RESUMO
Bacterial cytokinesis is mediated by the Z-ring, which is formed by the prokaryotic tubulin homolog FtsZ. Recent data indicate that the Z-ring is composed of small patches of FtsZ protofilaments that travel around the bacterial cell by treadmilling. Treadmilling involves a switch from a relaxed (R) state, favored for monomers, to a tense (T) conformation, which is favored upon association into filaments. The R conformation has been observed in numerous monomeric FtsZ crystal structures and the T conformation in Staphylococcus aureus FtsZ crystallized as assembled filaments. However, while Escherichia coli has served as a main model system for the study of the Z-ring and the associated divisome, a structure has not yet been reported for E. coli FtsZ. To address this gap, structures were determined of the E. coli FtsZ mutant FtsZ(L178E) with GDP and GTP bound to 1.35 and 1.40â Å resolution, respectively. The E. coli FtsZ(L178E) structures both crystallized as straight filaments with subunits in the R conformation. These high-resolution structures can be employed to facilitate experimental cell-division studies and their interpretation in E. coli.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Escherichia coli/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Conformação Proteica , Cristalografia por Raios X , Modelos MolecularesRESUMO
Interactions between innate antiviral factors at mucosal surfaces and HIV-1 virions contribute to the natural inefficiency of HIV-1 transmission and are a platform to inform the development of vaccine and nonvaccine strategies to block mucosal HIV-1 transmission. Tenascin-C (TNC) is a large, hexameric extracellular matrix glycoprotein identified in breast milk and genital fluids that broadly neutralizes HIV-1 via interaction with the HIV-1 Envelope (Env) variable 3 (V3) loop. In this report, we characterize the specific determinants of the interaction between TNC and the HIV-1 Env. We observed that TNC binding and neutralization of HIV-1 is dependent on the TNC fibrinogen-like globe (fbg) and fibronectin-type III (fn) domains, oligomerization, and its newly-mapped glycan structure. Moreover, we observed that TNC-mediated neutralization is also dependent on Env V3 residues 321/322 and 326/327, which surround the IGDIR motif of the V3 loop, as well the N332 glycan, which is critical to the broadly neutralizing activity of glycan-dependent V3-specific antibodies such as PGT128. Our results demonstrate a striking parallel between innate and adaptive immune mechanisms of broad HIV neutralization and provide further insight into the host protein-virus interactions responsible for the natural inefficiency of mucosal HIV-1 transmission.
Assuntos
HIV-1/metabolismo , Tenascina/química , Tenascina/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Aminoácidos , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Glicosilação , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/imunologia , Humanos , Modelos Moleculares , Testes de Neutralização , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
To study fibronectin (FN) conformation and assembly, we generated several deletion mutants: FNΔI1-5, FNΔIII1-3, FNΔIII4-8, and FNΔIII11-14. A monomeric form, FNmono, which lacked the C-terminal dimerization region, was also created. FNtnA-D was generated by swapping FNIII domains 1-8 in FNΔIII11-14 with seven FNIII domains from tenascin-C. The conformations of these mutants were analyzed by glycerol gradient sedimentation under low-salt (20 mM NaCl) and high-salt (200 mM NaCl) conditions. Surprisingly, most of the mutants showed a compact conformation under low-salt conditions, except for FNtnA-D. When we tested these mutants in cell culture, FNΔI1-5, FNΔIII1-3, and FNtnA-D were unable to form a matrix. Interestingly, FNΔIII1-3 and FNtnA-D were capable of co-assembly with full-length FN, while FNΔI1-5 was not. This indicates that the segment I1-5 is crucial for matrix assembly and segment III1-3 is also important. Mutations in FN are associated with glomerulopathy, but when we studied mutant proteins, the single-nucleotide mutations had only minor effects on conformation and matrix assembly. The mutations may destabilize their FNIII domains or generate dimers of dimers by disulfide cross-linking.
Assuntos
Fibronectinas/metabolismo , Glomerulonefrite Membranoproliferativa/metabolismo , Mutação INDEL , Multimerização Proteica , Fibronectinas/química , Fibronectinas/genética , Glomerulonefrite Membranoproliferativa/genética , Células HEK293 , Humanos , Domínios ProteicosRESUMO
Globular proteins are not permanently folded but spontaneously unfold and refold on time scales that can span orders of magnitude for different proteins. A longstanding debate in the protein-folding field is whether unfolding rates or folding rates correlate to the stability of a protein. In the present study, we have determined the unfolding and folding kinetics of 10 FNIII domains. FNIII domains are one of the most common protein folds and are present in 2% of animal proteins. FNIII domains are ideal for this study because they have an identical seven-strand ß-sandwich structure, but they vary widely in sequence and thermodynamic stability. We assayed thermodynamic stability of each domain by equilibrium denaturation in urea. We then assayed the kinetics of domain opening and closing by a technique known as thiol exchange. For this we introduced a buried Cys at the identical location in each FNIII domain and measured the kinetics of labeling with DTNB over a range of urea concentrations. A global fit of the kinetics data gave the kinetics of spontaneous unfolding and refolding in zero urea. We found that the folding rates were relatively similar, â¼0.1-1 s-1, for the different domains. The unfolding rates varied widely and correlated with thermodynamic stability. Our study is the first to address this question using a set of domains that are structurally homologous but evolved with widely varying sequence identity and thermodynamic stability. These data add new evidence that thermodynamic stability correlates primarily with unfolding rate rather than folding rate. The study also has implications for the question of whether opening of FNIII domains contributes to the stretching of fibronectin matrix fibrils.
Assuntos
Fibronectinas/química , Redobramento de Proteína , Desdobramento de Proteína , Ureia/química , Humanos , Domínios Proteicos , Estabilidade Proteica , TermodinâmicaRESUMO
Tenascin-C (TNC) is a newly identified innate HIV-1-neutralizing protein present in breast milk, yet its presence and potential HIV-inhibitory function in other mucosal fluids is unknown. In this study, we identified TNC as a component of semen and cervical fluid of HIV-1-infected and uninfected individuals, although it is present at a significantly lower concentration and frequency compared to that of colostrum and mature breast milk, potentially due to genital fluid protease degradation. However, TNC was able to neutralize HIV-1 after exposure to low pH, suggesting that TNC could be active at low pH in the vaginal compartment. As mucosal fluids are complex and contain a number of proteins known to interact with the HIV-1 envelope, we further studied the relationship between the concentration of TNC and neutralizing activity in breast milk. The amount of TNC correlated only weakly with the overall innate HIV-1-neutralizing activity of breast milk of uninfected women and negatively correlated with neutralizing activity in milk of HIV-1 infected women, indicating that the amount of TNC in mucosal fluids is not adequate to impede HIV-1 transmission. Moreover, the presence of polyclonal IgG from milk of HIV-1 infected women, but not other HIV-1 envelope-binding milk proteins or monoclonal antibodies, blocked the neutralizing activity of TNC. Finally, as exogenous administration of TNC would be necessary for it to mediate measurable HIV-1 neutralizing activity in mucosal compartments, we established that recombinantly produced TNC has neutralizing activity against transmitted/founder HIV-1 strains that mimic that of purified TNC. Thus, we conclude that endogenous TNC concentration in mucosal fluids is likely inadequate to block HIV-1 transmission to uninfected individuals.
Assuntos
Líquido Extracelular/imunologia , Genitália , Infecções por HIV/imunologia , HIV-1/imunologia , Proteínas do Leite/imunologia , Leite Humano/imunologia , Tenascina/imunologia , Anticorpos Neutralizantes/imunologia , Colo do Útero/imunologia , Feminino , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Imunidade Inata , Imunoglobulina G/imunologia , Masculino , Proteínas do Leite/farmacologia , Mucosa/imunologia , Mucosa/metabolismo , Testes de Neutralização , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes , Sêmen/imunologia , Tenascina/farmacologiaRESUMO
The Ndc80 complex (Ndc80, Nuf2, Spc24 and Spc25) is a highly conserved kinetochore protein essential for end-on anchorage to spindle microtubule plus ends and for force generation coupled to plus-end polymerization and depolymerization. Spc24/Spc25 at one end of the Ndc80 complex binds the kinetochore. The N-terminal tail and CH domains of Ndc80 bind microtubules, and an internal domain binds microtubule-associated proteins (MAPs) such as the Dam1 complex. To determine how the microtubule- and MAP-binding domains of Ndc80 contribute to force production at the kinetochore in budding yeast, we have inserted a FRET tension sensor into the Ndc80 protein about halfway between its microtubule-binding and internal loop domains. The data support a mechanical model of force generation at metaphase where the position of the kinetochore relative to the microtubule plus end reflects the relative strengths of microtubule depolymerization, centromere stretch and microtubule-binding interactions with the Ndc80 and Dam1 complexes.
Assuntos
Centrômero/metabolismo , Cromossomos Fúngicos/metabolismo , Cinetocoros/metabolismo , Proteínas Luminescentes , Microtúbulos/metabolismo , Sítios de Ligação/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Imagem com Lapso de TempoRESUMO
The myokine irisin is supposed to be cleaved from a transmembrane precursor, FNDC5 (fibronectin type III domain containing 5), and to mediate beneficial effects of exercise on human metabolism. However, evidence for irisin circulating in blood is largely based on commercial ELISA kits which are based on polyclonal antibodies (pAbs) not previously tested for cross-reacting serum proteins. We have analyzed four commercial pAbs by Western blotting, which revealed prominent cross-reactivity with non-specific proteins in human and animal sera. Using recombinant glycosylated and non-glycosylated irisin as positive controls, we found no immune-reactive bands of the expected size in any biological samples. A FNDC5 signature was identified at ~20â kDa by mass spectrometry in human serum but was not detected by the commercial pAbs tested. Our results call into question all previous data obtained with commercial ELISA kits for irisin, and provide evidence against a physiological role for irisin in humans and other species.
Assuntos
Artefatos , Ensaio de Imunoadsorção Enzimática/métodos , Exercício Físico/fisiologia , Fibronectinas/sangue , Músculo Esquelético/metabolismo , Animais , Análise Química do Sangue/métodos , Citocinas/sangue , Humanos , Camundongos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Haemophilus influenzae is a Gram-negative cocco-bacillus that initiates infection by colonizing the upper respiratory tract. Hap is an H. influenzae serine protease autotransporter protein that mediates adherence, invasion and microcolony formation in assays with human epithelial cells and is presumed to facilitate the process of colonization. Additionally, Hap mediates adherence to fibronectin, laminin and collagen IV, extracellular matrix (ECM) proteins that are present in the respiratory tract and are probably important targets for H. influenzae colonization. The region of Hap responsible for adherence to ECM proteins has been localized to the C-terminal 511 aa of the Hap passenger domain (HapS). In this study, we characterized the structural determinants of the interaction between HapS and fibronectin. Using defined fibronectin fragments, we established that Hap interacts with the fibronectin repeat fragment called FNIII(1-2). Using site-directed mutagenesis, we found a series of motifs in the C-terminal region of HapS that contribute to the interaction with fibronectin. Most of these motifs are located on the F1 and F3 faces of the HapS structure, suggesting that the F1 and F3 faces may be responsible for the HapS-fibronectin interaction.
Assuntos
Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Haemophilus influenzae/fisiologia , Domínios e Motivos de Interação entre Proteínas , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Ligação Proteica , Mapeamento de Interação de ProteínasRESUMO
In this issue, Lin et al. report the discovery of P12, a 14 amino acid peptide from the first fibronectin (FN) type III domain of FN, which has the capability of enhancing cell survival in culture and improving wound healing in rat skin. P12 belongs to a new class of bioactive peptides that they have named epiviosamines. Epiviosamines may have clinical applications.
Assuntos
Sobrevivência Celular , Fibroblastos/metabolismo , Fibronectinas/química , Peptídeos/química , Proteínas Proto-Oncogênicas c-sis/química , Pele/metabolismo , Animais , Becaplermina , HumanosRESUMO
Achieving an AIDS-free generation will require elimination of postnatal transmission of HIV-1 while maintaining the nutritional and immunologic benefits of breastfeeding for infants in developing regions. Maternal/infant antiretroviral prophylaxis can reduce postnatal HIV-1 transmission, yet toxicities and the development of drug-resistant viral strains may limit the effectiveness of this strategy. Interestingly, in the absence of antiretroviral prophylaxis, greater than 90% of infants exposed to HIV-1 via breastfeeding remain uninfected, despite daily mucosal exposure to the virus for up to 2 y. Moreover, milk of uninfected women inherently neutralizes HIV-1 and prevents virus transmission in animal models, yet the factor(s) responsible for this anti-HIV activity is not well-defined. In this report, we identify a primary HIV-1-neutralizing protein in breast milk, Tenascin-C (TNC). TNC is an extracellular matrix protein important in fetal development and wound healing, yet its antimicrobial properties have not previously been established. Purified TNC captured and neutralized multiclade chronic and transmitted/founder HIV-1 variants, and depletion of TNC abolished the HIV-1-neutralizing activity of milk. TNC bound the HIV-1 Envelope protein at a site that is induced upon engagement of its primary receptor, CD4, and is blocked by V3 loop- (19B and F39F) and chemokine coreceptor binding site-directed (17B) monoclonal antibodies. Our results demonstrate the ability of an innate mucosal host protein found in milk to neutralize HIV-1 via binding to the chemokine coreceptor site, potentially explaining why the majority of HIV-1-exposed breastfed infants are protected against mucosal HIV-1 transmission.
Assuntos
Síndrome da Imunodeficiência Adquirida/transmissão , HIV-1/efeitos dos fármacos , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Leite Humano/química , Tenascina/farmacologia , Síndrome da Imunodeficiência Adquirida/prevenção & controle , Western Blotting , Linhagem Celular , Cromatografia por Troca Iônica , Relação Dose-Resposta a Droga , Feminino , Humanos , Imunoprecipitação , Concentração Inibidora 50 , Espectrometria de Massas , Tenascina/metabolismo , Proteínas do Envelope Viral/metabolismoRESUMO
Irisin was recently identified as a putative myokine that is induced by exercise. Studies suggest that it is produced by cleavage of the FNDC5 (fibronectin domain-containing protein 5) receptor; irisin corresponds to the extracellular receptor ectodomain. Data suggesting that irisin stimulates white-to-brown fat conversion have led to the hypothesis that it does so by binding an unknown receptor, thus functioning as a myokine. As brown fat promotes energy dissipation, myokines that elicit the transformation of white to brown fat have potentially profound benefits in the treatment of obesity and metabolic disorders. Understanding the molecular basis for such exercise-induced phenomena is thus of considerable interest. Moreover, FNDC5-like receptors are highly conserved and have been shown to be critical for neuronal development. However, the structural and molecular mechanisms utilized by these proteins are currently unknown. Here, we describe the crystal structure and biochemical characterization of the FNDC5 ectodomain, corresponding to the irisin myokine. The 2.28 Å structure shows that irisin consists of an N-terminal fibronectin III (FNIII)-like domain attached to a flexible C-terminal tail. Strikingly, the FNIII-like domain forms a continuous intersubunit ß-sheet dimer, previously unobserved for any FNIII protein. Biochemical data confirm that irisin is a dimer and that dimerization is unaffected by glycosylation. This finding suggests a possible mechanism for receptor activation by the irisin domain as a preformed myokine dimer ligand or as a paracrine or autocrine dimerization module on FNDC5-like receptors.
Assuntos
Fibronectinas/química , Multimerização Proteica/fisiologia , Cristalografia por Raios X , Fibronectinas/genética , Fibronectinas/metabolismo , Glicosilação , Humanos , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Chronic subdural hematoma (CSDH) is generally treated by burr hole irrigation. However, sometimes repeated recurrence is observed, and treatment may consequently become difficult. We examined the efficacy of embolization of the middle meningeal artery (MMA) for such cases. METHODS: We considered embolization of the MMA for three patients who had refractory CSDH with repeated recurrence and two CSDH patients who were at risk of recurrence and showed signs of recurrence after surgery. A microcatheter was advanced through the MMA as peripherally as possible, and embolization was performed with 15-20% n-butyl-2-cyanoacrylate or 200 µm polyvinyl alcohol particles. RESULTS: EMBOLIZATION WAS PERFORMED IN THE THREE PATIENTS WHO HAD REFRACTORY CSDH WITH REPEATED RECURRENCE: The procedure was performed after burr hole irrigation of the hematoma in two patients and before the irrigation in one patient. In the two CSDH patients at risk of recurrence, embolization was performed when signs of recurrence appeared. The timing of embolization differed for each patient. However, in all the patients, the hematoma tended to decrease in size, and no recurrence was observed. CONCLUSION: Embolization of the MMA is effective for refractory CSDH or CSDH patients with a risk of recurrence, and is considered an effective therapeutic method to stop hematoma enlargement and promote resolution.
RESUMO
The mechanism of fibronectin (FN) assembly and the self-association sites are still unclear and contradictory, although the N-terminal 70-kDa region ((I)1-9) is commonly accepted as one of the assembly sites. We previously found that (I)1-9 binds to superfibronectin, which is an artificial FN aggregate induced by anastellin. In the present study, we found that (I)1-9 bound to the aggregate formed by anastellin and a small FN fragment, (III)1-2. An engineered disulfide bond in (III)2, which stabilizes folding, inhibited aggregation, but a disulfide bond in (III)1 did not. A gelatin precipitation assay showed that (I)1-9 did not interact with anastellin, (III)1, (III)2, (III)1-2, or several (III)1-2 mutants including (III)1-2KADA. (In contrast to previous studies, we found that the (III)1-2KADA mutant was identical in conformation to wild-type (III)1-2.) Because (I)1-9 only bound to the aggregate and the unfolding of (III)2 played a role in aggregation, we generated a (III)2 domain that was destabilized by deletion of the G strand. This mutant bound (I)1-9 as shown by the gelatin precipitation assay and fluorescence resonance energy transfer analysis, and it inhibited FN matrix assembly when added to cell culture. Next, we introduced disulfide mutations into full-length FN. Three disulfide locks in (III)2, (III)3, and (III)11 were required to dramatically reduce anastellin-induced aggregation. When we tested the disulfide mutants in cell culture, only the disulfide bond in (III)2 reduced the FN matrix. These results suggest that the unfolding of (III)2 is one of the key factors for FN aggregation and assembly.
Assuntos
Fibronectinas/química , Dobramento de Proteína , Dissulfetos/química , Dissulfetos/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Gelatina/química , Células HEK293 , Humanos , Mutação , Estabilidade Proteica , Estrutura Terciária de ProteínaRESUMO
Fibronectin (FN) is an extracellular matrix protein that is assembled into fibrils by cells during tissue morphogenesis and wound healing. FN matrix fibrils are highly elastic, but the mechanism of elasticity has been debated: it may be achieved by mechanical unfolding of FN-III domains or by a conformational change of the molecule without domain unfolding. Here, we investigate the folded state of FN-III domains in FN fibrils by measuring the accessibility of buried cysteines. Four of the 15 FN-III domains (III-2, -3, -9, and -11) appear to unfold in both stretched fibrils and in solution, suggesting that these domains spontaneously open and close even in the absence of tension. Two FN-III domains (III-6 and -12) appear to unfold only in fibrils and not in solution. These results suggest that domain unfolding can at best contribute partially to the 4-fold extensibility of fibronectin fibrils.
Assuntos
Cisteína/química , Fibronectinas/química , Dobramento de Proteína , Animais , Cisteína/genética , Cisteína/metabolismo , Elasticidade , Fibronectinas/genética , Fibronectinas/metabolismo , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Estrutura Terciária de ProteínaRESUMO
Haemophilus influenzae is a gram-negative bacterium that initiates infection by colonizing the upper respiratory tract. The H. influenzae Hap autotransporter protein mediates adherence, invasion, and microcolony formation in assays with respiratory epithelial cells and presumably facilitates colonization. The serine protease activity of Hap is associated with autoproteolytic cleavage and extracellular release of the HapS passenger domain, leaving the Hapbeta C-terminal domain embedded in the outer membrane. Cleavage occurs most efficiently at the LN1036-37 peptide bond and to a lesser extent at three other sites. In this study, we utilized site-directed mutagenesis, homology modeling, and assays with a peptide library to characterize the structural determinants of Hap proteolytic activity and cleavage specificity. In addition, we used homology modeling to predict the S1, S2, and S4 subsite residues of the Hap substrate groove. Our results indicate that the P1 and P2 positions at the Hap cleavage sites are critical for cleavage, with leucine preferred over larger hydrophobic residues or other amino acids in these positions. The substrate groove is formed by L263 and N274 at the S1 subsite, R264 at the S2 subsite, and E265 at the S4 subsite. This information may facilitate design of approaches to block Hap activity and interfere with H. influenzae colonization.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Haemophilus influenzae/enzimologia , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Western Blotting , Haemophilus influenzae/genética , Haemophilus influenzae/patogenicidade , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Quaternária de Proteína , Homologia de Sequência de Aminoácidos , Serina Endopeptidases/genéticaRESUMO
We previously reported that the fibronectin (FN) type III domains of FN may unfold to interact with anastellin and form FN aggregates. In the present study, we have focused on the interaction between anastellin and the third FN type III domain (III3), which is a key anastellin binding site on FN. Anastellin binding to III3 was monitored by 8-anilino-1-naphthalene sulfonate (ANS) fluorescence. ANS binding to anastellin dramatically increased its emission intensity, but this was reduced to half by the addition of III3, suggesting that ANS and III3 share a common hydrophobic binding site on anastellin. An engineered mutant of III3 that was stabilized by an intrachain disulfide bond did not interact with anastellin, as seen by its failure to interfere with ANS binding to anastellin. We also mutated hydrophobic core residues to destabilize III3 and found that these mutants were still capable of interacting with anastellin. Anastellin binding to III3 was also monitored using an intramolecular green fluorescent protein (GFP)-based fluorescence resonance energy transfer (FRET) construct, in which III3 was flanked by two GFP variants (III3-FRET). Anastellin bound to III3-FRET and caused an increase in the FRET signal. The dissociation constant was estimated to be approximately 210 nM. The binding kinetics of anastellin to III3-FRET fit a first-order reaction with a half-time of approximately 30 s; the kinetics with destabilized III3 mutants were even faster. Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry suggested that the middle part of III3 became destabilized and protease sensitive upon anastellin binding. Thus, the stability of III3 seems to be a key factor in anastellin binding.
Assuntos
Fibronectinas/química , Fibronectinas/metabolismo , Naftalenossulfonato de Anilina/metabolismo , Sítios de Ligação/genética , Fibronectinas/genética , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Cinética , Modelos Moleculares , Mutação , Fragmentos de Peptídeos , Ligação Proteica/genética , Dobramento de Proteína , Estrutura Terciária de Proteína/genética , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Fibronectin (FN) matrix fibrils have long been thought to be formed by disulfide-bonded FN multimers, although there is no direct evidence that they are covalently linked with each other. To understand the biochemical properties of these fibrils, we extracted a crude FN matrix from FN-YPet transfected 3T3 cell culture using 0.2% deoxycholate and DNase. The insoluble extracted matrix preserved fibrillar structures and a major portion of the extracted proteins migrated as FN monomers on an SDS gel under reducing conditions. Under non-reducing conditions, some FN molecules appeared to be trapped at the top of the stacking gel. We tested this by mixing fluorescently labeled FN dimers with the extracted matrix just before loading on an SDS gel, and found that most of them were trapped with the extracted proteins at the top of the stacking gel. These results suggested that some components of the extracted matrix plugged the stacking gel and FN dimers were trapped with them. Rotary shadowing electron microscopy showed that the extracted matrix had some fibers that resembled fibrillin microfibrils. Peptide mass fingerprinting confirmed the presence of fibrillin in the extracted matrix. Fibrillin is known to form disulfide-bonded multimers and it is likely to be one of the components that plug the stacking gel and trap FN molecules in this system. The phenomenon by which FN molecules appear to migrate as multimers on SDS gels is thus an artifact rising from the presence of other large components in the extract. We conclude that FN matrix fibrils are made of FN dimers that are further cross-linked by non-covalent protein-protein bonds.
Assuntos
Fenômenos Químicos , Matriz Extracelular/química , Fibronectinas/química , Multimerização Proteica , Animais , Detergentes/química , Eletroforese em Gel de Poliacrilamida , Matriz Extracelular/ultraestrutura , Fibrilinas , Fibronectinas/isolamento & purificação , Camundongos , Proteínas dos Microfilamentos/química , Microscopia Eletrônica , Células NIH 3T3RESUMO
Fibronectin (FN) is secreted as a disulfide-bonded FN dimer. Each subunit contains three types of repeating modules: FN-I, FN-II, and FN-III. The interactions of alpha5beta1 or alphav integrins with the RGD motif of FN-III repeat 10 (FN-III10) are considered an essential step in the assembly of FN fibrils. To test this hypothesis in vivo, we replaced the RGD motif with the inactive RGE in mice. FN-RGE homozygous embryos die at embryonic day 10 with shortened posterior trunk, absent tail bud-derived somites, and severe vascular defects resembling the phenotype of alpha5 integrin-deficient mice. Surprisingly, the absence of a functional RGD motif in FN did not compromise assembly of an FN matrix in mutant embryos or on mutant cells. Matrix assembly assays and solid-phase binding assays reveal that alphavbeta3 integrin assembles FN-RGE by binding an isoDGR motif in FN-I5, which is generated by the nonenzymatic rearrangement of asparagines (N) into an iso-aspartate (iso-D). Our findings demonstrate that FN contains a novel motif for integrin binding and fibril formation whose activity is controlled by amino acid modification.
Assuntos
Ácido Aspártico/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Oligopeptídeos/química , Reticulina/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular Transformada , Dimerização , Dissulfetos/química , Embrião de Mamíferos , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibronectinas/genética , Heterozigoto , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Camundongos , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , SolubilidadeRESUMO
We have experimentally studied the fluorescence resonance energy transfer (FRET) between green fluorescent protein (GFP) molecules by inserting folded or intrinsically unstructured proteins between CyPet and Ypet. We discovered that most of the enhanced FRET signal previously reported for this pair was due to enhanced dimerization, so we engineered a monomerizing mutation into each. An insert containing a single fibronectin type III domain (3.7 nm end-to-end) gave a moderate FRET signal while a two-domain insert (7.0 nm) gave no FRET. We then tested unstructured proteins of various lengths, including the charged-plus-PQ domain of ZipA, the tail domain of alpha-adducin, and the C-terminal tail domain of FtsZ. The structures of these FRET constructs were also studied by electron microscopy and sedimentation. A 12 amino acid linker and the N-terminal 33 amino acids of the charged domain of the ZipA gave strong FRET signals. The C-terminal 33 amino acids of the PQ domain of the ZipA and several unstructured proteins with 66-68 amino acids gave moderate FRET signals. The 150 amino acid charged-plus-PQ construct gave a barely detectable FRET signal. FRET efficiency was calculated from the decreased donor emission to estimate the distance between donor and acceptor. The donor-acceptor distance varied for unstructured inserts of the same length, suggesting that they had variable stiffness (persistence length). We conclude that GFP-based FRET can be useful for studying intrinsically unstructured proteins, and we present a range of calibrated protein inserts to experimentally determine the distances that can be studied.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/química , Proteínas/química , Algoritmos , Eletroforese em Gel de Poliacrilamida , Proteínas Luminescentes/química , Modelos Moleculares , Conformação ProteicaRESUMO
The extracellular matrix proteins tenascin and fibronectin experience significant mechanical forces in vivo. Both contain a number of tandem repeating homologous fibronectin type III (fnIII) domains, and atomic force microscopy experiments have demonstrated that the mechanical strength of these domains can vary significantly. Previous work has shown that mutations in the core of an fnIII domain from human tenascin (TNfn3) reduce the unfolding force of that domain significantly: The composition of the core is apparently crucial to the mechanical stability of these proteins. Based on these results, we have used rational redesign to increase the mechanical stability of the 10th fnIII domain of human fibronectin, FNfn10, which is directly involved in integrin binding. The hydrophobic core of FNfn10 was replaced with that of the homologous, mechanically stronger TNfn3 domain. Despite the extensive substitution, FNoTNc retains both the three-dimensional structure and the cell adhesion activity of FNfn10. Atomic force microscopy experiments reveal that the unfolding forces of the engineered protein FNoTNc increase by approximately 20% to match those of TNfn3. Thus, we have specifically designed a protein with increased mechanical stability. Our results demonstrate that core engineering can be used to change the mechanical strength of proteins while retaining functional surface interactions.