RESUMO
Snow mold fungus, Typhula ishikariensis, secretes seven antifreeze protein isoforms (denoted TisAFPs) that assist in the survival of the mold under snow cover. Here, the X-ray crystal structure of a hyperactive isoform, TisAFP8, at 1.0 Å resolution is presented. TisAFP8 folds into a right-handed ß-helix accompanied with a long α-helix insertion. TisAFP8 exhibited significantly high antifreeze activity that is comparable with other hyperactive AFPs, despite its close structural and sequence similarity with the moderately active isoform TisAFP6. A series of mutations introduced into the putative ice-binding sites (IBSs) in the ß-sheet and adjacent loop region reduced antifreeze activity. A double-mutant A20T/A212S, which comprises a hydrophobic patch between the ß-sheet and loop region, caused the greatest depression of antifreeze activity of 75%, when compared with that of the wild-type protein. This shows that the loop region is involved in ice binding and hydrophobic residues play crucial functional roles. Additionally, bound waters around the ß-sheet and loop region IBSs were organized into an ice-like network and can be divided into two groups that appear to mediate separately TisAFP and ice. The docking model of TisAFP8 with the basal plane via its loop region IBS reveals a better shape complementarity than that of TisAFP6. In conclusion, we present new insights into the ice-binding mechanism of TisAFP8 by showing that a higher hydrophobicity and better shape complementarity of its IBSs, especially the loop region, may render TisAFP8 hyperactive to ice binding.
Assuntos
Proteínas Anticongelantes/química , Proteínas Anticongelantes/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Neve/microbiologia , Proteínas Anticongelantes/genética , Sítios de Ligação , Cristalografia por Raios X , Proteínas Fúngicas/genética , Fungos/genética , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação/genéticaRESUMO
Antifreeze proteins (AFPs) are found in organisms ranging from fish to bacteria, where they serve different functions to facilitate survival of their host. AFPs that protect freeze-intolerant fish and insects from internal ice growth bind to ice using a regular array of well-conserved residues/motifs. Less is known about the role of AFPs in freeze-tolerant species, which might be to beneficially alter the structure of ice in or around the host. Here we report the 0.95-Å high-resolution crystal structure of a 223-residue secreted AFP from the snow mold fungus Typhula ishikariensis. Its main structural element is an irregular ß-helix with six loops of 18 or more residues that lies alongside an α-helix. ß-Helices have independently evolved as AFPs on several occasions and seem ideally structured to bind to several planes of ice, including the basal plane. A novelty of the ß-helical fold is the nonsequential arrangement of loops that places the N- and C termini inside the solenoid of ß-helical coils. The ice-binding site (IBS), which could not be predicted from sequence or structure, was located by site-directed mutagenesis to the flattest surface of the protein. It is remarkable for its lack of regularity and its poor conservation in homologs from psychrophilic diatoms and bacteria and other fungi.
Assuntos
Proteínas Anticongelantes/metabolismo , Sequência Conservada , Fungos/metabolismo , Gelo , Neve , Sequência de Aminoácidos , Proteínas Anticongelantes/química , Sítios de Ligação , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Homologia de Sequência de AminoácidosRESUMO
We previously reported that a silkworm hemolymph protein, apolipophorin (ApoLp), binds to the cell surface of Staphylococcus aureus and inhibits expression of the saePQRS operon encoding a two-component system, SaeRS, and hemolysin genes. In this study, we investigated the inhibitory mechanism of ApoLp on S. aureus hemolysin gene expression. ApoLp bound to lipoteichoic acids (LTA), an S. aureus cell surface component. The addition of purified LTA to liquid medium abolished the inhibitory effect of ApoLp against S. aureus hemolysin production. In an S. aureus knockdown mutant of ltaS encoding LTA synthetase, the inhibitory effects of ApoLp on saeQ expression and hemolysin production were attenuated. Furthermore, the addition of anti-LTA monoclonal antibody to liquid medium decreased the expression of S. aureus saeQ and hemolysin genes. In S. aureus strains expressing SaeS mutant proteins with a shortened extracellular domain, ApoLp did not decrease saeQ expression. These findings suggest that ApoLp binds to LTA on the S. aureus cell surface and inhibits S. aureus hemolysin gene expression via a two-component regulatory system, SaeRS.
Assuntos
Apolipoproteínas , Bombyx/química , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas Hemolisinas/biossíntese , Proteínas de Insetos , Lipopolissacarídeos/metabolismo , Staphylococcus aureus/metabolismo , Ácidos Teicoicos/metabolismo , Animais , Apolipoproteínas/química , Apolipoproteínas/isolamento & purificação , Apolipoproteínas/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/farmacologia , Lipopolissacarídeos/genética , Ligação Proteica , Proteínas Quinases/biossíntese , Proteínas Quinases/genética , Staphylococcus aureus/genética , Ácidos Teicoicos/genética , Fatores de TranscriçãoRESUMO
The Antarctic sea ice diatom Navicular glaciei produced ice-binding protein (NagIBP) that is similar to the antifreeze protein (TisAFP) from snow mold Typhula ishikariensis. In the thermal hysteresis range of NagIBP, ice growth was completely inhibited. At the freezing point, the ice grew in a burst to 6 direction perdicular to the c-axis of ice crystal. This burst pattern is similar to TisAFP and other hyperactive AFPs. The thermal hysteresis of NagIBP and TisAFP could be increased by decreasing a cooling rate to allow more time for the proteins to bind ice. This suggests the possible second binding of proteins occurs on the ice surface, which might increase the hysteresises to a sufficient level to prevent freezing of the brine pockets which habitat of N. glaciei. The secondary ice binding was described as that after AFP molecules bind onto the flat ice plane irreversibly, which was based on adsorption-inhibition mechanism model at the ice-water interface, convex ice front was formed and overgrew during normal TH measurement (no annealing) until uncontrolled growth at the nonequilibrium freezing point. The results suggested that NagIBP is a hyperactive AFP that is expressed for freezing avoidance.
Assuntos
Proteínas de Algas/química , Proteínas Anticongelantes/química , Diatomáceas/química , Gelo/análise , Proteínas de Algas/isolamento & purificação , Regiões Antárticas , Proteínas Anticongelantes/isolamento & purificação , Cristalização , Diatomáceas/fisiologia , Congelamento , Proteínas Fúngicas/química , Cinética , Mimetismo Molecular , Ligação ProteicaRESUMO
The F region downstream of the mecI gene in the SCCmec element in hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) contains two bidirectionally overlapping open reading frames (ORFs), the fudoh ORF and the psm-mec ORF. The psm-mec ORF encodes a cytolysin, phenol-soluble modulin (PSM)-mec. Transformation of the F region into the Newman strain, which is a methicillin-sensitive S. aureus (MSSA) strain, or into the MW2 (USA400) and FRP3757 (USA300) strains, which are community-acquired MRSA (CA-MRSA) strains that lack the F region, attenuated their virulence in a mouse systemic infection model. Introducing the F region to these strains suppressed colony-spreading activity and PSMα production, and promoted biofilm formation. By producing mutations into the psm-mec ORF, we revealed that (i) both the transcription and translation products of the psm-mec ORF suppressed colony-spreading activity and promoted biofilm formation; and (ii) the transcription product of the psm-mec ORF, but not its translation product, decreased PSMα production. These findings suggest that both the psm-mec transcript, acting as a regulatory RNA, and the PSM-mec protein encoded by the gene on the mobile genetic element SCCmec regulate the virulence of Staphylococcus aureus.
Assuntos
Sequências Repetitivas Dispersas/fisiologia , Perforina/genética , Perforina/metabolismo , Staphylococcus aureus/patogenicidade , Animais , Sequência de Bases , Feminino , Sequências Repetitivas Dispersas/genética , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Organismos Geneticamente Modificados , Perforina/fisiologia , Biossíntese de Proteínas/fisiologia , Staphylococcus aureus/genética , Transcrição Gênica/fisiologia , Transformação Bacteriana/fisiologia , Virulência/genéticaRESUMO
Differentiated embryo chondrocyte-2 (DEC2), also known as bHLHE41 or Sharp1, is a pleiotropic transcription repressor that controls the expression of genes involved in cellular differentiation, hypoxia responses, apoptosis, and circadian rhythm regulation. Although a previous study demonstrated that DEC2 participates in the circadian control of hepatic metabolism by regulating the expression of cytochrome P450, the molecular mechanism is not fully understood. We reported previously that brief exposure of HepG2 cells to 50% serum resulted in 24-h oscillation in the expression of CYP3A4 as well as circadian clock genes. In this study, we found that the expression of CYP2D6, a major drug-metabolizing enzyme in humans, also exhibited a significant oscillation in serum-shocked HepG2 cells. DEC2 interacted with CCAAT/enhancer-binding protein (C/EBPα), accompanied by formation of a complex with histone deacetylase-1, which suppressed the transcriptional activity of C/EBPα to induce the expression of CYP2D6. The oscillation in the protein levels of DEC2 in serum-shocked HepG2 cells was nearly antiphase to that in the mRNA levels of CYP2D6. Transfection of cells with small interfering RNA against DEC2 decreased the amplitude of CYP2D6 mRNA oscillation in serum-shocked cells. These results suggest that DEC2 periodically represses the promoter activity of CYP2D6, resulting in its circadian expression in serum-shocked cells. DEC2 seems to constitute a molecular link through which output components from the circadian clock are associated with the time-dependent expression of hepatic drug-metabolizing enzyme.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Proteína alfa Estimuladora de Ligação a CCAAT/fisiologia , Ritmo Circadiano , Citocromo P-450 CYP2D6/genética , Proteínas CLOCK/fisiologia , Células Hep G2 , Fator 4 Nuclear de Hepatócito/fisiologia , Humanos , RNA Mensageiro/análise , Soro , Fatores de TempoRESUMO
Silkworm hemolymph inhibits hemolysin production by Staphylococcus aureus. We purified a factor in the silkworm hemolymph responsible for this inhibitory activity. The final fraction with the greatest specific activity contained 220- and 74-kDa proteins. Determination of the N-terminal amino acid sequence revealed that the 220- and 74-kDa proteins were apolipophorin I and apolipophorin II, respectively, indicating that the factor was apolipophorin (ApoLp). The purified ApoLp fraction showed decreased expression of S. aureus hla encoding α-hemolysin, hlb encoding ß-hemolysin, saeRS, and RNAIII, which activate the expression of these hemolysin genes. Injection of an anti-ApoLp antibody into the hemolymph increased the sensitivity of silkworms to the lethal effect of S. aureus. Hog gastric mucin, a mammalian homologue of ApoLp, decreased the expression of S. aureus hla and hlb. These findings suggest that ApoLp in the silkworm hemolymph inhibits S. aureus virulence and contributes to defense against S. aureus infection and that its activity is conserved in mammalian mucin.
Assuntos
Apolipoproteínas/farmacologia , Toxinas Bacterianas/metabolismo , Bombyx/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/farmacologia , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Animais , Apolipoproteínas/genética , Apolipoproteínas/metabolismo , Sequência de Bases , Bombyx/genética , Bombyx/microbiologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Dados de Sequência MolecularRESUMO
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the dysregulation of various cell types and immunological pathways. Autoantibodies play an important role in its pathogenesis. The presence of autoantibodies suggests that self-antigen presentation through major histocompatibility complex (MHC) class II on antigen presenting cells is involved in the pathogenesis of autoimmune diseases, including SLE. Cathepsin S (CatS) is a key protease for antigen peptide loading onto lysosomal/endosomal MHC class II molecules through invariant chain degradation to promote antigen presentation. Inhibition of CatS is therefore expected to suppress antigen presentation via MHC class II, T and B cell activation, and antibody production from B cells. Here, we report the pharmacological profile of ASP1617, a novel CatS inhibitor. ASP1617 induced invariant chain accumulation and decreased the expression level of MHC class ΙΙ on the cell surface in both mouse and human B cells. Further, ASP1617 prevented DO11.10 mice T cell proliferation to ovalbumin antigen. We investigated the effects of ASP1617 and mycophenolate mofetil (MMF) on the development of lupus-like nephritis in NZB/W F1 mice, a widely used SLE mouse model. Oral administration of ASP1617 suppressed anti-dsDNA IgG, prevented progression of lupus-like glomerulonephritis, and significantly prevented proteinuria excretion. In contrast, MMF did not suppress anti-dsDNA IgG. Further, we found that plasma and/or urine CatS levels were increased in specimens from NZB/W F1 mice and several SLE patients. These results indicate that CatS may be an attractive therapeutic target for the treatment of SLE.
Assuntos
Catepsinas/antagonistas & inibidores , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Inibidores de Proteases/farmacologia , Administração Oral , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Endogâmicos NZB , Inibidores de Proteases/administração & dosagem , Inibidores de Proteases/uso terapêuticoRESUMO
Previously, we reported the fundamental pharmacological characteristics of a novel Igß and Fc gamma receptor IIB cross-linking antibody, ASP2713, as a new treatment option for systemic lupus erythematosus. The aims of the present study were to investigate ASP2713's characteristics with regard to pharmacological effect, pharmacokinetics (PK), and receptor occupancy, and to predict its human PK and clinically effective dose. The relationship between the concentration and receptor occupancy of ASP2713 for Igß of B cell receptors was examined using whole blood B cells. Calculated EC50 values in cynomolgus monkeys and healthy volunteers were 0.35 and 0.058 µg/mL, respectively. Dose-dependent inhibition of anti-tetanus toxoid (TTx) antibody production, PK, and receptor occupancy of ASP2713 in TTx-sensitized cynomolgus monkeys suggested a minimally effective dose of 1 mg/kg by single intravenous (IV) administration. Scaling-up of monkey PK parameters to humans by allometric scaling predicted a clinically effective dose of 0.4 mg/kg IV administration at 4-week intervals to maintain a trough concentration in humans which achieved the same receptor occupancy expected at the effective dose in monkeys. This study aids in understanding the characteristics of ASP2713 and can be used as a basis for clinical dose setting.
Assuntos
Anticorpos Monoclonais , Imunoglobulina G , Animais , Humanos , Macaca fascicularisRESUMO
The basidiomycetous yeast, Glaciozyma antarctica, was isolated from various terrestrial materials collected from the Sôya coast, East Antarctica, and formed frost-columnar colonies on agar plates frozen at -1 °C. Thawed colonies were highly viscous, indicating that the yeast produced a large number of extracellular polysaccharides (EPS). G. antarctica was then cultured on frozen media containing red food coloring to observe the dynamics of solutes in unfrozen water; pigments accumulated in frozen yeast colonies, indicating that solutes were concentrated in unfrozen water of yeast colonies. Moreover, the yeast produced a small quantity of ice-binding proteins (IBPs) which inhibited ice crystal growth. Solutes in unfrozen water were considered to accumulate in the pore of frozen colonies. The extracellular IBPs may have held an unfrozen state of medium water after accumulation in the frost-columnar colony.
RESUMO
A supersoluble 40-residue type I antifreeze protein (AFP) was discovered in a righteye flounder, the barfin plaice (bp). Unlike all other AFPs characterized to date, bpAFP transitions from moderately-active to hyperactive with increasing concentration. At sub-mM concentrations, bpAFP bound to pyramidal planes of ice to shape it into a bi-pyramidal hexagonal trapezohedron, similarly to the other moderately-active AFPs. At mM concentrations, bpAFP uniquely underwent further binding to the whole ice crystal surface including the basal planes. The latter caused a bursting ice crystal growth normal to c-axis, 3 °C of high thermal hysteresis, and alteration of an ice crystal into a smaller lemon-shaped morphology, all of which are well-known properties of hyperactive AFPs. Analytical ultracentrifugation showed this activity transition is associated with oligomerization to form tetramer, which might be the forerunner of a naturally occurring four-helix-bundle AFP in other flounders.
Assuntos
Proteínas Anticongelantes/química , Proteínas Anticongelantes/imunologia , Peptídeos/química , Peptídeos/imunologia , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Alérgenos/química , Alérgenos/imunologia , Animais , Proteínas de Peixes/química , Proteínas de Peixes/imunologia , Concentração de Íons de Hidrogênio , Estabilidade Proteica , SolubilidadeRESUMO
Post-transcriptional modifications in rRNA, such as methylation, are observed in functionally important regions of the ribosome. The methyltransferases responsible for these modifications work as housekeeping enzymes to fine-tune ribosomal function, and the roles of some methyltransferases become more evident under stress conditions. Recently, the 16S rRNA methyltransferases RsmI and RsmH, which are responsible for cytidine dimethylation at the decoding center of the ribosome, were identified in Escherichia coli. The physiological relevance of the rRNA modification, however, remains obscure. We identified rsmI and rsmH as novel virulence genes in Staphylococcus aureus using a silkworm infection model. These genes induced 2'-O- and N(4)-methylations, respectively, of m(4) Cm1412 of S. aureus 16S rRNA. Deletion of either rsmI or rsmH in S. aureus attenuated the virulence of S. aureus in silkworms, and led to its sensitivity to oxidative stress. Dual luciferase assay revealed that the double-knockout strain exhibited decreased translational fidelity under oxidative stress conditions. In addition, administration of N-acetyl-L-cysteine, a free-radical scavenger, restored the killing ability of the double-knockout strain against silkworms. These findings suggest that the methyl-modifications of cytidine at position 1412 in 16S rRNA contribute to S. aureus animal killing by conferring resistance to oxidative stress in host animals.
Assuntos
Proteínas de Escherichia coli/fisiologia , Escherichia coli/enzimologia , Metiltransferases/fisiologia , Staphylococcus aureus/patogenicidade , Sequestradores de Radicais Livres/farmacologia , Macrófagos/microbiologia , Estresse Oxidativo , Biossíntese de Proteínas , VirulênciaRESUMO
Fourteen Arctic bacterial strains belonging to five genera, Cryobacterium, Leifsonia, Polaromonas, Pseudomonas, and Subtercola isolated from sediments found in cryoconite holes of Arctic glaciers, were subjected to screening for antifreeze proteins (AFPs). Eight strains showed AFP activity, and six strains of four species were further characterized. Pseudomonas ficuserectae exhibited a high thermal hysteresis (TH) activity. Ice recrystallization inhibition (IRI) activity was observed in most cultures at low protein concentration. Bacterial AFPs produced rounded shape of ice crystals that did not change their size and morphology within the TH window. Cry-g (P. ficuserectae) failed to inhibit ice recrystallization, indicating that the IRI activity of the AFPs does not relate to the strength of TH activity. SDS-PAGE analysis of the AFPs suggests their apparent molecular weights to be around 23 kDa. This study is significant as it screens several species of Arctic bacterial strains for AFP activity. So far, only one species of bacteria, Pseudomonas putida, was reported from the Arctic to produce AFPs. N-terminal amino acid sequence analysis shows that the bacterial AFPs isolated belong to the AFP family IBP-1, which is known to have an important physiological role in the cold environment. AFPs of glacier cryoconite habitat have been discussed.
RESUMO
UNLABELLED: Antifreeze proteins (AFPs) are structurally diverse macromolecules that bind to ice crystals and inhibit their growth to protect the organism from injuries caused by freezing. An AFP identified from the Antarctic bacterium Colwellia sp. strain SLW05 (ColAFP) is homologous to AFPs from a wide variety of psychrophilic microorganisms. To understand the antifreeze function of ColAFP, we have characterized its antifreeze activity and determined the crystal structure of this protein. The recombinant ColAFP exhibited thermal hysteresis activity of approximately 4 °C at a concentration of 0.14 mm, and induced rapid growth of ice crystals in the hexagonal direction. Fluorescence-based ice plane affinity analysis showed that ColAFP binds to multiple planes of ice, including the basal plane. These observations show that ColAFP is a hyperactive AFP. The crystal structure of ColAFP determined at 1.6 Å resolution revealed an irregular ß-helical structure, similar to known homologs. Mutational and molecular docking studies showed that ColAFP binds to ice through a compound ice-binding site (IBS) located at a flat surface of the ß-helix and the adjoining loop region. The IBS of ColAFP lacks the repetitive sequences that are characteristic of hyperactive AFPs. These results suggest that ColAFP exerts antifreeze activity through a compound IBS that differs from the characteristic IBSs shared by other hyperactive AFPs. This study demonstrates a novel method for protection from freezing by AFPs in psychrophilic microorganisms. DATABASE: Structural data for ColAFP have been submitted to the Protein Data Bank (PDB) under accession number 3WP9.
Assuntos
Alteromonadaceae/química , Proteínas Anticongelantes/química , Proteínas de Bactérias/química , Camada de Gelo/microbiologia , Alteromonadaceae/genética , Substituição de Aminoácidos , Regiões Antárticas , Proteínas Anticongelantes/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Sequências Repetitivas de AminoácidosRESUMO
A total of 71 isolates were collected from lake sediment and soil surrounding lakes in the Skarvsnes area, Antarctica. Based on ITS region sequence similarity, these isolates were classified to 10 genera. Twenty-three isolates were categorized as ascomycetous fungi from five genera (Embellisia, Phoma, Geomyces, Tetracladium or Thelebolus) and 48 isolates were categorized as basidiomycetous fungi in five genera (Mrakia, Cryptococcus, Dioszegia, Rhodotorula or Leucosporidium). Thirty-five percent of culturable fungi were of the genus Mrakia. Eighteen isolates from eight genera were selected and tested for both antifreeze activity and capacity for growth under temperatures ranging from -1 to 25 °C. Rhodotorula sp. NHT-2 possessed a high degree of sequence homology with R. gracialis, while Leucosporidium sp. BSS-1 possessed a high degree of sequence homology with Leu. antarcticum (Glaciozyma antarctica), and these two isolates demonstrated antifreeze activity. All isolates examined were capable of growth at -1 °C. Mrakia spp., while capable of growth at -1 °C, did not demonstrate any antifreeze activity and exhibited only limited secretion of extracellular polysaccharides. Species of the genus Mrakia possessed high amounts of unsaturated fatty acids, suggesting that members of this genus have adapted to cold environments by increasing their membrane fluidity.