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1.
Physiol Rev ; 99(4): 1655-1699, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31313981

RESUMO

Integrins are heterodimeric cell surface receptors ensuring the mechanical connection between cells and the extracellular matrix. In addition to the anchorage of cells to the extracellular matrix, these receptors have critical functions in intracellular signaling, but are also taking center stage in many physiological and pathological conditions. In this review, we provide some historical, structural, and physiological notes so that the diverse functions of these receptors can be appreciated and put into the context of the emerging field of mechanobiology. We propose that the exciting journey of the exploration of these receptors will continue for at least another new generation of researchers.


Assuntos
Adesão Celular , Membrana Celular/metabolismo , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Mecanotransdução Celular , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proliferação de Células , Humanos , Integrinas/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Fosfoproteínas/metabolismo , Conformação Proteica , Relação Estrutura-Atividade , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAP
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.
FASEB J ; 34(2): 2227-2237, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31916632

RESUMO

Cyanidin-3-glucoside (C3G) is a natural pigment, found in many colorful fruits and vegetables. It has many health benefits, including anti-inflammation, cancer prevention, and anti-diabetes. Although C3G is assumed to be an antioxidant, it has been reported to affect cell-matrix adhesions. However, the underlying molecular mechanism is unknown. Here, we show that the expression of talin1, a key regulator of integrins and cell adhesions, negatively correlated with the survival rate of colon cancer patients and that depletion of talin1 inhibited 3D spheroid growth in colon cancer cells. Interestingly, C3G bound to talin and promoted the interaction of talin with ß1A-integrin. Molecular docking analysis shows that C3G binds to the interface of the talin-ß-integrin complex, acting as an allosteric regulator and altering the interaction between talin and integrin. Moreover, C3G promoted colon cancer cell attachment to fibronectin. While C3G had no significant effect on colon cancer cell proliferation, it significantly inhibited 3D spheroid growth in fibrin gel assays. Since C3G has no or very low toxicity, it could be potentially used for colon cancer prevention or therapy.


Assuntos
Antocianinas/farmacocinética , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo , Glucosídeos/farmacocinética , Proteínas de Neoplasias , Talina , Animais , Células CHO , Técnicas de Cultura de Células , Neoplasias do Colo/química , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Cricetinae , Cricetulus , Células HCT116 , Humanos , Simulação de Dinâmica Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Talina/química , Talina/metabolismo
5.
J Biol Chem ; 288(51): 36610-23, 2013 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-24178295

RESUMO

Proprotein convertase subtilisin/kexin (PCSK) enzymes convert proproteins into bioactive end products. Although other PCSK enzymes are known to be essential for biological processes ranging from cholesterol metabolism to host defense, the in vivo importance of the evolutionarily ancient PCSK7 has remained enigmatic. Here, we quantified the expressions of all pcsk genes during the 1st week of fish development and in several tissues. pcsk7 expression was ubiquitous and evident already during the early development. To compare mammalian and zebrafish PCSK7, we prepared homology models, which demonstrated remarkable structural conservation. When the PCSK7 function in developing larvae was inhibited, we found that PCSK7-deficient fish have defects in various organs, including the brain, eye, and otic vesicle, and these result in mortality within 7 days postfertilization. A genome-wide analysis of PCSK7-dependent gene expression showed that, in addition to developmental processes, several immune system-related pathways are also regulated by PCSK7. Specifically, the PCSK7 contributed to the mRNA expression and proteolytic cleavage of the cytokine TGFß1a. Consequently, tgfß1a morphant fish displayed phenotypical similarities with pcsk7 morphants, underscoring the importance of this cytokine in the zebrafish development. Targeting PCSK activity has emerged as a strategy for treating human diseases. Our results suggest that inhibiting PCSK7 might interfere with normal vertebrate development.


Assuntos
Subtilisinas/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Domínio Catalítico , Sequência Conservada , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Larva/crescimento & desenvolvimento , Larva/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos , Proteólise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência , Subtilisinas/química , Subtilisinas/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética
6.
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
7.
BMC Genomics ; 12: 618, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22185580

RESUMO

BACKGROUND: Subtilisin/kexin-like proprotein convertase (PCSK) enzymes have important regulatory function in a wide variety of biological processes. PCSKs proteolytically process at a target sequence that contains basic amino acids arginine and lysine, which results in functional maturation of the target protein. In vitro assays have showed significant biochemical redundancy between the seven family members, but the phenotypes of PCSK deficient mice and patients carrying an inactive PCSK allele argue for a specific biological function. Modeling the structures of individual PCSK enzymes has offered little insights into the specificity determinants. However, previous studies have shown that there can be a coordinated expression between a PCSK and its target molecule. Here, we have surveyed the putative PCSK target proteins using genome-wide expression correlation analysis and cleavage site prediction algorithms. RESULTS: We first performed a gene expression correlation analysis over the whole genome for all PCSK enzymes. PCSKs were found to cluster differently based on the strength of correlations. The screen for putative PCSK target proteins showed a significant enrichment (p-values from 1.2e-4 to < 1.0e-10) of putative targets among the most positively correlating genes for most PCSKs. Interestingly, there was no enrichment in putative targets among the genes that correlated positively with the biologically redundant PCSK7, whereas PCSK5 showed an inverse correlation. PCSKs also showed a highly variable degree of shared target genes that were identified by expression correlation and cleavage site prediction. Multiple alignments were used to evaluate the putative targets to pinpoint the important residues for the substrate recognition. Finally, we validated our approach and identified biochemically PAPPA1 and ADAMTS6 as novel targets for FURIN proteolytic activity. CONCLUSIONS: Most PCSK enzymes display strong positive expression correlation with predicted target proteins in our genome-wide analysis. We also show that expression correlation screen combined with a cleavage site-prediction analysis can be used to identify novel bona fide target molecules for PCSKs. Exploring the positively correlating genes can thus offer additional insights into the biology of proprotein convertases.


Assuntos
Estudo de Associação Genômica Ampla , Pró-Proteína Convertases/metabolismo , Animais , Camundongos , Modelos Moleculares , Especificidade por Substrato
8.
BMC Ecol Evol ; 21(1): 53, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33836663

RESUMO

BACKGROUND: Avidins are biotin-binding proteins commonly found in the vertebrate eggs. In addition to streptavidin from Streptomyces avidinii, a growing number of avidins have been characterized from divergent bacterial species. However, a systematic research concerning their taxonomy and ecological role has never been done. We performed a search for avidin encoding genes among bacteria using available databases and classified potential avidins according to taxonomy and the ecological niches utilized by host bacteria. RESULTS: Numerous avidin-encoding genes were found in the phyla Actinobacteria and Proteobacteria. The diversity of protein sequences was high and several new variants of genes encoding biotin-binding avidins were found. The living strategies of bacteria hosting avidin encoding genes fall mainly into two categories. Human and animal pathogens were overrepresented among the found bacteria carrying avidin genes. The other widespread category were bacteria that either fix nitrogen or live in root nodules/rhizospheres of plants hosting nitrogen-fixing bacteria. CONCLUSIONS: Bacterial avidins are a taxonomically and ecologically diverse group mainly found in Actinobacteria, Proteobacteria and Bacteroidetes, associated often with plant invasiveness. Avidin encoding genes in plasmids hint that avidins may be horizontally transferred. The current survey may be used as a basis in attempts to understand the ecological significance of biotin-binding capacity.


Assuntos
Actinobacteria , Avidina , Actinobacteria/genética , Animais , Bacteroidetes/genética , Humanos , Proteobactérias/genética , Streptomyces
9.
PeerJ ; 5: e4128, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29230365

RESUMO

BACKGROUND: Carbonic anhydrases (CAs) are ubiquitous, essential enzymes which catalyze the conversion of carbon dioxide and water to bicarbonate and H+ ions. Vertebrate genomes generally contain gene loci for 15-21 different CA isoforms, three of which are enzymatically inactive. CA VI is the only secretory protein of the enzymatically active isoforms. We discovered that non-mammalian CA VI contains a C-terminal pentraxin (PTX) domain, a novel combination for both CAs and PTXs. METHODS: We isolated and sequenced zebrafish (Danio rerio) CA VI cDNA, complete with the sequence coding for the PTX domain, and produced the recombinant CA VI-PTX protein. Enzymatic activity and kinetic parameters were measured with a stopped-flow instrument. Mass spectrometry, analytical gel filtration and dynamic light scattering were used for biophysical characterization. Sequence analyses and Bayesian phylogenetics were used in generating hypotheses of protein structure and CA VI gene evolution. A CA VI-PTX antiserum was produced, and the expression of CA VI protein was studied by immunohistochemistry. A knock-down zebrafish model was constructed, and larvae were observed up to five days post-fertilization (dpf). The expression of ca6 mRNA was quantitated by qRT-PCR in different developmental times in morphant and wild-type larvae and in different adult fish tissues. Finally, the swimming behavior of the morphant fish was compared to that of wild-type fish. RESULTS: The recombinant enzyme has a very high carbonate dehydratase activity. Sequencing confirms a 530-residue protein identical to one of the predicted proteins in the Ensembl database (ensembl.org). The protein is pentameric in solution, as studied by gel filtration and light scattering, presumably joined by the PTX domains. Mass spectrometry confirms the predicted signal peptide cleavage and disulfides, and N-glycosylation in two of the four observed glycosylation motifs. Molecular modeling of the pentamer is consistent with the modifications observed in mass spectrometry. Phylogenetics and sequence analyses provide a consistent hypothesis of the evolutionary history of domains associated with CA VI in mammals and non-mammals. Briefly, the evidence suggests that ancestral CA VI was a transmembrane protein, the exon coding for the cytoplasmic domain was replaced by one coding for PTX domain, and finally, in the therian lineage, the PTX-coding exon was lost. We knocked down CA VI expression in zebrafish embryos with antisense morpholino oligonucleotides, resulting in phenotype features of decreased buoyancy and swim bladder deflation in 4 dpf larvae. DISCUSSION: These findings provide novel insights into the evolution, structure, and function of this unique CA form.

10.
ACS Chem Biol ; 11(1): 211-21, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26550684

RESUMO

Proteins with high specificity, affinity, and stability are needed for biomolecular recognition in a plethora of applications. Antibodies are powerful affinity tools, but they may also suffer from limitations such as low stability and high production costs. Avidin and streptavidin provide a promising scaffold for protein engineering, and due to their ultratight binding to D-biotin they are widely used in various biotechnological and biomedical applications. In this study, we demonstrate that the avidin scaffold is suitable for use as a novel receptor for several biologically active small molecules: Artificial, chicken avidin-based proteins, antidins, were generated using a directed evolution method for progesterone, hydrocortisone, testosterone, cholic acid, ketoprofen, and folic acid, all with micromolar to nanomolar affinity and significantly reduced biotin-binding affinity. We also describe the crystal structure of an antidin, sbAvd-2(I117Y), a steroid-binding avidin, which proves that the avidin scaffold can tolerate significant modifications without losing its characteristic tetrameric beta-barrel structure, helping us to further design avidin-based small molecule receptors.


Assuntos
Avidina/metabolismo , Bioensaio/métodos , Receptores Artificiais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/metabolismo , Animais , Avidina/química , Varredura Diferencial de Calorimetria , Galinhas , Cristalografia por Raios X , Fluorometria , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Molecular , Receptores Artificiais/química , Bibliotecas de Moléculas Pequenas/química
11.
J Phys Chem B ; 119(38): 12381-9, 2015 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-26309152

RESUMO

Integrins are major players in cell adhesion and migration, and malfunctions in controlling their activity are associated with various diseases. Nevertheless, the details of integrin activation are not completely understood, and the role of lipids in the process is largely unknown. Herein, we show using atomistic molecular dynamics simulations that the interplay of phosphatidylinositol 4,5-bisphosphate (PIP2) and talin may directly alter the conformation of integrin αIIbß3. Our results provide a new perspective on the role of PIP2 in integrin activation and indicate that the charged PIP2 lipid headgroup can perturb a clasp at the cytoplasmic face of the integrin heterodimer.


Assuntos
Fosfatidilinositol 4,5-Difosfato/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Talina/química , Animais , Ligação de Hidrogênio , Bicamadas Lipídicas/química , Camundongos , Simulação de Dinâmica Molecular
12.
Protein Eng Des Sel ; 28(1): 23-8, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25445152

RESUMO

Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries.


Assuntos
Clonagem de Organismos/métodos , Embaralhamento de DNA/métodos , Biblioteca Gênica , Sequência de Aminoácidos , Técnicas de Visualização da Superfície Celular , Evolução Molecular Direcionada , Escherichia coli/genética , Dados de Sequência Molecular , Alinhamento de Sequência
13.
Biologics ; 8: 59-65, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24596454

RESUMO

BACKGROUND: The milk casein-derived bioactive tripeptides isoleucine-proline-proline (IPP) and valine-proline-proline (VPP) have been shown to prevent development of hypertension in animal models and to lower blood pressure in moderately hypertensive subjects in most but not all clinical trials. Inhibition of angiotensin-converting enzyme 1 (ACE-1) has been suggested as the explanation for these antihypertensive and beneficial vascular effects. Previously, human umbilical vein endothelial cells (HUVEC) have not been used to test ACE-1 inhibiting properties of casein derived tripeptides in vasculature. PURPOSE: We focused on the cis/trans configurations of the peptide bonds in proline-containing tripeptides in order to discover whether the different structural properties of these peptides influence their activity in ACE-1 inhibition. We hypothesized that the configuration of proline-containing peptides plays a significant role in enzyme inhibition. METHODS: AutoDock 4.2 docking software was used to predict suitable peptide bond configurations of the tripeptides. Besides modeling studies, we completed ACE-1 activity measurements in vitro using HUVEC cultures. RESULTS: In HUVEC cells, both IPP and VPP inhibited ACE-1. Based on molecular docking studies, we propose that in ACE-1 inhibition IPP and VPP share a similar cis configuration between the first aliphatic (isoleucine or valine) and the second (proline) amino acid residues and more different configurations between two proline residues. In vivo experiments are needed to validate the significance of the present findings.

14.
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
15.
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
16.
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
17.
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
18.
PLoS One ; 6(5): e20535, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21655240

RESUMO

BACKGROUND: Avidin is a chicken egg-white protein with high affinity to vitamin H, also known as D-biotin. Many applications in life science research are based on this strong interaction. Avidin is a homotetrameric protein, which promotes its modification to symmetrical entities. Dual-chain avidin, a genetically engineered avidin form, has two circularly permuted chicken avidin monomers that are tandem-fused into one polypeptide chain. This form of avidin enables independent modification of the two domains, including the two biotin-binding pockets; however, decreased yields in protein production, compared to wt avidin, and complicated genetic manipulation of two highly similar DNA sequences in the tandem gene have limited the use of dual-chain avidin in biotechnological applications. PRINCIPAL FINDINGS: To overcome challenges associated with the original dual-chain avidin, we developed chimeric dual-chain avidin, which is a tandem fusion of avidin and avidin-related protein 4 (AVR4), another member of the chicken avidin gene family. We observed an increase in protein production and better thermal stability, compared with the original dual-chain avidin. Additionally, PCR amplification of the hybrid gene was more efficient, thus enabling more convenient and straightforward modification of the dual-chain avidin. When studied closer, the generated chimeric dual-chain avidin showed biphasic biotin dissociation. SIGNIFICANCE: The improved dual-chain avidin introduced here increases its potential for future applications. This molecule offers a valuable base for developing bi-functional avidin tools for bioseparation, carrier proteins, and nanoscale adapters. Additionally, this strategy could be helpful when generating hetero-oligomers from other oligomeric proteins with high structural similarity.


Assuntos
Avidina/química , Avidina/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Animais , Avidina/genética , Técnicas Biossensoriais , Biotina/genética , Biotina/metabolismo , Galinhas , Cromatografia em Gel , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Simulação de Dinâmica Molecular , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/genética , Ressonância de Plasmônio de Superfície
19.
Biochimie ; 92(8): 1072-80, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20493921

RESUMO

Carbonic anhydrase (CA) enzymes are expressed in all organs of the mammalian body where they participate in important physiological functions. CA VII is a cytosolic isozyme which may be expressed as two forms according to the recent GenBank data. We designed a present study to express and characterize the human CA VII forms: full-length CA VII and short form (predicted to lack 56 residues from the N-terminus). Reverse transcriptase PCR analysis revealed mRNAs for both CA VII forms in the human brain. These different forms were expressed as recombinant proteins to investigate their biochemical properties. The full-length CA VII was used to raise a polyclonal antiserum in a rabbit, and the antiserum was then employed in western blot analyses and immunohistochemistry of mouse tissues. Data from mass spectrometry and comparative modeling showed that CA VII protein contains a single intramolecular disulfide bridge (Cys-56 to Cys-180) which is lacking in the short form. The computer model suggested distinctly different folding for the different forms. The more exposed structure and the absence of the disulfide bridge in the short form could make this protein more susceptible to degradation. In fact, this was realized in several protein purification efforts in which the short form readily degraded during the experimental procedures. From these results, we conclude that the full-length CA VII is a predominant active form in human brain and also in other tissues. In addition to the brain, CA VII is expressed in several other organs including the stomach, duodenum, colon, liver, and skeletal muscle. The distribution pattern suggests multiple functions for CA VII in different organs.


Assuntos
Anidrases Carbônicas/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Primers do DNA , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray
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