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1.
Nat Commun ; 14(1): 1143, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36854761

RESUMO

The protein phosphatase 2A (PP2A) heterotrimer PP2A-B56α is a human tumour suppressor. However, the molecular mechanisms inhibiting PP2A-B56α in cancer are poorly understood. Here, we report molecular level details and structural mechanisms of PP2A-B56α inhibition by an oncoprotein CIP2A. Upon direct binding to PP2A-B56α trimer, CIP2A displaces the PP2A-A subunit and thereby hijacks both the B56α, and the catalytic PP2Ac subunit to form a CIP2A-B56α-PP2Ac pseudotrimer. Further, CIP2A competes with B56α substrate binding by blocking the LxxIxE-motif substrate binding pocket on B56α. Relevant to oncogenic activity of CIP2A across human cancers, the N-terminal head domain-mediated interaction with B56α stabilizes CIP2A protein. Functionally, CRISPR/Cas9-mediated single amino acid mutagenesis of the head domain blunted MYC expression and MEK phosphorylation, and abrogated triple-negative breast cancer in vivo tumour growth. Collectively, we discover a unique multi-step hijack and mute protein complex regulation mechanism resulting in tumour suppressor PP2A-B56α inhibition. Further, the results unfold a structural determinant for the oncogenic activity of CIP2A, potentially facilitating therapeutic modulation of CIP2A in cancer and other diseases.


Assuntos
Carcinogênese , Proteína Fosfatase 2 , Processamento de Proteína Pós-Traducional , Neoplasias de Mama Triplo Negativas , Humanos , Aminoácidos , Carcinogênese/genética , Carcinogênese/metabolismo , Domínio Catalítico , Fosforilação , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/ultraestrutura , Neoplasias de Mama Triplo Negativas/metabolismo
2.
Proc Natl Acad Sci U S A ; 117(51): 32402-32412, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33288722

RESUMO

Binding of the intracellular adapter proteins talin and its cofactor, kindlin, to the integrin receptors induces integrin activation and clustering. These processes are essential for cell adhesion, migration, and organ development. Although the talin head, the integrin-binding segment in talin, possesses a typical FERM-domain sequence, a truncated form has been crystallized in an unexpected, elongated form. This form, however, lacks a C-terminal fragment and possesses reduced ß3-integrin binding. Here, we present a crystal structure of a full-length talin head in complex with the ß3-integrin tail. The structure reveals a compact FERM-like conformation and a tightly associated N-P-L-Y motif of ß3-integrin. A critical C-terminal poly-lysine motif mediates FERM interdomain contacts and assures the tight association with the ß3-integrin cytoplasmic segment. Removal of the poly-lysine motif or disrupting the FERM-folded configuration of the talin head significantly impairs integrin activation and clustering. Therefore, structural characterization of the FERM-folded active talin head provides fundamental understanding of the regulatory mechanism of integrin function.


Assuntos
Integrina beta3/metabolismo , Talina/química , Talina/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Humanos , Integrina beta3/química , Leucina/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Mutagênese , Polilisina/química , Domínios Proteicos , Dobramento de Proteína , Talina/genética
3.
J Cell Sci ; 133(19)2020 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-33046605

RESUMO

Integrin activation and clustering by talin are early steps of cell adhesion. Membrane-bound talin head domain and kindlin bind to the ß integrin cytoplasmic tail, cooperating to activate the heterodimeric integrin, and the talin head domain induces integrin clustering in the presence of Mn2+ Here we show that kindlin-1 can replace Mn2+ to mediate ß3 integrin clustering induced by the talin head, but not that induced by the F2-F3 fragment of talin. Integrin clustering mediated by kindlin-1 and the talin head was lost upon deletion of the flexible loop within the talin head F1 subdomain. Further mutagenesis identified hydrophobic and acidic motifs in the F1 loop responsible for ß3 integrin clustering. Modeling, computational and cysteine crosslinking studies showed direct and catalytic interactions of the acidic F1 loop motif with the juxtamembrane domains of α- and ß3-integrins, in order to activate the ß3 integrin heterodimer, further detailing the mechanism by which the talin-kindlin complex activates and clusters integrins. Moreover, the F1 loop interaction with the ß3 integrin tail required the newly identified compact FERM fold of the talin head, which positions the F1 loop next to the inner membrane clasp of the talin-bound integrin heterodimer.This article has an associated First Person interview with the first author of the paper.


Assuntos
Integrina beta3 , Talina , Adesão Celular , Análise por Conglomerados , Integrina beta3/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Talina/genética , Talina/metabolismo
4.
Viruses ; 11(12)2019 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-31795245

RESUMO

Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevented with a calpain inhibitor and was dependent on optimal calcium concentration, especially for calpain 2. In addition, calpain cleavage at the VP3-VP1 interface was supported by a competition assay using a peptide containing the VP3-VP1 cleavage site. Moreover, a mass spectrometry analysis showed that calpains can cleave this same peptide at the VP3-VP1 interface, the cutting site being two amino acids aside from 3C's cutting site. Furthermore, we show that calpains cannot cleave between P1 and 2A. In conclusion, we show that cellular proteases, calpains, can cleave structural proteins from enterovirus polyprotein in vitro. Whether they assist polyprotein processing in infected cells remains to be shown.


Assuntos
Calpaína/metabolismo , Proteínas do Capsídeo/metabolismo , Infecções por Enterovirus/virologia , Enterovirus/metabolismo , Poliproteínas/metabolismo , Animais , Capsídeo/metabolismo , Células Cultivadas , Glicoproteínas/farmacologia , Humanos , Espectrometria de Massas , Peptídeos/metabolismo , Proteólise , Ratos , Proteínas Virais/metabolismo
5.
PLoS One ; 14(2): e0212339, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30785944

RESUMO

Chicken avidin (Avd) and streptavidin from Streptomyces avidinii are extensively used in bionanotechnology due to their extremely tight binding to biotin (Kd ~ 10-15 M for chicken Avd). We previously reported engineered Avds known as antidins, which have micro- to nanomolar affinities for steroids, non-natural ligands of Avd. Here, we report the 2.8 Å X-ray structure of the sbAvd-2 (I117Y) antidin co-crystallized with progesterone. We describe the creation of new synthetic phage display libraries and report the experimental as well as computational binding analysis of progesterone-binding antidins. We introduce a next-generation antidin with 5 nM binding affinity for progesterone, and demonstrate the use of antidins for measuring progesterone in serum samples. Our data give insights on how to engineer and alter the binding preferences of Avds and to develop better molecular tools for modern bionanotechnological applications.


Assuntos
Avidina/metabolismo , Biotina/metabolismo , Progesterona/sangue , Progesterona/metabolismo , Animais , Avidina/química , Sítios de Ligação , Bioensaio , Biotina/química , Cães , Ligantes , Modelos Moleculares , Progesterona/química , Ligação Proteica
6.
Sci Rep ; 9(1): 995, 2019 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-30700796

RESUMO

Tuberculosis is a multifactorial bacterial disease, which can be modeled in the zebrafish (Danio rerio). Abdominal cavity infection with Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, leads to a granulomatous disease in adult zebrafish, which replicates the different phases of human tuberculosis, including primary infection, latency and spontaneous reactivation. Here, we have carried out a transcriptional analysis of zebrafish challenged with low-dose of M. marinum, and identified intelectin 3 (itln3) among the highly up-regulated genes. In order to clarify the in vivo significance of Itln3 in immunity, we created nonsense itln3 mutant zebrafish by CRISPR/Cas9 mutagenesis and analyzed the outcome of M. marinum infection in both zebrafish embryos and adult fish. The lack of functional itln3 did not affect survival or the mycobacterial burden in the zebrafish. Furthermore, embryonic survival was not affected when another mycobacterial challenge responsive intelectin, itln1, was silenced using morpholinos either in the WT or itln3 mutant fish. In addition, M. marinum infection in dexamethasone-treated adult zebrafish, which have lowered lymphocyte counts, resulted in similar bacterial burden in both WT fish and homozygous itln3 mutants. Collectively, although itln3 expression is induced upon M. marinum infection in zebrafish, it is dispensable for protective mycobacterial immune response.


Assuntos
Citocinas/metabolismo , Lectinas/metabolismo , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/microbiologia , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Códon sem Sentido/genética , Citocinas/genética , Dexametasona/farmacologia , Resistência à Doença/imunologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Embrião não Mamífero/microbiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genoma , Lectinas/genética , Depleção Linfocítica , Morfolinos/farmacologia , Mutação/genética , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/imunologia , Mycobacterium marinum/efeitos dos fármacos , Análise de Sobrevida , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
7.
J Virol Methods ; 255: 29-37, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29425680

RESUMO

Enteroviruses (EVs), such as the Coxsackie B-viruses (CVBs), are common human pathogens, which can cause severe diseases including meningitis, myocarditis and neonatal sepsis. EVs encode two proteases (2Apro and 3Cpro), which perform the proteolytic cleavage of the CVB polyprotein and also cleave host cell proteins to facilitate viral replication. The 2Apro cause direct damage to the infected heart and tools to investigate 2Apro and 3Cpro expression may contribute new knowledge on virus-induced pathologies. Here, we developed new antibodies to CVB-encoded 2Apro and 3Cpro; Two monoclonal 2Apro antibodies and one 3Cpro antibody were produced. Using cells infected with selected viruses belonging to the EV A, B and C species and immunocytochemistry, we demonstrate that the 3Cpro antibody detects all of the EV species B (EV-B) viruses tested and that the 2Apro antibody detects all EV-B viruses apart from Echovirus 9. We furthermore show that the new antibodies work in Western blotting, immunocyto- and immunohistochemistry, and flow cytometry to detect CVBs. Confocal microscopy demonstrated the expression kinetics of 2Apro and 3Cpro, and revealed a preferential cytosolic localization of the proteases in CVB3 infected cells. In summary, the new antibodies detect proteases that belong to EV species B in cells and tissue using multiple applications.


Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Cisteína Endopeptidases/imunologia , Enterovirus Humano B/imunologia , Infecções por Enterovirus/diagnóstico , Infecções por Enterovirus/imunologia , Proteínas Virais/imunologia , Proteases Virais 3C , Animais , Antígenos Virais/genética , Células Cultivadas , Clonagem Molecular , Cisteína Endopeptidases/genética , Enterovirus Humano B/enzimologia , Enterovirus Humano B/genética , Infecções por Enterovirus/virologia , Expressão Gênica , Células HeLa , Humanos , Imuno-Histoquímica , Camundongos , Sorogrupo , Proteínas Virais/genética
8.
Vaccine ; 35(30): 3718-3725, 2017 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-28579231

RESUMO

Coxsackie B viruses are among the most common enteroviruses, causing a wide range of diseases. Recent studies have also suggested that they may contribute to the development of type 1 diabetes. Vaccination would provide an effective way to prevent CVB infections, and the objective of this study was to develop an efficient vaccine production protocol for the generation of novel CVB vaccines. Various steps in the production of a formalin-inactivated Coxsackievirus B1 (CVB1) vaccine were optimized including the Multiplicity Of Infection (MOI) used for virus amplification, virus cultivation time, type of cell growth medium, virus purification method and formulation of the purified virus. Safety and immunogenicity of the formalin inactivated CVB1 vaccine was characterized in a mouse model. Two of the developed methods were found to be optimal for virus purification: the first employed PEG-precipitation followed by gelatin-chromatography and sucrose cushion pelleting (three-step protocol), yielding 19-fold increase in virus concentration (0.06µg/cm2) as compared to gold standard method. The second method utilized tandem sucrose pelleting without a PEG precipitation step, yielding 83-fold increase in virus concentration (0.24µg/cm2), but it was more labor-intensive and cannot be efficiently scaled up. Both protocols provide radically higher virus yields compared with traditional virus purification protocols involving PEG-precipitation and sucrose gradient ultracentrifugation. Formalin inactivation of CVB1 produced a vaccine that induced a strong, virus-neutralizing antibody response in vaccinated mice, which protected against challenge with CVB1 virus. Altogether, these results provide valuable information for the development of new enterovirus vaccines.


Assuntos
Infecções por Coxsackievirus/prevenção & controle , Enterovirus Humano A/imunologia , Imunogenicidade da Vacina , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Infecções por Coxsackievirus/imunologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Enterovirus Humano A/crescimento & desenvolvimento , Enterovirus Humano A/isolamento & purificação , Feminino , Formaldeído/farmacologia , Camundongos , Polissorbatos/farmacologia , Vacinação , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/isolamento & purificação , Células Vero , Vacinas Virais/administração & dosagem , Vacinas Virais/isolamento & purificação , Cultura de Vírus
9.
PLoS One ; 12(4): e0176086, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28426764

RESUMO

Bradavidin is a tetrameric biotin-binding protein similar to chicken avidin and bacterial streptavidin, and was originally cloned from the nitrogen-fixing bacteria Bradyrhizobium diazoefficiens. We have previously reported the crystal structure of the full-length, wild-type (wt) bradavidin with 138 amino acids, where the C-terminal residues Gly129-Lys138 ("Brad-tag") act as an intrinsic ligand (i.e. Gly129-Lys138 bind into the biotin-binding site of an adjacent subunit within the same tetramer) and has potential as an affinity tag for biotechnological purposes. Here, the X-ray structure of core-bradavidin lacking the C-terminal residues Gly114-Lys138, and hence missing the Brad-tag, was crystallized in complex with biotin at 1.60 Å resolution [PDB:4BBO]. We also report a homology model of rhodavidin, an avidin-like protein from Rhodopseudomonas palustris, and of an avidin-like protein from Bradyrhizobium sp. Ai1a-2, both of which have the Brad-tag sequence at their C-terminus. Moreover, core-bradavidin V1, an engineered variant of the original core-bradavidin, was also expressed at high levels in E. coli, as well as a double mutant (Cys39Ala and Cys69Ala) of core-bradavidin (CC mutant). Our data help us to further engineer the core-bradavidin-Brad-tag pair for biotechnological assays and chemical biology applications, and provide deeper insight into the biotin-binding mode of bradavidin.


Assuntos
Biotina/química , Proteínas de Transporte/química , Marcadores de Afinidade , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica
10.
Amino Acids ; 49(3): 529-540, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27503559

RESUMO

Coeliac disease is hallmarked by an abnormal immune reaction against ingested wheat-, rye- and barley-derived gluten and the presence of transglutaminase 2 (TG2)-targeted autoantibodies. The small-bowel mucosal damage characteristic of the disorder develops gradually from normal villus morphology to inflammation and finally to villus atrophy with crypt hyperplasia. Patients with early-stage coeliac disease have TG2-autoantibodies present in serum and small-intestinal mucosa and they may already suffer from abdominal symptoms before the development of villus atrophy. Previously, we have shown that intraperitoneal injections of coeliac patient-derived sera or purified immunoglobulin fraction into mice induce a condition mimicking early-stage coeliac disease. In the current study, we sought to establish whether recombinantly produced patient-derived TG2-targeted autoantibodies are by themselves sufficient for the development of such an experimentally induced condition in immune-compromised mice. Interestingly, mice injected with coeliac patient TG2-antibodies had altered small-intestinal mucosal morphology, increased lamina propria cellular infiltration and disease-specific autoantibodies deposited in the small bowel, but did not evince clinical features of the disease. Thus, coeliac patient-derived TG2-specific autoantibodies seem to be sufficient for the induction of subtle small-bowel mucosal alterations in mice, but the development of clinical features probably requires additional factors such as other antibody populations relevant in coeliac disease.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Autoanticorpos/biossíntese , Doença Celíaca/imunologia , Proteínas de Ligação ao GTP/imunologia , Hospedeiro Imunocomprometido , Mucosa Intestinal/efeitos dos fármacos , Intestino Delgado/efeitos dos fármacos , Transglutaminases/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Células CHO , Doença Celíaca/genética , Doença Celíaca/patologia , Cricetulus , Feminino , Proteínas de Ligação ao GTP/genética , Expressão Gênica , Glutens/química , Glutens/imunologia , Humanos , Imunoglobulina A/biossíntese , Imuno-Histoquímica , Inflamação , Injeções Intraperitoneais , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Intestino Delgado/imunologia , Intestino Delgado/patologia , Camundongos , Camundongos Nus , Proteína 2 Glutamina gama-Glutamiltransferase , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transglutaminases/genética
11.
J Virol ; 89(15): 7593-603, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25972558

RESUMO

UNLABELLED: Thermus thermophilus bacteriophage P23-77 is the type member of a new virus family of icosahedral, tailless, inner-membrane-containing double-stranded DNA (dsDNA) viruses infecting thermophilic bacteria and halophilic archaea. The viruses have a unique capsid architecture consisting of two major capsid proteins assembled in various building blocks. We analyzed the function of the minor capsid protein VP11, which is the third known capsid component in bacteriophage P23-77. Our findings show that VP11 is a dynamically elongated dimer with a predominantly α-helical secondary structure and high thermal stability. The high proportion of basic amino acids in the protein enables electrostatic interaction with negatively charged molecules, including nucleic acid and large unilamellar lipid vesicles (LUVs). The plausible biological function of VP11 is elucidated by demonstrating the interactions of VP11 with Thermus-derived LUVs and with the major capsid proteins by means of the dynamic-light-scattering technique. In particular, the major capsid protein VP17 was able to link VP11-complexed LUVs into larger particles, whereas the other P23-77 major capsid protein, VP16, was unable to link VP11-comlexed LUVs. Our results rule out a previously suggested penton function for VP11. Instead, the electrostatic membrane association of VP11 triggers the binding of the major capsid protein VP17, thus facilitating a controlled incorporation of the two different major protein species into the assembling capsid. IMPORTANCE: The study of thermophilic viruses with inner membranes provides valuable insights into the mechanisms used for stabilization and assembly of protein-lipid systems at high temperatures. Our results reveal a novel way by which an internal membrane and outer capsid shell are linked in a virus that uses two different major protein species for capsid assembly. We show that a positive protein charge is important in order to form electrostatic interactions with the lipid surface, thereby facilitating the incorporation of other capsid proteins on the membrane surface. This implies an alternative function for basic proteins present in the virions of other lipid-containing thermophilic viruses, whose proposed role in genome packaging is based on their capability to bind DNA. The unique minor capsid protein of bacteriophage P23-77 resembles in its characteristics the scaffolding proteins of tailed phages, though it constitutes a substantial part of the mature virion.


Assuntos
Bacteriófagos/metabolismo , Proteínas do Capsídeo/metabolismo , Lipídeos/química , Thermus/metabolismo , Montagem de Vírus , Sequência de Aminoácidos , Bacteriófagos/química , Bacteriófagos/genética , Capsídeo/química , Capsídeo/metabolismo , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Metabolismo dos Lipídeos , Modelos Moleculares , Dados de Sequência Molecular , Eletricidade Estática , Thermus/química , Thermus/virologia , Vírion/química , Vírion/genética , Vírion/metabolismo
12.
Bioconjug Chem ; 25(12): 2233-43, 2014 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-25405260

RESUMO

Switchavidin is a chicken avidin mutant displaying reversible binding to biotin, an improved binding affinity toward conjugated biotin, and low nonspecific binding due to reduced surface charge. These properties make switchavidin an optimal tool in biosensor applications for the reversible immobilization of biotinylated proteins on biotinylated sensor surfaces. Furthermore, switchavidin opens novel possibilities for patterning, purification, and labeling.


Assuntos
Avidina/química , Avidina/metabolismo , Técnicas Biossensoriais , Biotina/química , Células 3T3 , Animais , Avidina/genética , Sítios de Ligação , Biotinilação , Varredura Diferencial de Calorimetria , Galinhas , Camundongos , Mutação , Ressonância de Plasmônio de Superfície
13.
Mol Biosyst ; 10(12): 3217-28, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25277990

RESUMO

The major mechanical function of talin is to couple the ß-integrin cytoplasmic tails to actin filaments. A variety of ß-integrin tails contain conserved binding motifs for talin, and recent research shows that ß-integrins differ both in affinity to talin and preferences for other cytoplasmic adaptor proteins. While talin predominantly links ß3 integrins to actin filaments within the peripheral cell adhesion sites, talin can become replaced by other integrin adaptor proteins through their overlapping binding sites on integrin tails. Although the NPxY motif in the ß-integrin tail is important for talin recognition, our simulations suggest considerably smaller contribution of the NPxY motif in the force resistance of the talin-integrin complex than for the residues upstream of the NPxY. It might thus be possible for the NPxY motif to detach from talin and interact with other integrin binding proteins while the ß-integrin still remains bound to talin. The epithelial integrin ß6 reportedly activates latent TGFß1, and we propose that its function may involve direct interaction with talin.


Assuntos
Integrinas/química , Estresse Mecânico , Talina/química , Sequência de Aminoácidos , Técnicas Biossensoriais , Glutamina/química , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química
14.
PLoS One ; 9(6): e100564, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24959850

RESUMO

Chimeric avidin (ChiAVD) is a product of rational protein engineering remarkably resistant to heat and harsh conditions. In quest of the fundamentals behind factors affecting stability we have elucidated the solution NMR spectroscopic structure of the biotin-bound form of ChiAVD and characterized the protein dynamics through 15N relaxation and hydrogen/deuterium (H/D) exchange of this and the biotin-free form. To surmount the challenges arising from the very large size of the protein for NMR spectroscopy, we took advantage of its high thermostability. Conventional triple resonance experiments for fully protonated proteins combined with methyl-detection optimized experiments acquired at 58°C were adequate for the structure determination of this 56 kDa protein. The model-free parameters derived from the 15N relaxation data reveal a remarkably rigid protein at 58°C in both the biotin-bound and the free forms. The H/D exchange experiments indicate a notable increase in hydrogen protection upon biotin binding.


Assuntos
Avidina/química , Avidina/metabolismo , Biotina/química , Biotina/metabolismo , Modelos Moleculares , Peso Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Engenharia de Proteínas , Multimerização Proteica , Termodinâmica
15.
J Cell Biol ; 205(2): 265-81, 2014 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-24778313

RESUMO

Integrin-dependent cell adhesion and spreading are critical for morphogenesis, tissue regeneration, and immune defense but also tumor growth. However, the mechanisms that induce integrin-mediated cell spreading and provide mechanosensing on different extracellular matrix conditions are not fully understood. By expressing ß3-GFP-integrins with enhanced talin-binding affinity, we experimentally uncoupled integrin activation, clustering, and substrate binding from its function in cell spreading. Mutational analysis revealed Tyr747, located in the first cytoplasmic NPLY(747) motif, to induce spreading and paxillin adapter recruitment to substrate- and talin-bound integrins. In addition, integrin-mediated spreading, but not focal adhesion localization, was affected by mutating adjacent sequence motifs known to be involved in kindlin binding. On soft, spreading-repellent fibronectin substrates, high-affinity talin-binding integrins formed adhesions, but normal spreading was only possible with integrins competent to recruit the signaling adapter protein paxillin. This proposes that integrin-dependent cell-matrix adhesion and cell spreading are independently controlled, offering new therapeutic strategies to modify cell behavior in normal and pathological conditions.


Assuntos
Integrina beta3/metabolismo , Mecanotransdução Celular/fisiologia , Talina/metabolismo , Motivos de Aminoácidos , Animais , Células COS , Adesão Celular/fisiologia , Chlorocebus aethiops , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Integrina beta3/genética , Camundongos , Células NIH 3T3 , Paxilina/genética , Paxilina/metabolismo , Talina/genética
16.
PLoS One ; 8(10): e77207, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24204770

RESUMO

The avidin protein family members are well known for their high affinity towards D-biotin and high structural stability. These properties make avidins valuable tools for a wide range of biotechnology applications. We have identified a new member of the avidin family in the zebrafish (Danio rerio) genome, hereafter called zebavidin. The protein is highly expressed in the gonads of both male and female zebrafish and in the gills of male fish, but our data suggest that zebavidin is not crucial for the developing embryo. Biophysical and structural characterisation of zebavidin revealed distinct properties not found in any previously characterised avidins. Gel filtration chromatography and native mass spectrometry suggest that the protein forms dimers in the absence of biotin at low ionic strength, but assembles into tetramers upon binding biotin. Ligand binding was analysed using radioactive and fluorescently labelled biotin and isothermal titration calorimetry. Moreover, the crystal structure of zebavidin in complex with biotin was solved at 2.4 Å resolution and unveiled unique ligand binding and subunit interface architectures; the atomic-level details support our physicochemical observations.


Assuntos
Avidina/química , Proteínas de Peixes/química , Genoma , Glicoproteínas/química , Proteínas de Peixe-Zebra/química , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Avidina/genética , Avidina/metabolismo , Biotina/química , Biotina/metabolismo , Cristalografia por Raios X , Embrião não Mamífero , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Expressão Gênica , Brânquias/embriologia , Brânquias/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Gônadas/embriologia , Gônadas/metabolismo , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
17.
Biochim Biophys Acta ; 1834(10): 1988-97, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23856547

RESUMO

T-cell protein tyrosine phosphatase (TCPTP) is a ubiquitously expressed non-receptor protein tyrosine phosphatase. It is involved in the negative regulation of many cellular signaling pathways. Thus, activation of TCPTP could have important therapeutic applications in diseases such as cancer and inflammation. We have previously shown that the α-cytoplasmic tail of integrin α1ß1 directly binds and activates TCPTP. In addition, we have identified in a large-scale high-throughput screen six small molecules that activate TCPTP. These small molecule activators include mitoxantrone and spermidine. In this study, we have investigated the molecular mechanism behind agonist-induced TCPTP activation. By combining several molecular modeling and biochemical techniques, we demonstrate that α1-peptide and mitoxantrone activate TCPTP via direct binding to the catalytic domain, whereas spermidine does not interact with the catalytic domain of TCPTP in vitro. Furthermore, we have identified a hydrophobic groove surrounded by negatively charged residues on the surface of TCPTP as a putative binding site for the α1-peptide and mitoxantrone. Importantly, these data have allowed us to identify a new molecule that binds to TCPTP, but interestingly cannot activate its phosphatase activity. Accordingly, we describe here mechanism of TCPTP activation by mitoxantrone, the cytoplasmic tail of α1-integrin, and a mitoxantrone-like molecule at the atomic level. These data provide invaluable insight into the development of novel TCPTP activators, and may facilitate the rational discovery of small-molecule cancer therapeutics.


Assuntos
Antineoplásicos/química , Integrina alfa1beta1/química , Mitoxantrona/química , Peptídeos/química , Proteína Tirosina Fosfatase não Receptora Tipo 2/química , Bibliotecas de Moléculas Pequenas/química , Espermidina/química , Bases de Dados de Proteínas , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Eletricidade Estática , Termodinâmica
18.
Protein Sci ; 22(7): 980-94, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23661323

RESUMO

Bradavidin II is a biotin-binding protein from Bradyrhizobium japonicum that resembles chicken avidin and bacterial streptavidin. A biophysical characterization was carried out using dynamic light scattering, native mass spectrometry, differential scanning calorimetry, and isothermal titration calorimetry combined with structural characterization using X-ray crystallography. These observations revealed that bradavidin II differs from canonical homotetrameric avidin protein family members in its quaternary structure. In contrast with the other avidins, bradavidin II appears to have a dynamic (transient) oligomeric state in solution. It is monomeric at low protein concentrations but forms higher oligomeric assemblies at higher concentrations. The crystal structure of bradavidin II revealed an important role for Phe42 in shielding the bound ligand from surrounding water molecules, thus functionally replacing the L7,8 loop essential for tight ligand binding in avidin and streptavidin. This bradavidin II characterization opens new avenues for oligomerization-independent biotin-binding protein development.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Sequência de Aminoácidos , Animais , Biotina/química , Biotina/metabolismo , Galinhas , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Desdobramento de Proteína , Alinhamento de Sequência , Temperatura
19.
Biomol NMR Assign ; 7(1): 35-8, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22392339

RESUMO

Avidin is a homotetrameric ~56 kDa protein found in chicken egg white. Avidin's ability to bind biotin with a very high affinity has widely been exploited in biotechnological applications. Protein engineering has further diversified avidin's feasibility. ChiAVD(I117Y) is a product of rational protein engineering. It is a hyperthermostable synthetic hybrid of avidin and avidin-related protein 4 (AVR4). In this chimeric protein a 23-residue segment in avidin has been replaced with the corresponding sequence found in AVR4, and a point mutation at subunit interface 1-3 (and 2-4) has been introduced. Here we report the backbone and sidechain resonance assignments of the biotin-bound form of ChiAVD(I117Y) as well as the backbone resonance assignments of the free form.


Assuntos
Avidina/química , Avidina/metabolismo , Biotina/metabolismo , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Ligantes , Peso Molecular , Ligação Proteica
20.
ACS Appl Mater Interfaces ; 4(8): 4067-77, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22786654

RESUMO

Stabilized bioreceptor layers are of great importance in the design of novel biosensors. In earlier work, chimeric avidins enabled immobilization of biotinylated antibodies onto gold surfaces with greater stability compared to more conventional avidins (wild-type avidin and streptavidin). In the present study, the applicability of chimeric avidins as a general binding scaffold for biotinylated antibodies on spin-coated functionalized polythiophene thin films has been studied by surface plasmon resonance and atomic force microscopy. Novel chimeric avidins showed remarkably increased binding characteristics compared with other avidins, such as wild-type avidin, streptavidin, and bacterial avidin when merely physically adsorbed onto the polythiophene surface. They gave the highest binding capacities, the highest affinity constant, and the highest stability for biotinylated probe immobilization. Introduction of carboxylic acid groups to polythiophene layer further enhanced the binding level of the avidins. Polythiophene layers functionalized with chimeric avidins thus offered a promising generic platform for biosensor applications.


Assuntos
Avidina/química , Técnicas Biossensoriais/métodos , Polímeros/química , Tiofenos/química , Adsorção , Anticorpos/química , Ouro/química , Imunoglobulina G/química , Microscopia de Força Atômica/métodos , Modelos Químicos , Conformação Molecular , Ligação Proteica , Estreptavidina/química , Ressonância de Plasmônio de Superfície
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