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1.
Comput Biol Chem ; 84: 107168, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31791808

RESUMO

The cyclotides are the largest known family of cyclic proteins, which are found in several plant families including Violaceae. They are circular bioactive peptides consisting of 28-37 amino acids, which possess a cyclic cystine knot (CCK) motif and could be useful in biotechnology and drug design as scaffolds for peptide-based drugs. This study describes our finding of a potentially novel gene transcript from the petals of the Iranian Viola tricolor (V. tricolor) flowers. This study is based on the cDNA screening method employed for isolation of cyclotide precursor genes and in silico analysis. Our study resulted in the finding of a novel cyclotide-like precursor from V. tricolor, which is documented in the NCBI by GenBank accession number: KP065812. The in silico analysis revealed that there are lots of similar sequences in many other plant families and they all exhibit some different features from previously discovered cyclotide precursors. The differences occur particularly in the main cyclotide domain that exists without the usual CCK structure. All of these hypothetical precursors have a conserved ER-signal sequence, a Cysteine (C)-rich sequence forming two zinc finger motifs and a cyclotide-like region containing several conserved elements including two highly conserved C residues. In conclusion, using the cDNA screening method we found a potentially new cyclotide-like precursor gene and in silico studies revealed its significant characteristics that may open up a new research line on the distribution and evolution of cyclotides.


Assuntos
Ciclotídeos/análise , Proteínas de Plantas/análise , Precursores de Proteínas/análise , Viola/química , Sequência de Aminoácidos , Ciclotídeos/química , Ciclotídeos/genética , Flores/química , Genes de Plantas , Irã (Geográfico) , Folhas de Planta/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Precursores de Proteínas/química , Precursores de Proteínas/genética , Alinhamento de Sequência , Análise de Sequência de DNA
2.
J Synchrotron Radiat ; 26(Pt 4): 967-979, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31274418

RESUMO

Dissociation of transforming growth factor beta-1 (TGFß-1) from the inhibitory protein latency-associated peptide (LAP) can occur from low doses of X-ray irradiation of the LAP-TGFß-1 complex, resulting in the activation of TGFß-1, and can have health-related consequences. Using the tools and knowledge developed in the study of radiation damage in the crystallographic setting, small-angle X-ray scattering (SAXS) and complementary techniques suggest an activation process that is initiated but not driven by the initial X-ray exposure. LAP is revealed to be extended when not bound to TGFß-1 and has a different structural conformation compared to the bound state. These studies pave the way for the structural understanding of systems impacted at therapeutic X-ray doses and show the potential impact of radiation damage studies beyond their original intent.


Assuntos
Peptídeos/química , Precursores de Proteínas/química , Fator de Crescimento Transformador beta1/química , Fator de Crescimento Transformador beta/química , Raios X , Relação Dose-Resposta à Radiação , Humanos , Conformação Proteica , Espalhamento a Baixo Ângulo , Difração de Raios X
3.
Biochim Biophys Acta Proteins Proteom ; 1867(11): 140252, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31325636

RESUMO

Intrinsically disordered proteins (IDPs) explore diverse conformations in their free states and, a few of them, also in their molecular complexes. This functional plasticity is essential for the function of IDPs, although their dynamics in both free and bound states is poorly understood. NUPR1 is a protumoral multifunctional IDP, activated during the acute phases of pancreatitis. It interacts with DNA and other IDPs, such as prothymosin α (ProTα), with dissociation constants of ~0.5 µM, and a 1:1 stoichiometry. We studied the structure and picosecond-to-nanosecond (ps-ns) dynamics by using both NMR and SAXS in: (i) isolated NUPR1; (ii) the NUPR1/ProTα complex; and (iii) the NUPR1/double stranded (ds) GGGCGCGCCC complex. Our SAXS findings show that NUPR1 remained disordered when bound to either partner, adopting a worm-like conformation; the fuzziness of bound NUPR1 was also pinpointed by NMR. Residues with the largest values of the relaxation rates (R1, R1ρ, R2 and ηxy), in the free and bound species, were mainly clustered around the 30s region of the sequence, which agree with one of the protein hot-spots already identified by site-directed mutagenesis. Not only residues in this region had larger relaxation rates, but they also moved slower than the rest of the molecule, as indicated by the reduced spectral density approach (RSDA). Upon binding, the energy landscape of NUPR1 was not funneled down to a specific, well-folded conformation, but rather its backbone flexibility was kept, with distinct motions occurring at the hot-spot region.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , DNA/química , Complexos Multiproteicos/química , Proteínas de Neoplasias/química , Precursores de Proteínas/química , Timosina/análogos & derivados , Humanos , Domínios Proteicos , Espalhamento a Baixo Ângulo , Timosina/química , Difração de Raios X
4.
Mater Sci Eng C Mater Biol Appl ; 103: 109762, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31349478

RESUMO

Early detection is the most effective mean of improving prognosis for many fatal diseases such as cancer. In this context, the Surface Enhanced Resonance Raman Scattering (SERRS) technique is being proposed as alternative to fluorescent methods in detection of biomarkers, because SERRS nanostructures are bright as fluorescent tags but more stable and clearly detectable using the narrow Raman "fingerprints" of a suitable reporter. Here we show that biocompatible SERRS active gold nanostructures, functionalized with an engineered PreS1 peptide (AuNP@PEG-PreS1), detect the presence of the SerpinB3 antigen overexpressed on liver tumor cells, a biomarker of the onset of liver cell carcinomatous transformation. A proper engineering of the targeting unit, linked to the nanostructure by a polymer chain, affords a sensitivity and specificity larger than 80%, at subnanomolar concentrations. Taking into account the high sensitivity of SERRS and that SB3 overexpression is an early event in liver cell carcinomatous transformation, AuNP@PEG-PreS1 nanostructures could be used in routine diagnostic activities, to improve the accuracy of HCC detection in particular in patients with chronic liver diseases.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Ouro , Antígenos de Superfície da Hepatite B , Neoplasias Hepáticas/tratamento farmacológico , Nanopartículas Metálicas , Peptídeos , Precursores de Proteínas , Animais , Antígenos de Neoplasias/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Ouro/química , Ouro/farmacologia , Células Hep G2 , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/farmacologia , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Nanopartículas Metálicas/química , Nanopartículas Metálicas/uso terapêutico , Camundongos , Camundongos Transgênicos , Proteínas de Neoplasias/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Precursores de Proteínas/química , Precursores de Proteínas/farmacologia , Serpinas/metabolismo , Análise Espectral Raman
5.
Biopolymers ; 110(6): e23277, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30972750

RESUMO

Surfactant protein C (SP-C) is an important constituent of lung surfactant (LS) and, along with SP-B, is included in exogenous surfactant replacement therapies for treating respiratory distress syndrome (RDS). SP-C's biophysical activity depends upon the presence of a rigid C-terminal helix, of which the secondary structure is more crucial to functionality than precise side-chain chemistry. SP-C is highly sequence-conserved, suggesting that the ß-branched, aliphatic side chains of the helix are also important. Nonnatural mimics of SP-C were created using a poly-N-substituted glycine, or "peptoid," backbone. The mimics included varying amounts of α-chiral, aliphatic side chains and α-chiral, aromatic side chains in the helical region, imparting either biomimicry or structural rigidity. Biophysical studies confirmed that the peptoids mimicked SP-C's secondary structure and replicated many of its surface-active characteristics. Surface activity was optimized by incorporating both structurally rigid and biomimetic side chain chemistries in the helical region indicating that both characteristics are important for activity. By balancing these features in one mimic, a novel analogue was created that emulates SP-C's in vitro surface activity while overcoming many of the challenges related to natural SP-C. Peptoid-based analogues hold great potential for use in a synthetic, biomimetic LS formulation for treating RDS.


Assuntos
Materiais Biomiméticos/química , Peptoides/química , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/metabolismo , Dicroísmo Circular , Desenho de Drogas , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Microscopia de Fluorescência , Peptoides/síntese química , Peptoides/metabolismo , Conformação Proteica em alfa-Hélice , Precursores de Proteínas/química , Proteolipídeos/química , Propriedades de Superfície
6.
Proteins ; 87(1): 81-90, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30367523

RESUMO

The translocase of the outer membrane (TOM) mediates the membrane permeation of mitochondrial matrix proteins. Tom20 is a subunit of the TOM complex and binds to the N-terminal region (ie, presequence) in mitochondrial matrix precursor proteins. Previous experimental studies indicated that the presequence recognition by Tom20 was achieved in a dynamic-equilibrium among multiple bound states of the α-helical presequence. Accordingly, the co-crystallization of Tom20 and a presequence peptide required a disulfide-bond cross-linking. A 3-residue spacer sequence (XAG) was inserted between the presequence and the anchoring Cys residue at the C-terminus to not disturb the movement of the presequence peptide in the binding site of Tom20. Two crystalline forms were obtained according to Ala or Tyr at the X position of the spacer sequence, which may reflect the dynamic-equilibrium of the presequence. Here, we have performed replica-exchange molecular dynamics (REMD) simulations to study the effect of disulfide-bond linker and single amino acid difference in the spacer region of the linker on the conformational dynamics of Tom20-presequence complex. Free energy and network analyses of the REMD simulations were compared against previous simulations of non-tethered system. We concluded that the disulfide-bond tethering did not strongly affect the conformational ensemble of the presequence peptide in the complex. Further investigation showed that the choice of Ala or Tyr at the X position did not affect the most distributions of the conformational ensemble of the presequence. The present study provides a rational basis for the disulfide-bond tethering to study the dynamics of weakly binding complexes.


Assuntos
/química , Biologia Computacional/métodos , Proteínas de Membrana Transportadoras/química , Fragmentos de Peptídeos/química , Precursores de Proteínas/química , Receptores de Superfície Celular/química , /metabolismo , Animais , Cristalização , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Precursores de Proteínas/metabolismo , Estrutura Terciária de Proteína , Ratos , Receptores de Superfície Celular/metabolismo
7.
Structure ; 27(1): 78-89.e3, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30393051

RESUMO

Nerve growth factor (NGF) is an important neurotrophic factor involved in the regulation of cell differentiation and survival of target neurons. Expressed as a proNGF precursor, NGF is matured by furin-mediated protease cleavage. Increasing evidence suggests that NGF and proNGF have distinct functional roles. While the structure of mature NGF is available, little is known about that of the pro-domain because of its dynamical structural features. We exploited an ad hoc hybrid strategy based on nuclear magnetic resonance and modeling validated by small-angle X-ray scattering to gain novel insights on the pro-domain, both in isolation and in the context of proNGF. We show that the isolated pro-domain is intrinsically unstructured but forms transient intramolecular contacts with mature NGF and has per se the ability to induce growth cone collapse, indicating functional independence. Our data represent an important step toward the structural and functional characterization of the properties of proNGF.


Assuntos
Fator de Crescimento Neural/química , Precursores de Proteínas/química , Animais , Células Cultivadas , Cones de Crescimento/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Simulação de Dinâmica Molecular , Fator de Crescimento Neural/metabolismo , Domínios Proteicos , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Espalhamento a Baixo Ângulo , Difração de Raios X
8.
Methods ; 157: 80-87, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30419336

RESUMO

Protein-protein interactions are essential for cellular structure and function. To delineate how the intricate assembly of protein interactions contribute to cellular processes in health and disease, new methodologies that are both highly sensitive and can be applied at large scale are needed. Here, we develop HiPLA (high-throughput imaging proximity ligation assay), a method that employs the well-established antibody-based proximity ligation assay in a high-throughput imaging screening format as a novel means to systematically visualize protein interactomes. Using HiPLA with a library of antibodies targeting nuclear proteins, we probe the interaction of 60 proteins and associated post-translational modifications (PTMs) with the nuclear lamina in a model of the premature aging disorder Hutchinson-Gilford progeria syndrome (HGPS). We identify a subset of proteins that differentially interact with the nuclear lamina in HGPS. Using HiPLA in combination with quantitative indirect immunofluorescence, we find that the majority of differential interactions are accompanied by corresponding changes in expression of the interacting protein. Taken together, HiPLA offers a novel approach to probe cellular protein-protein interaction at a large scale and reveals mechanistic insights into the assembly of protein complexes.


Assuntos
Núcleo Celular/genética , Lamina Tipo A/genética , Progéria/genética , Mapeamento de Interação de Proteínas/métodos , Núcleo Celular/patologia , Humanos , Lamina Tipo A/química , Mutação , Progéria/patologia , Precursores de Proteínas/química , Precursores de Proteínas/genética
9.
Biophys J ; 115(12): 2368-2385, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30527337

RESUMO

The cadherin superfamily of proteins is defined by the presence of extracellular cadherin (EC) "repeats" that engage in protein-protein interactions to mediate cell-cell adhesion, cell signaling, and mechanotransduction. The extracellular domains of nonclassical cadherins often have a large number of EC repeats along with other subdomains of various folds. Protocadherin-15 (PCDH15), a protein component of the inner-ear tip link filament essential for mechanotransduction, has 11 EC repeats and a membrane adjacent domain (MAD12) of atypical fold. Here we report the crystal structure of a pig PCDH15 fragment including EC10, EC11, and MAD12 in a parallel dimeric arrangement. MAD12 has a unique molecular architecture and folds as a ferredoxin-like domain similar to that found in the nucleoporin protein Nup54. Analytical ultracentrifugation experiments along with size-exclusion chromatography coupled to multiangle laser light scattering and small-angle x-ray scattering corroborate the crystallographic dimer and show that MAD12 induces parallel dimerization of PCDH15 near its membrane insertion point. In addition, steered molecular dynamics simulations suggest that MAD12 is mechanically weak and may unfold before tip-link rupture. Sequence analyses and structural modeling predict the existence of similar domains in cadherin-23, protocadherin-24, and the "giant" FAT and CELSR cadherins, indicating that some of them may also exhibit MAD-induced parallel dimerization.


Assuntos
Caderinas/química , Espaço Extracelular/metabolismo , Fenômenos Mecânicos , Multimerização Proteica , Precursores de Proteínas/química , Animais , Fenômenos Biomecânicos , Caderinas/metabolismo , Camundongos , Simulação de Dinâmica Molecular , Domínios Proteicos , Dobramento de Proteína , Precursores de Proteínas/metabolismo , Estrutura Quaternária de Proteína , Suínos
10.
Elife ; 72018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30558715

RESUMO

New strategies are urgently required to develop antibiotics. The siderophore uptake system has attracted considerable attention, but rational design of siderophore antibiotic conjugates requires knowledge of recognition by the cognate outer-membrane transporter. Acinetobacter baumannii is a serious pathogen, which utilizes (pre)acinetobactin to scavenge iron from the host. We report the structure of the (pre)acinetobactin transporter BauA bound to the siderophore, identifying the structural determinants of recognition. Detailed biophysical analysis confirms that BauA recognises preacinetobactin. We show that acinetobactin is not recognised by the protein, thus preacinetobactin is essential for iron uptake. The structure shows and NMR confirms that under physiological conditions, a molecule of acinetobactin will bind to two free coordination sites on the iron preacinetobactin complex. The ability to recognise a heterotrimeric iron-preacinetobactin-acinetobactin complex may rationalize contradictory reports in the literature. These results open new avenues for the design of novel antibiotic conjugates (trojan horse) antibiotics.


Assuntos
Acinetobacter baumannii/metabolismo , Imidazóis/metabolismo , Ferro/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Oxazóis/metabolismo , Precursores de Proteínas/metabolismo , Cristalografia por Raios X , Imidazóis/química , Espectroscopia de Ressonância Magnética , Proteínas de Membrana Transportadoras/química , Oxazóis/química , Ligação Proteica , Multimerização Proteica , Precursores de Proteínas/química , Oligoelementos/metabolismo
11.
Cell ; 175(5): 1365-1379.e25, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30445040

RESUMO

The exchange of metabolites between the mitochondrial matrix and the cytosol depends on ß-barrel channels in the outer membrane and α-helical carrier proteins in the inner membrane. The essential translocase of the inner membrane (TIM) chaperones escort these proteins through the intermembrane space, but the structural and mechanistic details remain elusive. We have used an integrated structural biology approach to reveal the functional principle of TIM chaperones. Multiple clamp-like binding sites hold the mitochondrial membrane proteins in a translocation-competent elongated form, thus mimicking characteristics of co-translational membrane insertion. The bound preprotein undergoes conformational dynamics within the chaperone binding clefts, pointing to a multitude of dynamic local binding events. Mutations in these binding sites cause cell death or growth defects associated with impairment of carrier and ß-barrel protein biogenesis. Our work reveals how a single mitochondrial "transfer-chaperone" system is able to guide α-helical and ß-barrel membrane proteins in a "nascent chain-like" conformation through a ribosome-free compartment.


Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Membranas Intracelulares/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Domínios Proteicos , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência
12.
Biochemistry ; 57(48): 6645-6648, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30430826

RESUMO

It was recently reported that human linker histone H1.0 and its chaperone prothymosin-α (ProTα) form an extremely disordered 1:1 complex with an ultrahigh affinity (equilibrium dissociation constant KD of ∼2 × 10-12 M) measured using a single-molecule Förster resonance energy transfer method. It was hypothesized that the ultrahigh affinity and extreme disorder may be required for the chaperone function of ProTα, in which it displaces the linker histone from condensed chromatin. Here, we measure the binding affinity for the ProTα-H1.0 complex using isothermal titration calorimetry and report a KD value of (4.6 ± 0.5) × 10-7 M. In addition, we show that ProTα facilitates the formation of the H1.0-nucleosome complex in vitro. The results of our study contrast with those of the previous report and provide new insights into the chaperone function of ProTα. Possible causes for the observed discrepancy in binding affinity are discussed.


Assuntos
Histonas/metabolismo , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Sequência de Aminoácidos , Calorimetria , Transferência Ressonante de Energia de Fluorescência , Histonas/química , Histonas/genética , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Ligação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Timosina/química , Timosina/genética , Timosina/metabolismo
13.
J Am Chem Soc ; 140(47): 16213-16221, 2018 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-30387998

RESUMO

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are ubiquitous natural products. Bioactive RiPPs are produced from a precursor peptide, which is modified by enzymes. Usually, a single product is encoded in a precursor peptide. However, in cyanobactins and several other RiPP pathways, a single precursor peptide encodes multiple bioactive products flanking with recognition sequences known as "cassettes". The role of multiple cassettes in one peptide is mysterious, but in general their presence is a marker of biosynthetic plasticity. Here, we show that in cyanobactin biosynthesis the presence of multiple cassettes confers distributive enzyme processing to multiple steps of the pathway, a feature we propose to be a hallmark of multicassette RiPPs. TruD heterocyclase is stochastic and distributive. Although a canonical biosynthetic route is favored with certain substrates, every conceivable biosynthetic route is accepted. Together, these factors afford greater plasticity to the biosynthetic pathway by equalizing the processing of each cassette, enabling access to chemical diversity.


Assuntos
Peptídeos Cíclicos/biossíntese , Precursores de Proteínas/metabolismo , Alquilação , Sequência de Aminoácidos , Ciclização , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Transferases Intramoleculares/química , Transferases Intramoleculares/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Domínios Proteicos , Precursores de Proteínas/química , Processamento de Proteína Pós-Traducional , Processos Estocásticos , Especificidade por Substrato
14.
J Microbiol Biotechnol ; 28(8): 1376-1383, 2018 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-30301315

RESUMO

The hepatitis B virus (HBV) envelope contains small (S), middle (M), and large (L) proteins. PreS1 of the L protein contains a receptor-binding motif crucial for HBV infection. This motif is highly conserved among 10 HBV genotypes (A-J), making it a potential target for the prevention of HBV infection. In this study, we successfully generated a neutralizing human monoclonal antibody (mAb), 1A8 (IgG1), that recognizes the receptor-binding motif of preS1 using a phage-displayed human synthetic Fab library. Analysis of the antigen-binding activity of 1A8 for different genotypes indicated that it can specifically bind to the preS1 of major HBV genotypes (A-D). Based on Bio-Layer interferometry, the affinity (KD) of 1A8 for the preS1 of genotype C was 3.55 nM. 1A8 immunoprecipitated the hepatitis B virions of genotypes C and D. In an in vitro neutralization assay using HepG2 cells overexpressing the cellular receptor sodium taurocholate cotransporting polypeptide, 1A8 effectively neutralized HBV infection with genotype D. Taken together, the results suggest that 1A8 may neutralize the four HBV genotypes. Considering that genotypes A-D are most prevalent, 1A8 may be a neutralizing human mAb with promising potential in the prevention and treatment of HBV infection.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Biblioteca de Peptídeos , Precursores de Proteínas/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Bacteriófagos/genética , Genótipo , Células HEK293 , Células Hep G2 , Anticorpos Anti-Hepatite B/imunologia , Anticorpos Anti-Hepatite B/isolamento & purificação , Antígenos de Superfície da Hepatite B/química , Antígenos de Superfície da Hepatite B/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/genética , Testes de Neutralização , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/imunologia , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo
15.
J Biol Chem ; 293(46): 17953-17970, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30262666

RESUMO

Connective tissue growth factor (CTGF; now often referred to as CCN2) is a secreted protein predominantly expressed during development, in various pathological conditions that involve enhanced fibrogenesis and tissue fibrosis, and in several cancers and is currently an emerging target in several early-phase clinical trials. Tissues containing high CCN2 activities often display smaller degradation products of full-length CCN2 (FL-CCN2). Interpretation of these observations is complicated by the fact that a uniform protein structure that defines biologically active CCN2 has not yet been resolved. Here, using DG44 CHO cells engineered to produce and secrete FL-CCN2 and cell signaling and cell physiological activity assays, we demonstrate that FL-CCN2 is itself an inactive precursor and that a proteolytic fragment comprising domains III (thrombospondin type 1 repeat) and IV (cystine knot) appears to convey all biologically relevant activities of CCN2. In congruence with these findings, purified FL-CCN2 could be cleaved and activated following incubation with matrix metalloproteinase activities. Furthermore, the C-terminal fragment of CCN2 (domains III and IV) also formed homodimers that were ∼20-fold more potent than the monomeric form in activating intracellular phosphokinase cascades. The homodimer elicited activation of fibroblast migration, stimulated assembly of focal adhesion complexes, enhanced RANKL-induced osteoclast differentiation of RAW264.7 cells, and promoted mammosphere formation of MCF-7 mammary cancer cells. In conclusion, CCN2 is synthesized and secreted as a preproprotein that is autoinhibited by its two N-terminal domains and requires proteolytic processing and homodimerization to become fully biologically active.


Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Precursores de Proteínas/metabolismo , Animais , Células CHO , Linhagem Celular Tumoral , Fator de Crescimento do Tecido Conjuntivo/química , Cricetulus , Proteína Rica em Cisteína 61/química , Proteína Rica em Cisteína 61/metabolismo , Humanos , Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Camundongos , Proteína Sobre-Expressa em Nefroblastoma/química , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Domínios Proteicos , Precursores de Proteínas/química , Proteólise , Células RAW 264.7 , Ratos , Proteínas Recombinantes de Fusão/metabolismo
16.
Int J Mol Sci ; 19(10)2018 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-30261697

RESUMO

Among multiple distinct isoforms, Nrf1D is synthesized from a de novo translation of an alternatively-spliced transcript of Nrf1 mRNA, as accompanied by a naturally-occurring deletion of its stop codon-flanking 1466 nucleotides. This molecular event leads to the generation of a reading frameshift mutation, which results in a constitutive substitution of the intact Nrf1's C-terminal 72 amino acids (aa, covering the second half of the leucine zipper motif to C-terminal Neh3L domain) by an additional extended 80-aa stretch to generate a unique variant Nrf1D. The C-terminal extra 80-aa region of Nrf1D was herein identified to be folded into a redox-sensitive transmembrane domain, enabling it to be tightly integrated within the endoplasmic reticulum (ER) membranes. Notably, the salient feature of Nrf1D enables it to be distinguishable from prototypic Nrf1, such that Nrf1D is endowed with a lesser ability than wild-type Nrf1 to mediate target gene expression. Further evidence has also been presented revealing that both mRNA and protein levels of Nrf1D, together with other isoforms similar to those of Nrf1, were detected to varying extents in hemopoietic and somatic tissues. Surprisingly, we found the existence of Nrf1D-derived isoforms in blood plasma, implying that it is a candidate secretory transcription factor, albeit its precursor acts as an integral transmembrane-bound CNC-bZIP protein that entails dynamic topologies across membranes, before being unleashed from the ER to enter the blood.


Assuntos
Células da Medula Óssea/metabolismo , Fator 1 Nuclear Respiratório/metabolismo , Estresse Oxidativo , Precursores de Proteínas/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina Básica/química , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Encéfalo/metabolismo , Células COS , Feminino , Células Hep G2 , Humanos , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fator 1 Nuclear Respiratório/sangue , Fator 1 Nuclear Respiratório/química , Fator 1 Nuclear Respiratório/genética , Domínios Proteicos , Precursores de Proteínas/química , Precursores de Proteínas/genética , Pele/metabolismo , Testículo/metabolismo
17.
Diabetes Obes Metab ; 20 Suppl 2: 28-50, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30230185

RESUMO

Insulin synthesis in pancreatic ß-cells is initiated as preproinsulin. Prevailing glucose concentrations, which oscillate pre- and postprandially, exert major dynamic variation in preproinsulin biosynthesis. Accompanying upregulated translation of the insulin precursor includes elements of the endoplasmic reticulum (ER) translocation apparatus linked to successful orientation of the signal peptide, translocation and signal peptide cleavage of preproinsulin-all of which are necessary to initiate the pathway of proper proinsulin folding. Evolutionary pressures on the primary structure of proinsulin itself have preserved the efficiency of folding ("foldability"), and remarkably, these evolutionary pressures are distinct from those protecting the ultimate biological activity of insulin. Proinsulin foldability is manifest in the ER, in which the local environment is designed to assist in the overall load of proinsulin folding and to favour its disulphide bond formation (while limiting misfolding), all of which is closely tuned to ER stress response pathways that have complex (beneficial, as well as potentially damaging) effects on pancreatic ß-cells. Proinsulin misfolding may occur as a consequence of exuberant proinsulin biosynthetic load in the ER, proinsulin coding sequence mutations, or genetic predispositions that lead to an altered ER folding environment. Proinsulin misfolding is a phenotype that is very much linked to deficient insulin production and diabetes, as is seen in a variety of contexts: rodent models bearing proinsulin-misfolding mutants, human patients with Mutant INS-gene-induced Diabetes of Youth (MIDY), animal models and human patients bearing mutations in critical ER resident proteins, and, quite possibly, in more common variety type 2 diabetes.


Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/biossíntese , Precursores de Proteínas/biossíntese , Animais , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Humanos , Insulina/química , Camundongos , Mutação/genética , Proinsulina/biossíntese , Proinsulina/química , Proinsulina/genética , Dobramento de Proteína , Precursores de Proteínas/química , Sistemas de Translocação de Proteínas/metabolismo
18.
J Phys Chem B ; 122(49): 11262-11270, 2018 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-30230839

RESUMO

The malleability of intrinsically disordered proteins (IDPs) has generated great interest in understanding how their conformations respond to crowded cellular environments. Experiments can report gross properties such as fluorescence resonance energy transfer (FRET) efficiency but cannot resolve the conformational ensembles of IDPs and their interactions with macromolecular crowders. Computation can in principle provide the latter information but in practice has been hampered by the enormous expense for realistic modeling of IDPs and crowders and for sufficient conformational sampling. Here, taking advantage of a powerful method called FMAP (fast Fourier transform-based modeling of atomistic protein-crowder interactions), we computed how the conformational ensembles of three IDPs are modified in concentrated polyethylene glycol (PEG) 6000 solutions. We represented the IDPs at the all-atom level and the PEG molecules at a coarse-grained level and calculated the experimental observable, i.e., FRET efficiency. Whereas accounting for only steric repulsion of PEG led to overestimation of crowding effects, quantitative agreement with experimental data was obtained upon including mild IDP-PEG attraction. The present work demonstrates that realistic modeling of IDPs under crowded conditions for direct comparison with experiments is now achievable.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Transferência Ressonante de Energia de Fluorescência , Integrase de HIV/química , HIV-1/química , Simulação de Dinâmica Molecular , Coativador 3 de Receptor Nuclear/química , Polietilenoglicóis/química , Conformação Proteica , Domínios Proteicos , Precursores de Proteínas/química , Timosina/análogos & derivados
19.
J Biol Inorg Chem ; 23(8): 1255-1263, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30187212

RESUMO

Prothymosin-α is a small, multifunctional intrinsically disordered protein associated with cell survival and proliferation which binds multiple Zn2+ ions and undergoes partial folding. The interaction between prothymosin-α and at least two of its protein targets is significantly enhanced in the presence of Zn2+ ions, suggesting that Zn2+ binding plays a role in the protein's function. The primary sequence of prothymosin-α is highly acidic, with almost 50% comprised of Asp and Glu, and is unusual for a Zn2+-binding protein as it lacks Cys and His residues. To gain a better understanding of the nature of the Zn2+-prothymosin-α interactions and the protein's ability to discriminate Zn2+ over other divalent cations (e.g., Ca2+, Co2+, Mg2+) we synthesized a set of three model peptides and characterized the effect of metal binding using electrospray ionization mass spectrometry (ESI MS) and circular dichroism (CD) spectroscopy. ESI MS data reveal that the native peptide model of the glutamic acid rich region binds 4 Zn2+ ions with apparent, stepwise Kd values that are, at highest, in the tens of micromolar range. A peptide model with the same amino acid composition as the native sequence, but with the residues arranged randomly, showed no evidence of structural change by CD upon introduction of Zn2+. These results suggest that the high net negative charge of the glutamic acid-rich region of prothymosin-α is not a sufficient criterion for Zn2+ to induce a structural change; rather, Zn2+ binding to prothymosin-α is sequence specific, providing important insight into the behavior of intrinsically disordered proteins.


Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Zinco/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Humanos , Proteínas Intrinsicamente Desordenadas/química , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ácido Poliglutâmico/síntese química , Ácido Poliglutâmico/química , Ácido Poliglutâmico/metabolismo , Ligação Proteica , Precursores de Proteínas/química , Espectrometria de Massas por Ionização por Electrospray , Temperatura Ambiente , Timosina/química , Timosina/metabolismo
20.
Elife ; 72018 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-30070639

RESUMO

Hearing and balance involve the transduction of mechanical stimuli into electrical signals by deflection of bundles of stereocilia linked together by protocadherin 15 (PCDH15) and cadherin 23 'tip links'. PCDH15 transduces tip link tension into opening of a mechano-electrical transduction (MET) ion channel. PCDH15 also interacts with LHFPL5, a candidate subunit of the MET channel. Here we illuminate the PCDH15-LHFPL5 structure, showing how the complex is composed of PCDH15 and LHFPL5 subunit pairs related by a 2-fold axis. The extracellular cadherin domains define a mobile tether coupled to a rigid, 2-fold symmetric 'collar' proximal to the membrane bilayer. LHFPL5 forms extensive interactions with the PCDH15 transmembrane helices and stabilizes the overall PCDH15-LHFPL5 assembly. Our studies illuminate the architecture of the PCDH15-LHFPL5 complex, localize mutations associated with deafness, and shed new light on how forces in the PCDH15 tether may be transduced into the stereocilia membrane.


Assuntos
Caderinas/química , Caderinas/metabolismo , Proteínas de Membrana/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Estereocílios/metabolismo , Sequência de Aminoácidos , Animais , Caderinas/ultraestrutura , Células HEK293 , Humanos , Imagem Tridimensional , Proteínas de Membrana/química , Proteínas de Membrana/ultraestrutura , Camundongos , Modelos Moleculares , Multimerização Proteica , Precursores de Proteínas/ultraestrutura , Células Sf9
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