RESUMO
Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). Here, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. By analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP ribosylation factor 1 (ARF1), is required for IBDV replication, since inhibiting its activity by treatment with brefeldin A (BFA) or golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative mutant T31N overexpression hampered IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnavirus-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, with the lack of a transcriptionally active core being the main differential feature. This structural trait, among others that resemble those of the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses has been argued. Here, we present original data showing that IBDV-induced GC reorganization and the cross talk between IBDV and the Rab1b-GBF1-ARF1 mediate the intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnavirus-host cell interactions and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Vírus da Doença Infecciosa da Bursa/fisiologia , Via Secretória/fisiologia , Replicação Viral/fisiologia , Proteínas rab1 de Ligação ao GTP/metabolismo , Fator 1 de Ribosilação do ADP/genética , Animais , Brefeldina A/farmacologia , Linhagem Celular , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Interações Hospedeiro-Patógeno , Piridinas/farmacologia , Quinolinas/farmacologia , Via Secretória/efeitos dos fármacos , Compartimentos de Replicação Viral/metabolismo , Replicação Viral/efeitos dos fármacos , Proteínas rab1 de Ligação ao GTP/genéticaRESUMO
The 14-3-3 protein family binds and regulates hundreds of serine/threonine phosphorylated proteins as an essential component of many signaling networks. Specific biological functions are currently been discovered for each of its seven isoforms in mammals. These proteins have been traditionally considered unregulated; however, its acetylation in an essential lysine residue, causing its inactivation, was recently published. Here, we studied the acetylation state of this lysine 49/51 during the osteogenic differentiation of human adipose-derived stem cells. We found that during this process, the levels of 14-3-3ß (but not its isoform 14-3-3γ) acK49/51 increase, representing the first report linking this PTM to a specific isoform and a cellular process. Our results suggested that this posttranslational modification could be catalyzed by the HBO1 acetyltransferase, as overexpression of HBO1 increased specifically 14-3-3 acK49/51 acetylation. Acetylated 14-3-3 proteins are located primarily in the nucleus, where their active state has been described to bind H3 histones and many transcription factors. The inhibition of the expression of different isoforms showed that the specific silencing of the 14-3-3ß gene, but not γ, increased significantly the osteogenic potential of the cells. This result correlated to the increase in acetylation of 14-3- 3ß Lys 49/51 during osteogenesis. The possible role of this PTM in osteogenesis is discussed.
Assuntos
Proteínas 14-3-3/metabolismo , Diferenciação Celular , Osteogênese , Células-Tronco/metabolismo , Células 3T3-L1 , Acetilação , Animais , Humanos , Camundongos , Células NIH 3T3RESUMO
The SARS-CoV-2 N protein binds several cell host proteins including 14-3-3γ, a well-characterized regulatory protein. However, the biological function of this interaction is not completely understood. We analyzed the variability of â¼90 000 sequences of the SARS-CoV-2 N protein, particularly, its mutations in disordered regions containing binding motifs for 14-3-3 proteins. We studied how these mutations affect the binding energy to 14-3-3γ and found that changes positively affecting the predicted interaction with 14-3-3γ are the most successfully spread, with the highest prevalence in the phylogenetic tree. Although most residues are highly conserved within the 14-3-3 binding site, compensatory mutations to maintain the interaction energy of N-14-3-3γ were found, including half of the current variants of concern and interest. Our results suggest that binding of N to 14-3-3γ is beneficial for the virus, thus targeting this viral-host protein-protein interaction seems an attractive approach to explore antiviral strategies.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/análise , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Sítios de Ligação , Proteínas do Nucleocapsídeo de Coronavírus/genética , Humanos , Mutação/genética , Fosfoproteínas/análise , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilação , Filogenia , Ligação ProteicaRESUMO
3D printing has emerged as vanguard technique of biofabrication to assemble cells, biomaterials and biomolecules in a spatially controlled manner to reproduce native tissues. In this work, gelatin methacrylate (GelMA)/alginate hydrogel scaffolds were obtained by 3D printing and 14-3-3ε protein was encapsulated in the hydrogel to induce osteogenic differentiation of human adipose-derived mesenchymal stem cells (hASC). GelMA/alginate-based grid-like structures were printed and remained stable upon photo-crosslinking. The viscosity of alginate allowed to control the pore size and strand width. A higher viscosity of hydrogel ink enhanced the printing accuracy. Protein-loaded GelMA/alginate-based hydrogel showed a clear induction of the osteogenic differentiation of hASC cells. The results are relevant for future developments of GelMA/alginate for bone tissue engineering given the positive effect of 14-3-3ε protein on both cell adhesion and proliferation.
Assuntos
Proteínas 14-3-3/química , Hidrogéis/química , Osteogênese/fisiologia , Impressão Tridimensional , Tecido Adiposo/metabolismo , Alginatos/química , Adesão Celular , Diferenciação Celular , Proliferação de Células , Reagentes de Ligações Cruzadas , Gelatina , Humanos , Tinta , Células-Tronco Mesenquimais/metabolismo , Metacrilatos/química , Osteogênese/efeitos dos fármacos , Proteínas Recombinantes/química , ViscosidadeRESUMO
Human Adipose-Derived Mesenchymal Stem/Stromal Cells (hAD-MSCs) have great potential for tissue regeneration. Since transplanted hAD-MSCs are likely to be placed in a hypoxic environment, culturing the cells under hypoxic conditions might improve their post-transplantation survival and regenerative performance. The combination of hAD-MSCs and PCL-nHA nanofibers synergically improves the contribution of both components for osteoblast differentiation. In this work, we hypothesized that this biomaterial constitutes a hypoxic environment for hAD-MSCs. We studied the cellular re-arrangement and the subcellular ultrastructure by Transmission Electron Microscopy (TEM) of hAD-MSCs grown into PCL-nHA nanofibers, and we compared them with the same cells grown in two-dimensional cultures, over tissue culture-treated plastic, or glass coverslips. Among the most evident changes, PCL-nHA grown cells showed enlarged mitochondria, and accumulation of glycogen granules, consistent with a hypoxic environment. We observed a 3.5 upregulation (p = 0.0379) of Hypoxia Inducible Factor (HIF)-1A gene expression in PCL-nHA grown cells. This work evidences for the first time intra-cellular changes in three-dimensional compared to two-dimensional cultures, which are adaptive responses of the cells to an environment more closely resembling that of the in vivo niche after transplantation, thus PCL-nHA nanofibers are adequate for hAD-MSCs pre-conditioning.
Assuntos
Células-Tronco Mesenquimais , Nanofibras , Humanos , Alicerces Teciduais/química , Durapatita/química , Durapatita/metabolismo , Poliésteres/química , Materiais Biocompatíveis/química , Diferenciação Celular , Nanofibras/química , Engenharia Tecidual/métodosRESUMO
Gumboro illness is caused by the highly contagious immunosuppressive infectious bursal disease virus (IBDV), which affects the poultry industry globally. We have previously shown that IBDV hijacks the endocytic pathway to construct viral replication complexes on endosomes linked to the Golgi complex (GC). Then, analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b, the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), and its substrate, the small GTPase ADP-ribosylation factor 1 (ARF1), for IBDV replication. In the current work, we focused on elucidating the IBDV assembly sites. We show that viral assembly occurs within single-membrane compartments closely associated with endoplasmic reticulum (ER) membranes, though we failed to elucidate the exact nature of the virus-wrapping membranes. Additionally, we show that IBDV infection promotes the stress of the ER, characterized by an accumulation of the chaperone binding protein (BiP) and lipid droplets (LDs) in the host cells. Overall, our results represent further original data showing the interplay between IBDV and the secretory pathway, making a substantial contribution to the field of birnaviruses-host cell interactions.
Assuntos
Infecções por Birnaviridae , Vírus da Doença Infecciosa da Bursa , Doenças das Aves Domésticas , Animais , Gotículas Lipídicas , Montagem de Vírus , Endossomos , Estresse do Retículo Endoplasmático , GalinhasRESUMO
ADP-ribosylation factors (ARFs) have crucial roles in vesicular trafficking. Brefeldin A-inhibited guanine nucleotide-exchange proteins (BIG)1 and BIG2 catalyze the activation of class I ARFs by accelerating replacement of bound GDP with GTP. Several additional and differing actions of BIG1 and BIG2 have been described. These include the presence in BIG2 of 3 A kinase-anchoring protein (AKAP) domains, one of which is identical in BIG1. Proteins that contain AKAP sequences act as scaffolds for the assembly of PKA with other enzymes, substrates, and regulators in complexes that constitute molecular machines for the reception, transduction, and integration of signals from cAMP or other sources, which are initiated, propagated, and transmitted by chemical, electrical, or mechanical means. Specific depletion of HeLa cell PDE3A with small interfering RNA significantly decreased membrane-associated BIG1 and BIG2, which by confocal immunofluorescence microscopy were widely dispersed from an initial perinuclear Golgi concentration. Concurrently, activated ARF1-GTP was significantly decreased. Selective inhibition of PDE3A by 1-h incubation of cells with cilostamide similarly decreased membrane-associated BIG1. We suggest that decreasing PDE3A allowed cAMP to accumulate in microdomains where its enzymatic activity limited cAMP concentration. There, cAMP-activated PKA phosphorylated BIG1 and BIG2 (AKAPs for assembly of PKA, PDE3A, and other molecules), which decreased their GEP activity and thereby amounts of activated ARF1-GTP. Thus, PDE3A in these BIG1 and BIG2 AKAP complexes may contribute to the regulation of ARF function via limitation of cAMP effects with spatial and temporal specificity.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/isolamento & purificação , Citosol/efeitos dos fármacos , Citosol/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/isolamento & purificação , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Espaço Intracelular/metabolismo , Inibidores da Fosfodiesterase 3 , Ligação Proteica , RNA Interferente Pequeno/genéticaRESUMO
BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein, activates class I ADP-ribosylation factors (ARF1-3) by catalyzing the replacement of bound GDP by GTP, an action critical for the regulation of protein transport in eukaryotic cells. Our earlier report [Padilla PI, Pancheco-Rodriguez G, Moss J, Vaughan M (2004) Proc Natl Acad Sci USA 101:2752-2757] that BIG1 concentrated in nucleoli of serum-starved HepG2 cells prompted us to identify molecules associated with BIG1 in dynamic nucleolar structures. Antibodies against BIG1 or nucleolin coprecipitated both proteins from nuclei, which was abolished by the incubation of nuclei with RNase A or DNase, indicating that the interaction depended on nucleic acids. (32)P labeling of RNAs immunoprecipitated with BIG1 or nucleolin from nuclei revealed bands of approximately 210 bases that also hybridized with U3 small nucleolar (sno)RNA-specific oligonucleotides. Clones of U3 snoRNA cDNAs from the material precipitated by antibodies against BIG1 or nucleolin yielded identical nucleotide sequences that also were found in genomic DNA. Later analyses revealed the presence of fibrillarin, nucleoporin p62, and La in BIG1 and nucleolin immunoprecipitates. Our data demonstrate that BIG1, nucleolin, U3, the U3-binding protein fibrillarin, and the RNA-binding protein La may exist together in nuclear complexes, consistent with a potential role for BIG1 in nucleolar processes. Evidence that BIG1 and nucleolin, but not fibrillarin, can be present with p62 at the nuclear envelope confirms the presence of BIG1 and nucleolin in dynamic molecular complexes that change in composition while moving through nuclei. Nuclear functions of BIG1 remain to be determined.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinoma Hepatocelular/química , Núcleo Celular/química , Proteínas Cromossômicas não Histona/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular Tumoral , Humanos , Membrana Nuclear , Ligação Proteica , RNA Nucleolar Pequeno , Proteína Sequestossoma-1 , NucleolinaRESUMO
Curvature-related processes are of major importance during protein-membrane interactions. The illusive simplicity of membrane reshaping masks a complex molecular process crucial for a wide range of biological functions like fusion, endo- and exocytosis, cell division, cytokinesis, and autophagy. To date, no functional expression of a reaction coordinate capable of biasing molecular dynamics simulations to produce membrane curvature has been reported. This represents a major drawback given that the adequate identification of proper collective variables to enhance sampling is fundamental for restrained dynamics techniques. In this work, we present a closed-form equation of a collective variable that induces bending in lipid bilayers in a controlled manner, allowing for straightforward calculation of free energy landscapes of important curvature-related events, using standard methods such as umbrella sampling and metadynamics. As a direct application of the collective variable, we calculate the bending free energies of a ternary lipid bilayer in the presence and the absence of a Bin/Amphiphysin/Rvs domain with an N-terminal amphipathic helix (N-BAR), a well-known peripheral membrane protein that induces curvature.
Assuntos
Entropia , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Autofagia , Divisão Celular , Modelos Moleculares , Proteínas/químicaRESUMO
Human Adipose-derived mesenchymal stem/stromal cells (hASCs) are of great interest because of their potential for therapeutic approaches. The method described here covers every single step necessary for hASCs isolation from subcutaneous abdominal adipose tissue, multicolor phenotyping by flow cytometry, and quantitative determination of adipogenic differentiation status by means of lipid droplets (LDs) accumulation, and Western blot analysis. Moreover, to simultaneously analyze both LDs accumulation and cellular proteins localization by fluorescence microscopy, we combined Oil Red O (ORO) staining with immunofluorescence detection. For LDs quantification we wrote a program for automatic ORO-stained digital image processing implemented in Octave, a freely available software package. Our method is based on the use of the traditional low cost neutral lipids dye ORO, which can be imaged both by bright-field and fluorescence microscopy. The utilization of ORO instead of other more expensive lipid-specific dyes, together with the fact that the whole method has been designed employing cost-effective culture reagents (standard culture medium and serum), makes it affordable for tight-budget research laboratories. These may be replaced, if necessary or desired, by defined xeno-free reagents for clinical research and applications.
RESUMO
Mitochondrial small subunit (mtSSU) rDNA variable (V1, V2, V4, V6, V8 and V9) domain sequences and rRNA secondary structures evidenced eight molecular groups within 32 strains of the Agrocybe aegerita multispecies complex from different continents. mtSSU-rRNA secondary structure evolution occurred mainly by insertion/deletion of sequences from 8 to 57nt long. Preferential insertion/deletion sites correlated with loops of the mtSSU-rRNA secondary structures, and suggested that these events occurred in regions without interactions in the ribosomal-protein assembly. Indels modified the stem length (V1 and V4 domains) or the size and loop number (V6 and V9 domains). Three indels inserted in the V1 and V4 domains had 76.5% to 94.7% identity with short sequences of the mitochondrial cytochrome c oxidase gene; this fact and the presence of inverted repeated motifs within indel sequences suggested a mechanism of evolution based on insertion/deletion of sequences from another region of the mitochondrial genome. Phylogenetic relationships inferred using both ribosomal DNA sequences and rRNA secondary structures were congruent and evidenced three clades within the A. aegerita complex: European, Argentinean, and a more distant Asian-American clade including A. aegerita and A. chaxingu strains. These results suggested that numerous genetic exchanges occurred between Asian-American strains after isolation of the European clade. V4-V6-V9 concatenated sequences of European and Argentinean clades had 86.1% identity, similar to the value calculated between two Agrocybe closely related species, suggesting that these clades could represent different species. A cleaved amplified polymorphic sequence test for rapid characterization of strains was developed.
Assuntos
Agaricales/genética , DNA Mitocondrial/genética , DNA Ribossômico/genética , RNA Ribossômico/genética , Agaricales/classificação , Pareamento de Bases , Sequência de Bases , DNA Fúngico/química , DNA Fúngico/genética , DNA Ribossômico/química , Evolução Molecular , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , RNA Ribossômico/química , Análise de Sequência de DNARESUMO
Twenty years ago, a novel concept in protein structural biology was discovered: the intrinsically disordered regions (IDRs). These regions remain largely unstructured under native conditions and the more are studied, more properties are attributed to them. Possibly, one of the most important is their ability to conform a new type of protein-protein interaction. Besides the classical domain-to-domain interactions, IDRs follow a 'fly-casting' model including 'induced folding'. Unfortunately, it is only possible to experimentally explore initial and final states. However, the complete movie of conformational changes of protein regions and their characterization can be addressed by in silico experiments. Here, we simulate the binding of two proteins to describe how the phosphorylation of a single residue modulates the entire process. 14-3-3 protein family is considered a master regulator of phosphorylated proteins and from a modern point-of-view, protein phosphorylation is a three component system, with writers (kinases), erasers (phosphatases) and readers. This later biological role is attributed to the 14-3-3 protein family. Our molecular dynamics results show that phosphorylation of the key residue Thr31 in a partner of 14-3-3, the aralkylamine N-acetyltransferase, releases the fly-casting mechanism during binding. On the other hand, the non-phosphorylation of the same residue traps the proteins, systematically and repeatedly driving the simulations into wrong protein-protein conformations.
Assuntos
Proteínas 14-3-3/metabolismo , Arilalquilamina N-Acetiltransferase/metabolismo , Fluorescência , Simulação de Dinâmica Molecular , FosforilaçãoRESUMO
Posttranslational regulation of protein function is an ubiquitous mechanism in eukaryotic cells. Here, we analyzed biological properties of nodes and edges of a human protein-protein interaction phosphorylation-based network, especially of those nodes critical for the network controllability. We found that the minimal number of critical nodes needed to control the whole network is 29%, which is considerably lower compared to other real networks. These critical nodes are more regulated by posttranslational modifications and contain more binding domains to these modifications than other kinds of nodes in the network, suggesting an intra-group fast regulation. Also, when we analyzed the edges characteristics that connect critical and non-critical nodes, we found that the former are enriched in domain-to-eukaryotic linear motif interactions, whereas the later are enriched in domain-domain interactions. Our findings suggest a possible structure for protein-protein interaction networks with a densely interconnected and self-regulated central core, composed of critical nodes with a high participation in the controllability of the full network, and less regulated peripheral nodes. Our study offers a deeper understanding of complex network control and bridges the controllability theorems for complex networks and biological protein-protein interaction phosphorylation-based networked systems.
Assuntos
Proteínas 14-3-3/metabolismo , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Humanos , Fosforilação , Ligação Proteica , Domínios ProteicosRESUMO
The 14-3-3 protein family interacts with more than 2000 different proteins in mammals, as a result of its specific phospho-serine/phospho-threonine binding activity. Seven paralogs are strictly conserved in mammalian species. Here, we show that during adipogenic differentiation of 3T3-L1 preadipocytes, the level of each 14-3-3 protein paralog is regulated independently. For instance 14-3-3ß, γ, and η protein levels are increased compared to untreated cells. In contrast, 14-3-3ε protein levels decreased after differentiation while others remained constant. In silico analysis of the promoter region of each gene showed differences that explain the results obtained at mRNA and protein levels.
RESUMO
The understanding of networks is a common goal of an unprecedented array of traditional disciplines. One of the protein network properties most influenced by the structural contents of its nodes is the inter-connectivity. Recent studies in which structural information was included into the topological analysis of protein networks revealed that the content of intrinsic disorder in the nodes could modulate the network topology, rewire networks, and change their inter-connectivity, which is defined by its clustering coefficient. Here, we review the role of intrinsic disorder present in the partners of the highly conserved 14-3-3 protein family on its interaction networks. The 14-3-3s are phospho-serine/threonine binding proteins that have strong influence in the regulation of metabolism and signal transduction networks. Intrinsic disorder increases the clustering coefficients, namely the inter-connectivity of the nodes within each 14-3-3 paralog networks. We also review two new ideas to measure intrinsic disorder independently of the primary sequence of proteins, a thermodynamic model and a method that uses protein structures and their solvent environment. This new methods could be useful to explain unsolved questions about versatility and fixation of intrinsic disorder through evolution. The relation between the intrinsic disorder and network topologies could be an interesting model to investigate new implicitness of the graph theory into biology.
RESUMO
The 14-3-3 protein family interacts with more than 700 different proteins in mammals, in part as a result of its specific phospho-serine/phospho-threonine binding activity. Upon binding to 14-3-3, the stability, subcellular localization and/or catalytic activity of the ligands are modified. Seven paralogs are strictly conserved in mammalian species. Although initially thought as redundant, the number of studies showing specialization is growing. We created a protein-protein interaction network for 14-3-3, kinases and their substrates signaling in human cells. We included information of phosphorylation, acetylation and other PTM sites, obtaining a complete representation of the 14-3-3 binding partners and their modifications. Using a computational system approach we found that networks of each 14-3-3 isoform are statistically different. It was remarkable to find that Tyr was the most phosphorylatable amino acid in domains of 14-3-3 epsilon partners. This, together with the over-representation of SH3 and Tyr_Kinase domains, suggest that epsilon could be involved in growth factors receptors signaling pathways particularly. We also found that within zeta's network, the number of acetylated partners (and the number of modify lysines) is significantly higher compared with each of the other isoforms. Our results imply previously unreported hidden differences of the 14-3-3 isoforms interaction networks. The phosphoproteome and lysine acetylome within each network revealed post-transcriptional regulation intertwining phosphorylation and lysine acetylation. A global understanding of these networks will contribute to predict what could occur when regulatory circuits become dysfunctional or are modified in response to external stimuli.
Assuntos
Proteínas 14-3-3/metabolismo , Lisina/metabolismo , Acetilação , Regulação da Expressão Gênica , Humanos , Fosforilação , Serina/metabolismo , Treonina/metabolismoRESUMO
14-3-3 proteins participate in many key cellular processes after binding to disordered phospho-partners. Usually, the phosphorylated state is an essential target for the binding. Here, we show for the first time residues other than those in the 14-3-3 binding motif that are essential for the binding between 14-3-3 and a phosphorylated partner. Results support that phosphorylation, although necessary, is not sufficient for 14-3-3's complex formation, as structurally constrained anchor residues play a critical function in stabilizing the protein-protein interaction.
Assuntos
Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Arilalquilamina N-Acetiltransferase/química , Arilalquilamina N-Acetiltransferase/metabolismo , Mapeamento de Interação de Proteínas , Animais , Sítios de Ligação , Cinética , Modelos Moleculares , Fosforilação , Ligação Proteica , OvinosRESUMO
To evaluate the importance of searching new naturally occurring strains to raise yields in mushroom production, eight wild and four commercial strains of Agrocybe cylindracea were cultivated on wheat straw. The highest biological efficiencies (BE) (54.5-72.4%) were obtained with three wild and two commercial strains when cultured on non-supplemented wheat straw. Rolled oats or soybean flour supplementation were tested using three selected strains, increasing BEs up to 1.2, 0.5 and 0.7-fold, respectively. This effect of supplementation was stronger in the Asiatic wild strain, yielding up to 41.1 and 30% more than the two other strains with rolled oats and soybean flour, respectively. The Asiatic wild strain cultivated with soybean flour supplementation achieved an average biological efficiency of 179%, to our knowledge, the highest reported for this species. These results show the importance of searching for new naturally occurring strains in combination with supplemented wheat straw substrate for raising yields in A. cylindracea cultivation.