key role in mammalian innate immunity. We show that RCD-1 localizes to the cell periphery and that cellular localization of RCD-1 was correlated with conserved positively charged residues on predicted amphipathic α-helices, as shown for murine gasdermin-D. Similar to gasdermin, RCD-1 binds acidic phospholipids in vitro, notably, cardiolipin and phosphatidylserine, and interacts with liposomes containing such lipids. The RCD-1 incompatibility system was reconstituted in human 293T cells, where coexpression of incompatible rcd-1-1/rcd-1-2 alleles triggered pyroptotic-like cell death. Oligomers of RCD-1 were associated with the cell death reaction, further supporting the evolutionary relationship between gasdermin and rcd-1 This report documents an ancient transkingdom relationship of cell death execution modules involved in organismal defense.


Assuntos
Proteínas Fúngicas , Proteínas de Neoplasias , Piroptose/fisiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/fisiologia , Células HEK293 , Humanos , Imunidade Inata/fisiologia , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Neurospora crassa/metabolismo
5.
Nat Commun ; 11(1): 3290, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620929

RESUMO

In mitochondria, ß-barrel outer membrane proteins mediate protein import, metabolite transport, lipid transport, and biogenesis. The Sorting and Assembly Machinery (SAM) complex consists of three proteins that assemble as a 1:1:1 complex to fold ß-barrel proteins and insert them into the mitochondrial outer membrane. We report cryoEM structures of the SAM complex from Myceliophthora thermophila, which show that Sam50 forms a 16-stranded transmembrane ß-barrel with a single polypeptide-transport-associated (POTRA) domain extending into the intermembrane space. Sam35 and Sam37 are located on the cytosolic side of the outer membrane, with Sam35 capping Sam50, and Sam37 interacting extensively with Sam35. Sam35 and Sam37 each adopt a GST-like fold, with no functional, structural, or sequence similarity to their bacterial counterparts. Structural analysis shows how the Sam50 ß-barrel opens a lateral gate to accommodate its substrates.


Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Biossíntese de Proteínas , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Microscopia Crioeletrônica , Detergentes/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Conformação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Sordariales/genética , Sordariales/metabolismo
6.
Mol Genet Genomics ; 295(5): 1269-1279, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32561986

RESUMO

Aspergillus nidulans nrtA encodes a nitrate transporter that plays an important role in the [Formula: see text] assimilatory process. Many studies have focused on protein functions rather than gene regulation. The knowledge of nrtA[Formula: see text] uptake process, particularly in the regulation mechanism of transcription factors AreA and NirA on nrtA transcription, is very limited. Herein, we investigated the transcriptional regulation of nrtA in response to various N-sources in detail and characterized the promoter activity of nrtA. We confirmed that nrtA was induced by [Formula: see text] and repressed by preferred N-sources. Additionally, for the first time, we found that the transcription of nrtA increased under N-starvation conditions. AreA mediates nrtA transcription under both [Formula: see text] and N-starvation conditions, while NirA is effective only under [Formula: see text] conditions. All of the proposed AreA and NirA binding sites in the promoter region were capable of binding to their corresponding transcription factors in vitro. In vivo, all of the NirA binding sites showed regulation activities, but to AreA, only several of the initiation-codon-proximal binding sites participated in nrtA transcription. Moreover, the active binding sites contributed in different degrees of regulation strength to nrtA transcription, which is unrelated to the distance between the binding sites and initiation codon. These results provided an extensive map of nrtA promoter, defining the functional regulatory elements of A. nidulans nrtA.


Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Aspergillus nidulans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Nitratos/metabolismo , Proteínas de Transporte de Ânions/química , Aspergillus nidulans/genética , Sítios de Ligação , Proteínas Fúngicas/química , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo
7.
Mol Genet Genomics ; 295(5): 1295-1304, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32566991

RESUMO

Pichia pastoris is able to metabolize methanol via a specific MUT (methanol utilization) pathway. Based on the powerful AOX1 (Alcohol Oxidase 1) promoter, the P. pastoris expression system has become one of the most widely used eukaryotic expression systems. The molecular mechanisms of methanol metabolic regulation remain unclearly understood, so it is important to identify and develop new transcriptional regulators. Our previous studies suggested that the expression of SUT2 could be induced by methanol but is repressed by glycerol, which indicates that SUT2 may be involved in methanol metabolism through an unknown mechanism. SUT2 encodes a putative transcription factor-like protein harboring a Gal4-like Zn2Cys6 DNA-binding domain in Pichia pastoris, and its homolog in Saccharomyces cerevisiae regulates sterol uptake and synthesis. This study shows that the overexpression of SUT2 promoted the expression of AOX1 and increases ergosterol content in cells. Furthermore, via truncation of the putative SUT2 promoter at diverse loci, the - 973 base pair (bp) to - 547 bp region to the ATG was shown to be the core element of the inducible promoter PSUT2, which strongly responds to the methanol signal. The transcriptional start site of SUT2, "A" at the 22nd bp upstream of ATG, was determined with 5'-rapid amplification of cDNA ends. A forward-loop cassette was constructed with MXR1 (Methanol Expression Regulator 1, a positive transcription factor of PAOX1) promoted by PSUT2, enabling moderate elevation in the expression level of Mxr1 and high activity of PAOX1 without damaging cellular robustness further boosting the production of heterologous proteins. The PAOX1-driven expression of enhanced green fluorescent protein in this novel system was improved by 18%, representing a promising method for extrinsic protein production. SUT2 may play roles in methanol metabolism by participating in sterol biosynthesis. PSUT2 was characterized as a novel inducible promoter in P. pastoris and a PSUT2-driven MXR1 forward-loop cassette was constructed to enhance the PAOX1 activity, laying a foundation for further development and application of P. pastoris expression system.


Assuntos
Metanol/metabolismo , Pichia/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Aldeído Oxidase/metabolismo , Sítios de Ligação , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Regiões Promotoras Genéticas , Deleção de Sequência , Fatores de Transcrição/química , Sítio de Iniciação de Transcrição
8.
PLoS Pathog ; 16(6): e1008652, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32574207

RESUMO

Plants trigger immune responses upon recognition of fungal cell wall chitin, followed by the release of various antimicrobials, including chitinase enzymes that hydrolyze chitin. In turn, many fungal pathogens secrete LysM effectors that prevent chitin recognition by the host through scavenging of chitin oligomers. We previously showed that intrachain LysM dimerization of the Cladosporium fulvum effector Ecp6 confers an ultrahigh-affinity binding groove that competitively sequesters chitin oligomers from host immune receptors. Additionally, particular LysM effectors are found to protect fungal hyphae against chitinase hydrolysis during host colonization. However, the molecular basis for the protection of fungal cell walls against hydrolysis remained unclear. Here, we determined a crystal structure of the single LysM domain-containing effector Mg1LysM of the wheat pathogen Zymoseptoria tritici and reveal that Mg1LysM is involved in the formation of two kinds of dimers; a chitin-dependent dimer as well as a chitin-independent homodimer. In this manner, Mg1LysM gains the capacity to form a supramolecular structure by chitin-induced oligomerization of chitin-independent Mg1LysM homodimers, a property that confers protection to fungal cell walls against host chitinases.


Assuntos
Ascomicetos/química , Quitina/química , Proteínas Fúngicas/química , Hifas/química , Multimerização Proteica , Ascomicetos/genética , Ascomicetos/metabolismo , Quitina/genética , Quitina/metabolismo , Cladosporium/química , Cladosporium/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Hifas/genética , Hifas/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Estrutura Quaternária de Proteína , Triticum/genética , Triticum/metabolismo , Triticum/microbiologia
9.
Nat Commun ; 11(1): 2830, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503980

RESUMO

The Spitzenkörper (SPK) constitutes a collection of secretory vesicles and polarity-related proteins intimately associated with polarized growth of fungal hyphae. Many SPK-localized proteins are known, but their assembly and dynamics remain poorly understood. Here, we identify protein-protein interaction cascades leading to assembly of two SPK scaffolds and recruitment of diverse effectors in Neurospora crassa. Both scaffolds are transported to the SPK by the myosin V motor (MYO-5), with the coiled-coil protein SPZ-1 acting as cargo adaptor. Neither scaffold appears to be required for accumulation of SPK secretory vesicles. One scaffold consists of Leashin-2 (LAH-2), which is required for SPK localization of the signalling kinase COT-1 and the glycolysis enzyme GPI-1. The other scaffold comprises a complex of Janus-1 (JNS-1) and the polarisome protein SPA-2. Via its Spa homology domain (SHD), SPA-2 recruits a calponin domain-containing F-actin effector (CCP-1). The SHD NMR structure reveals a conserved surface groove required for effector binding. Similarities between SPA-2/JNS-1 and the metazoan GIT/PIX complex identify foundational features of the cell polarity apparatus that predate the fungal-metazoan divergence.


Assuntos
Polaridade Celular , Proteínas Fúngicas/metabolismo , Miosina Tipo V/metabolismo , Neurospora crassa/metabolismo , Vesículas Secretórias/metabolismo , Proteínas Fúngicas/química , Hifas/citologia , Hifas/metabolismo , Miosina Tipo V/química , Neurospora crassa/citologia , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Mapas de Interação de Proteínas
10.
Food Chem ; 330: 127252, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32585587

RESUMO

Mycoprotein is a food ingredient from filamentous fungi rich in protein and fibre. This study investigated the protein bioaccessibility from the fungal cells by colourimetric assays in different mycoprotein formulations, following extraction methods and in vitro gastrointestinal digestion. The methods effects were further analysed by static laser light scattering, SDS-PAGE and optical-fluorescence microscopy. The extraction methods released a comparable proportion of protein (30 wt%) independent of sample concentration (10 wt% and 25 wt%), whereas the simulated digestions endpoints released a higher proportion of protein from the less concentrated (46 wt%). Furthermore, mechanical/physical processing had only a minor impact. Intestinal proteases promoted the most efficient protein release but without causing any apparent damage to the cell walls when viewed by microscopy. This suggested that the enzymes can diffuse through the cell walls, due to its porosity/permeability, and are the main factors responsible for the hydrolysis and bioaccessibility of protein from mycoprotein.


Assuntos
Proteínas Fúngicas/metabolismo , Animais , Disponibilidade Biológica , Parede Celular/química , Parede Celular/metabolismo , Fibras na Dieta/metabolismo , Digestão , Proteínas Fúngicas/química , Fungos/química , Fungos/metabolismo
11.
Am J Clin Nutr ; 112(2): 318-333, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32438401

RESUMO

BACKGROUND: Mycoprotein is a fungal-derived sustainable protein-rich food source, and its ingestion results in systemic amino acid and leucine concentrations similar to that following milk protein ingestion. OBJECTIVE: We assessed the mixed skeletal muscle protein synthetic response to the ingestion of a single bolus of mycoprotein compared with a leucine-matched bolus of milk protein, in rested and exercised muscle of resistance-trained young men. METHODS: Twenty resistance-trained healthy young males (age: 22 ± 1 y, body mass: 82 ± 2 kg, BMI: 25 ± 1 kg·m-2) took part in a randomized, double-blind, parallel-group study. Participants received primed, continuous infusions of L-[ring-2H5]phenylalanine and ingested either 31 g (26.2 g protein: 2.5 g leucine) milk protein (MILK) or 70 g (31.5 g protein: 2.5 g leucine) mycoprotein (MYCO) following a bout of unilateral resistance-type exercise (contralateral leg acting as resting control). Blood and m. vastus lateralis muscle samples were collected before exercise and protein ingestion, and following a 4-h postprandial period to assess mixed muscle fractional protein synthetic rates (FSRs) and myocellular signaling in response to the protein beverages in resting and exercised muscle. RESULTS: Mixed muscle FSRs increased following MILK ingestion (from 0.036 ± 0.008 to 0.052 ± 0.006%·h-1 in rested, and 0.035 ± 0.008 to 0.056 ± 0.005%·h-1 in exercised muscle; P <0.01) but to a greater extent following MYCO ingestion (from 0.025 ± 0.006 to 0.057 ± 0.004%·h-1 in rested, and 0.024 ± 0.007 to 0.072 ± 0.005%·h-1 in exercised muscle; P <0.0001) (treatment × time interaction effect; P <0.05). Postprandial FSRs trended to be greater in MYCO compared with MILK (0.065 ± 0.004 compared with 0.054 ± 0.004%·h-1, respectively; P = 0.093) and the postprandial rise in FSRs was greater in MYCO compared with MILK (Delta 0.040 ± 0.006 compared with Delta 0.018 ± 0.005%·h-1, respectively; P <0.01). CONCLUSIONS: The ingestion of a single bolus of mycoprotein stimulates resting and postexercise muscle protein synthesis rates, and to a greater extent than a leucine-matched bolus of milk protein, in resistance-trained young men. This trial was registered at clinicaltrials.gov as 660065600.


Assuntos
Proteínas Fúngicas/metabolismo , Proteínas do Leite/metabolismo , Proteínas Musculares/biossíntese , Adulto , Aminoácidos/metabolismo , Método Duplo-Cego , Exercício Físico , Proteínas Fúngicas/química , Humanos , Masculino , Proteínas do Leite/química , Músculo Esquelético/metabolismo , Treinamento de Resistência , Adulto Jovem
12.
Nat Commun ; 11(1): 2450, 2020 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-32415073

RESUMO

The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Deletion of a gene encoding a putative deacetylase, Agd3, leads to defects in GAG deacetylation, biofilm formation, and virulence. Here, we show that Agd3 deacetylates GAG in a metal-dependent manner, and is the founding member of carbohydrate esterase family CE18. The active site is formed by four catalytic motifs that are essential for activity. The structure of Agd3 includes an elongated substrate-binding cleft formed by a carbohydrate binding module (CBM) that is the founding member of CBM family 87. Agd3 homologues are encoded in previously unidentified putative bacterial exopolysaccharide biosynthetic operons and in other fungal genomes.


Assuntos
Amidoidrolases/química , Amidoidrolases/metabolismo , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/fisiologia , Biofilmes/crescimento & desenvolvimento , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Polissacarídeos/metabolismo , Acetilação , Sequência de Aminoácidos , Aspergillus fumigatus/genética , Domínio Catalítico , Sequência Conservada , Regulação Fúngica da Expressão Gênica , Glicosaminoglicanos/biossíntese , Metais/metabolismo , Domínios Proteicos , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Fatores de Tempo
13.
Biochim Biophys Acta Biomembr ; 1862(8): 183246, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32142818

RESUMO

The filamentous fungus Penicillium chrysogenum Q176 secretes the antimicrobial proteins (AMPs) PAF and PAFB, which share a compact disulfide-bond mediated, ß-fold structure rendering them highly stable. These two AMPs effectively inhibit the growth of human pathogenic fungi in micromolar concentrations and exhibit antiviral potential without causing cytotoxic effects on mammalian cells in vitro and in vivo. The antifungal mechanism of action of both AMPs is closely linked to - but not solely dependent on - the lipid composition of the fungal cell membrane and requires a strictly regulated protein uptake into the cell, indicating that PAF and PAFB are not canonical membrane active proteins. Variations in their antifungal spectrum and their killing dynamics point towards a divergent mode of action related to their physicochemical properties and surface charge distribution. In this review, we relate characteristic features of PAF and PAFB to the current knowledge about other AMPs of different sources. In addition, we present original data that have never been published before to substantiate our assumptions and provide evidences that help to explain and understand better the mechanistic function of PAF and PAFB. Finally, we underline the promising potential of PAF and PAFB as future antifungal therapeutics.


Assuntos
Antifúngicos/química , Peptídeos Catiônicos Antimicrobianos/química , Proteínas Fúngicas/química , Micoses/tratamento farmacológico , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/farmacologia , Apoptose/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Cisteína/genética , Proteínas Fúngicas/genética , Humanos , Lipídeos de Membrana/química , Micoses/genética , Micoses/microbiologia , Penicillium chrysogenum/química , Penicillium chrysogenum/genética
14.
Enzyme Microb Technol ; 135: 109507, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32146934

RESUMO

The azo dye Congo red is heavily used in textile industries and is actively present in the wastewater run-offs. Its structural complexity and physical characteristics make it resistant to the physicochemical treatments employed by the industry. Over time, application of the enzyme laccase has proved to be quite useful due to its ability to oxidize and eventually decolorize the dye. Moreover, the use of ABTS as the electron mediator also helps in enhancing the oxidizing capability of the enzyme with congo red. The present study involves establishing the role of the individual components i.e. laccase, ABTS and the dye, in the LMS electrochemically. Congo red doesn't have any form of electrochemical activity by itself, but the enzyme brings about a substantial change by increasing the rate of reduction. The effect of ABTS, though same, is concentration-dependent. For LMS, laccase helps in bringing about the rate of reduction much faster in the presence of the mediator, initiating the decolorization of the dye.


Assuntos
Vermelho Congo/metabolismo , Proteínas Fúngicas/metabolismo , Lacase/metabolismo , Trametes/metabolismo , Compostos Azo/química , Compostos Azo/metabolismo , Benzotiazóis/metabolismo , Biodegradação Ambiental , Proteínas Fúngicas/química , Cinética , Lacase/química , Ácidos Sulfônicos/metabolismo , Trametes/química , Trametes/enzimologia
15.
Enzyme Microb Technol ; 135: 109496, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32146939

RESUMO

Catechins in green tea possess various health benefits. Enzymatic treatment improves physiological activities by inducing bioconversion of catechins. Here, we investigated the effect of green tea infusion (GT) after tannase treatment, which transforms (-)-epigallocatechin gallate (EGCG) to gallic acid (GA) and (-)-epigallocatechin (EGC), on adipocyte differentiation and mature adipocyte metabolism. The optimal conditions for tannase-mediated improvement in GA and EGC yields in GT were investigated using response surface methodology. Yields of GA and EGC were 43-fold (0.43 mg/mL) and 1.66-fold higher (1.11 mg/mL), respectively, compared to GT without tannase treatment. The optimal reaction conditions for tannase-mediated biotransformation were observed on 0.54 mg mL-1 of tannase, reaction time (86.79 min), and reaction temperature at 22.59 °C. GT and tannase-treated GT (TANs) upregulated adiponectin, uncoupling protein 1, adipose triglyceride lipase, and hormone-sensitive lipase gene expression in differentiated 3T3-L1 adipocytes, with TAN inducing better effects than GT, which implies that tannase treatment improved the beneficial effect of GT on adipocyte metabolism. Thus, tannase-mediated bio-transformation is an attractive candidate for preparing GT with enhanced anti-obesity properties.


Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Hidrolases de Éster Carboxílico/química , Catequina/análogos & derivados , Proteínas Fúngicas/química , Extratos Vegetais/química , Adipócitos/citologia , Adiponectina/genética , Adiponectina/metabolismo , Animais , Aspergillus/enzimologia , Biocatálise , Camellia sinensis/química , Catequina/química , Catequina/farmacologia , Diferenciação Celular , Manipulação de Alimentos , Ácido Gálico/química , Ácido Gálico/farmacologia , Camundongos , Células NIH 3T3 , Extratos Vegetais/farmacologia , Folhas de Planta/química , Chá/química , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
16.
J Biosci Bioeng ; 130(1): 29-35, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32171656

RESUMO

Saccharomyces cerevisiae can obtain xylose utilization capacity via integration of heterogeneous xylose reductase (XR) and xylitol dehydrogenase (XDH) genes into its metabolic pathway, and XYL2 which encodes the XDH plays an essential role in this process. Herein, we reported that two hypothetical XYL2 genes from the multistress-tolerant yeasts of Issatchenkia orientalis and Torulaspora delbrueckii were cloned, and they encoded two XDHs, IoXyl2p and TdXyl2p, respectively, with the activities for oxidation of xylitol to xylulose. Comparative studies demonstrated that IoXyl2p and TdXyl2p, like the SsXyl2p from Scheffersomyces stipitis, were probably localized to the cytoplasm and strictly dependent on NAD+ rather than NADP+ as the cofactor for catalyzing the oxidation reaction of xylitol. IoXyl2p had the highest specific activity, maximum velocity (Vmax), affinity to xylitol (Km), and catalytic efficiency (kcat/Km) among the three XDHs. The optimum temperature for oxidation of xylitol were at 45 °C by IoXyl2p and at 35 °C by TdXyl2p and SsXyl2p, and the optimum pH of IoXyl2p, TdXyl2p and SsXyl2p for oxidation of xylitol was 8.0, 8.5 and 7.5, respectively. Mg2+ promoted the activities of IoXyl2p and TdXyl2p, but slightly inhibited the activity of SsXyl2p. Most metal ions had much weaker inhibition effects on IoXyl2p and TdXyl2p than SsXyl2p. IoXyl2p displayed the strongest salt resistance among the three XDHs. To summarize, IoXyl2p from I. orientalis and TdXyl2p from T. delbrueckii characterized in this study are considered to be the attractive candidates for the construction of genetically engineered S. cerevisiae for efficiently fermentation of carbohydrate in lignocellulosic hydrolysate.


Assuntos
D-Xilulose Redutase/genética , D-Xilulose Redutase/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Pichia/enzimologia , Torulaspora/enzimologia , Clonagem Molecular , D-Xilulose Redutase/química , Estabilidade Enzimática , Fermentação , Proteínas Fúngicas/química , Cinética , Pichia/genética , Pichia/metabolismo , Torulaspora/genética , Torulaspora/metabolismo , Xilitol/metabolismo , Xilose/metabolismo
17.
Food Chem ; 318: 126482, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32145543

RESUMO

We have examined the trans-resveratrol/lipase interaction by quantitative and qualitative analyses of fluorescence spectra, molecular docking and quantum-chemical calculations at DFT level. Interactions of CpLIP2 from C. parapsilosis CBS 604 and trans-resveratrol were confirmed with a major contribution of tryptophan residues to fluorescence quenching. A thermodynamic study across a wide temperature range was consistent with the presence of a single binding site with a binding free energy of -24 kJ/mol. Nevertheless, trans-resveratrol competitively inhibited CpLIP2 activity. Molecular docking and quantum-chemical calculations were consistent with a strong binding of trans-resveratrol to the CpLIP2 catalytic site via electrostatic and hydrophobic forces. The structural analysis quantitatively revealed an energy transfer from W51 and W350 to trans-resveratrol with a distance of 32 Å. Precise understanding of trans-resveratrol/CpLIP2 interactions has important implications on lipases for screening of stilbenoid.


Assuntos
Candida parapsilosis/enzimologia , Lipase/metabolismo , Resveratrol/metabolismo , Sítios de Ligação , Domínio Catalítico , Simulação por Computador , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Fluorescência , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Lipase/antagonistas & inibidores , Lipase/química , Simulação de Acoplamento Molecular , Resveratrol/química , Resveratrol/farmacocinética , Termodinâmica
18.
PLoS Pathog ; 16(3): e1008323, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32163521

RESUMO

Fusarium is a genus of filamentous fungi that includes species that cause devastating diseases in major staple crops, such as wheat, maize, rice, and barley, resulting in severe yield losses and mycotoxin contamination of infected grains. Phenamacril is a novel fungicide that is considered environmentally benign due to its exceptional specificity; it inhibits the ATPase activity of the sole class I myosin of only a subset of Fusarium species including the major plant pathogens F. graminearum, F. asiaticum and F. fujikuroi. To understand the underlying mechanisms of inhibition, species specificity, and resistance mutations, we have determined the crystal structure of phenamacril-bound F. graminearum myosin I. Phenamacril binds in the actin-binding cleft in a new allosteric pocket that contains the central residue of the regulatory Switch 2 loop and that is collapsed in the structure of a myosin with closed actin-binding cleft, suggesting that pocket occupancy blocks cleft closure. We have further identified a single, transferable phenamacril-binding residue found exclusively in phenamacril-sensitive myosins to confer phenamacril selectivity.


Assuntos
Cianoacrilatos/química , Proteínas Fúngicas/química , Fungicidas Industriais/química , Fusarium/enzimologia , Miosina Tipo I/química , Cianoacrilatos/farmacologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fungicidas Industriais/farmacologia , Fusarium/química , Fusarium/efeitos dos fármacos , Fusarium/genética , Miosina Tipo I/genética , Miosina Tipo I/metabolismo , Doenças das Plantas/microbiologia , Triticum/microbiologia , Zea mays/microbiologia
19.
J Agric Food Chem ; 68(9): 2757-2764, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32026695

RESUMO

Aspergillus oryzae 3.042 was mutagenized using atmospheric and room-temperature plasma (ARTP) technology to enhance its salt-tolerant proteases activity. Compared to the starting strain, mutant H8 subjected to 180 s of ARTP treatment exhibited excellent genetic stability (15 generations), growth rate, and significantly increased activities of neutral proteases, alkaline proteases, and aspartyl aminopeptidase during fermentation. Mutant H8 significantly enhanced the contents of 1-5 kDa peptides, aspartic acid, serine, threonine, and cysteine in soy sauce by 16.61, 7.69, 17.30, 8.61, and 45.00%, respectively, but it had no effects on the contents of the other 14 free amino acids (FAAs) due to its slightly enhanced acidic proteases activity. Analyses of transcriptional expressions of salt-tolerant alkaline protease gene (AP, gi: 217809) and aspartyl aminopeptidase gene (AAP, gi: 6165646) indicated that their expression levels were increased by approximately 30 and 27%, respectively. But no mutation was found in the sequences of AP and AAP expression cassettes, suggesting that the increased activities of proteases in mutant H8 should be partially attributed to the increased expression of proteases. ARTP technology showed great potential in enhancing the activities of salt-tolerant proteases from A. oryzae.


Assuntos
Aspergillus oryzae/enzimologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Cloreto de Sódio/metabolismo , Aspergillus oryzae/química , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Estabilidade Enzimática , Fermentação , Proteínas Fúngicas/química , Mutagênese , Peptídeo Hidrolases/química , Alimentos de Soja/análise , Alimentos de Soja/microbiologia
20.
J Agric Food Chem ; 68(9): 2702-2710, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32054270

RESUMO

d-Xylose is the most abundant hemicellulosic monomer on earth, but wild-type Saccharomyces cerevisiae has very limited d-xylose uptake capacity. We conducted bioprospecting for new sugar transporters from the d-xylose-consuming filamentous fungus Trichoderma reesei and identified three candidates belonging to the major facilitator superfamily. When they were expressed in yeast and assayed for d-xylose uptake, one of them, Xltr1p, had d-xylose transport activity that was more efficient than that of Gal2p, an endogenous yeast transporter. Site-directed mutagenesis was used to examine the functional contributions of 13 amino acid residues for the uptake of d-xylose, and these experiments identified particular amino acids that function distinctly in d-xylose vs glucose transport (e.g., F300). Excitingly, the yeast strain expressing the N326FXltr1p variant was able to carry a "high efficiency" transport for d-xylose but was nearly unable to utilize glucose; in contrast, the strain with the F300AXltr1p variant grew on glucose but lost d-xylose transport activity.


Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Saccharomyces cerevisiae/genética , Trichoderma/metabolismo , Xilose/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Transporte Biológico , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Expressão Gênica , Glucose/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Trichoderma/genética
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