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1.
Anal Chem ; 94(37): 12595-12603, 2022 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-36063091

RESUMEN

An absolute quantitative analysis of free radicals by combining the effective magnetic moment method and the quantitative electron paramagnetic resonance [qEPR] method is proposed. This combined method utilizes the advantages of both the analytical methods and compensates for their disadvantages. In the effective magnetic moment method, the magnetic moment under a constant magnetic field is measured accurately using a superconducting quantum interference device. The qEPR method compares a "primary standard sample" and "secondary standard sample". The effective magnetic moment method was used to determine the purity of the primary standard sample. The qEPR method realizes a simple purity analysis of free-radical reagents with traceability to the International System of Units (SI). The purity of the free radicals by the qEPR method for pure 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl benzoate [4HTB], 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine [TEMPOL], and di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium [DPPH] reagents was obtained with a relative expanded uncertainty of 0.7% for 4HTB to 1.5% for the DPPH. These small uncertainties were almost equal to those of the purity of the primary standard samples and were achieved by adopting in-plane positioning of the measured sample perpendicular to the cylindrical axis of the sample space of the superconducting quantum interference device. Some purity values of the free radicals for these reagents differed from those stated by the manufacturers. This combined method enables short-time quality control of pure radical reagents, instead of quality control by separation analytical methods or titrations.


Asunto(s)
Benzoatos , Fenómenos Magnéticos , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Indicadores y Reactivos
2.
Nucleic Acids Res ; 48(8): 4551-4561, 2020 05 07.
Artículo en Inglés | MEDLINE | ID: mdl-32187371

RESUMEN

Phosphorothioate modification is commonly introduced into therapeutic oligonucleotides, typically as a racemic mixture in which either of the two non-bridging phosphate oxygens is replaced by sulfur, which frequently increases affinities with proteins. Here, we used isothermal titration calorimetry and X-ray crystallography to investigate the thermodynamic and structural properties of the interaction between the primary DNA-binding domain (CUTr1) of transcription factor SATB1 and dodecamer DNAs with racemic phosphorothioate modifications at the six sites known to contact CUTr1 directly. For both the modified and unmodified DNAs, the binding reactions were enthalpy-driven at a moderate salt concentration (50 mM NaCl), while being entropy-driven at higher salt concentrations with reduced affinities. The phosphorothioate modifications lowered this susceptibility to salt, resulting in a significantly enhanced affinity at a higher salt concentration (200 mM NaCl), although only some DNA molecular species remained interacting with CUTr1. This was explained by unequal populations of the two diastereomers in the crystal structure of the complex of CUTr1 and the phosphorothioate-modified DNA. The preferred diastereomer formed more hydrogen bonds with the oxygen atoms and/or more hydrophobic contacts with the sulfur atoms than the other, revealing the origins of the enhanced affinity.


Asunto(s)
ADN/química , Proteínas de Unión a la Región de Fijación a la Matriz/química , Oligonucleótidos Fosforotioatos/química , Cristalografía por Rayos X , ADN/metabolismo , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Modelos Moleculares , Dominios Proteicos , Estereoisomerismo , Termodinámica
3.
Proc Natl Acad Sci U S A ; 116(16): 7957-7962, 2019 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-30923116

RESUMEN

Diffuse intrinsic pontine glioma (DIPG) remains an incurable childhood brain tumor for which novel therapeutic approaches are desperately needed. Previous studies have shown that the menin inhibitor MI-2 exhibits promising activity in preclinical DIPG and adult glioma models, although the mechanism underlying this activity is unknown. Here, using an integrated approach, we show that MI-2 exerts its antitumor activity in glioma largely independent of its ability to target menin. Instead, we demonstrate that MI-2 activity in glioma is mediated by disruption of cholesterol homeostasis, with suppression of cholesterol synthesis and generation of the endogenous liver X receptor ligand, 24,25-epoxycholesterol, resulting in cholesterol depletion and cell death. Notably, this mechanism is responsible for MI-2 activity in both DIPG and adult glioma cells. Metabolomic and biochemical analyses identify lanosterol synthase as the direct molecular target of MI-2, revealing this metabolic enzyme as a vulnerability in glioma and further implicating cholesterol homeostasis as an attractive pathway to target in this malignancy.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Tronco Encefálico , Glioma , Transferasas Intramoleculares/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Neoplasias del Tronco Encefálico/enzimología , Neoplasias del Tronco Encefálico/metabolismo , Colesterol/metabolismo , Glioma/enzimología , Glioma/metabolismo , Humanos , Redes y Vías Metabólicas/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo
4.
Br J Cancer ; 123(11): 1633-1643, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32921792

RESUMEN

BACKGROUND: Annexin A1 is expressed specifically on the tumour vasculature surface. Intravenously injected IF7 targets tumour vasculature via annexin A1. We tested the hypothesis that IF7 overcomes the blood-brain barrier and that the intravenously injected IF7C(RR)-SN38 eradicates brain tumours in the mouse. METHODS: (1) A dual-tumour model was generated by inoculating luciferase-expressing melanoma B16 cell line, B16-Luc, into the brain and under the skin of syngeneic C57BL/6 mice. IF7C(RR)-SN38 was injected intravenously daily at 7.0 µmoles/kg and growth of tumours was assessed by chemiluminescence using an IVIS imager. A similar dual-tumour model was generated with the C6-Luc line in immunocompromised SCID mice. (2) IF7C(RR)-SN38 formulated with 10% Solutol HS15 was injected intravenously daily at 2.5 µmoles/kg into two brain tumour mouse models: B16-Luc cells in C57BL/6 mice, and C6-Luc cells in nude mice. RESULTS: (1) Daily IF7C(RR)-SN38 injection suppressed tumour growth regardless of cell lines or mouse strains. (2) Daily injection of Solutol-formulated IF7C(RR)-SN38 led into complete disappearance of B16-Luc brain tumour in C57BL/6 mice, whereas this did not occur in C6-Luc in nude mice. CONCLUSIONS: IF7C(RR)-SN38 crosses the blood-brain barrier and suppresses growth of brain tumours in mouse models. Solutol HS15-formulated IF7C(RR)-SN38 may have promoted an antitumour immune response.


Asunto(s)
Anexina A1/metabolismo , Antineoplásicos/farmacología , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas , Portadores de Fármacos/farmacología , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Péptidos , Ratas
5.
Glycobiology ; 29(7): 576-587, 2019 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-30913288

RESUMEN

Fucosylation of the N-glycan core via the α1-6 linkage (core fucosylation) is detected in specific types of cancers and related diseases, and thereby serves for a relevant biomarker. The lectin from a mushroom Pholiota squarrosa (PhoSL) shows a clear specificity to core fucosylation, without recognizing those with other types of fucosylation, such as the H type via the α1-2 linkage or the Lewis type via the α1-3 or α1-4 linkage. Here we determined the crystal structure of the PhoSL trimer in complex with a disaccharide fucose(α1-6)N-acetylglucosamine (GlcNAc). In the three sugar-binding pockets of PhoSL, extensive hydrophobic and hydrogen-bonding contacts were formed with the fucose moiety. In contrast, the GlcNAc moiety showed only a few hydrophobic and hydrogen-bonding contacts. To elucidate the mechanism for the specificity, we performed molecular dynamics simulations on this disaccharide and a trisaccharide fucose(α1-6)[GlcNAc(ß1-4)]GlcNAc in complex with PhoSL. It was observed that the GlcNAc corresponding to the outer one of the N-glycan core entered the sugar-binding pocket with the N-acetyl group placed stably at the bottom, forming extensive hydrophobic and hydrogen-bonding interactions. In addition, these glycans adopted unstressed favorable conformations when bound to PhoSL. In contrast, H- and Lewis-types of fucosylated trisaccharides adopting favorable conformations caused inevitable steric hindrance with the steep edge of the binding pocket, when docked with PhoSL. Therefore, the specificity to core fucosylation of PhoSL was achieved by a combination of these preferential and exclusive mechanisms.


Asunto(s)
Fucosa/metabolismo , Pholiota/metabolismo , Polisacáridos/metabolismo , Conformación de Carbohidratos , Fucosa/química , Simulación de Dinámica Molecular , Polisacáridos/química
6.
Phys Chem Chem Phys ; 21(19): 9683-9693, 2019 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-30829349

RESUMEN

We theoretically and experimentally analyzed a conformational ensemble of a small, characteristic polypeptide consisting of positively- and negatively-charged amino acid residue clusters, (Lys)9(Glu)9(Lys)9, designed based on the supercoiled DNA-recognition (SDR) domain, with the capability of preferentially binding to supercoiled DNA. Advanced molecular dynamics (MD) simulations coupled with a generalized ensemble technique revealed that substantial amounts of ordered, helical structures were present at the boundaries of the Lys and Glu segments in the obtained conformational ensemble. In fact, the helical content of the peptide calculated from our MD simulations was consistent with that estimated from our experimental analysis employing circular dichroism (CD) spectroscopy. The statistical analysis of the structural ensemble revealed the metastable hydrophobic contact clusters, which were stabilized by closely cohesive residue contacts, formed through "hybrid" hydrophobic (methylene groups) and electrostatic (salt bridges) residue contacts. Both short-range and long-range residue contacts were involved in the metastable hydrophobic clusters, constituting the aforementioned local helical conformations and the compact entire structures, respectively. A significant helical propensity was also found in the (Lys)n and (Glu)m boundaries of other conventional protein structures deposited in the Protein Data Bank (PDB), thus indicating the generality of this conformational trend that has been identified herein.


Asunto(s)
Aminoácidos/química , Péptidos/química , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Conformación Proteica
7.
Biochem J ; 473(19): 3321-39, 2016 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-27462121

RESUMEN

Transcription factor SATB1 (special AT-rich sequence binding protein 1) contains multiple DNA-binding domains (DBDs), i.e. two CUT-domain repeats (CUTr1 and CUTr2 from the N-terminus) and a homeodomain, and binds to the matrix attachment region (MAR) of DNA. Although CUTr1 and the homeodomain, but not CUTr2, are known to contribute to DNA binding, different research groups have not reached a consensus on which DBD is responsible for recognition of the target sequence in MAR, 5'-TAATA-3'. Here, we used isothermal titration calorimetry to demonstrate that CUTr1 has binding specificity to this motif, whereas the homeodomain shows affinity for a variety of DNAs without specificity. In line with nonspecific DNA-binding properties of the homeodomain, a mutation of the invariant Asn at position 51 of the homeodomain (typically in contact with the A base in a sequence-specific binding mode) did not affect the binding affinity significantly. The NMR analyses and computational modeling of the homeodomain, however, revealed the tertiary structure and DNA-binding mode that are typical of homeodomains capable of sequence-specific binding. We believe that the lack of highly conserved basic residues in the helix relevant to the base recognition loosens its fitting into the DNA groove and impairs the specific binding. The two DBDs, when fused in tandem, showed strong binding to DNA containing the 5'-TAATA-3' motif with an affinity constant >10(8) M(-1) and retained nonspecific binding activity. The combination of the sequence-specific and nonspecific DNA-binding modes of SATB1 should be advantageous in a search for target loci during transcriptional regulation.


Asunto(s)
ADN/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Unión a la Región de Fijación a la Matriz/metabolismo , Secuencia de Aminoácidos , Calorimetría , Proteínas de Homeodominio/química , Humanos , Proteínas de Unión a la Región de Fijación a la Matriz/química , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Homología de Secuencia de Aminoácido
8.
J Biol Chem ; 289(6): 3394-404, 2014 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-24356963

RESUMEN

The understanding of how primordial proteins emerged has been a fundamental and longstanding issue in biology and biochemistry. For a better understanding of primordial protein evolution, we synthesized an artificial protein on the basis of an evolutionary hypothesis, segment-based elongation starting from an autonomously foldable short peptide. A 10-residue protein, chignolin, the smallest foldable polypeptide ever reported, was used as a structural support to facilitate higher structural organization and gain-of-function in the development of an artificial protein. Repetitive cycles of segment elongation and subsequent phage display selection successfully produced a 25-residue protein, termed AF.2A1, with nanomolar affinity against the Fc region of immunoglobulin G. AF.2A1 shows exquisite molecular recognition ability such that it can distinguish conformational differences of the same molecule. The structure determined by NMR measurements demonstrated that AF.2A1 forms a globular protein-like conformation with the chignolin-derived ß-hairpin and a tryptophan-mediated hydrophobic core. Using sequence analysis and a mutation study, we discovered that the structural organization and gain-of-function emerged from the vicinity of the chignolin segment, revealing that the structural support served as the core in both structural and functional development. Here, we propose an evolutionary model for primordial proteins in which a foldable segment serves as the evolving core to facilitate structural and functional evolution. This study provides insights into primordial protein evolution and also presents a novel methodology for designing small sized proteins useful for industrial and pharmaceutical applications.


Asunto(s)
Evolución Molecular , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Oligopéptidos/química , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Resonancia Magnética Nuclear Biomolecular , Oligopéptidos/genética , Estructura Secundaria de Proteína
9.
Analyst ; 140(17): 5881-4, 2015 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-26221631

RESUMEN

Imidazole was tethered to the C5 position of thymine in an ATP-binding DNA aptamer with two types of linkers, and the affinities of each aptamer for ATP and AMP were determined by surface plasmon resonance measurements. The imidazole-tethered aptamers exhibited higher affinity for ATP, almost independently of the linker structure or the modification site.


Asunto(s)
Adenosina Monofosfato/análisis , Adenosina Trifosfato/análisis , Aptámeros de Nucleótidos/química , Resonancia por Plasmón de Superficie , Secuencia de Bases , Sitios de Unión , ADN/química , Imidazoles/química , Espectrometría de Masa por Ionización de Electrospray
10.
J Phys Chem B ; 128(30): 7401-7406, 2024 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-39018377

RESUMEN

The indole ring of tryptophan can form NH/π hydrogen bonds, acting both as a hydrogen donor at the NH group in the pyrrole subring and as a hydrogen acceptor at the benzene subring. In the structural core of the trimeric stable protein Pholiota squarrosa lectin (PhoSL), three indoles are symmetrically arranged and form NH/π hydrogen bonds among each other. Here, we conducted quantum chemical calculations on this indole triad by using various methods and basis sets. The analyses revealed cooperativity in triad formation, with the many-body effect contributing approximately -2 kcal mol-1, which significantly stabilizes this protein. Symmetry-adapted perturbation theory ascribed this effect to the induced polarization. The electrostatic potential and atomic charges indeed revealed a charge redistribution through the NH/π hydrogen bond, which was favorable for triad formation.


Asunto(s)
Enlace de Hidrógeno , Triptófano , Triptófano/química , Teoría Cuántica , Estabilidad Proteica , Electricidad Estática , Conformación Proteica , Modelos Moleculares , Indoles/química
11.
J Biol Chem ; 287(10): 7683-91, 2012 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-22219184

RESUMEN

The WRKY family transcription factors regulate plant-specific reactions that are mostly related to biotic and abiotic stresses. They share the WRKY domain, which recognizes a DNA element (TTGAC(C/T)) termed the W-box, in target genes. Here, we determined the solution structure of the C-terminal WRKY domain of Arabidopsis WRKY4 in complex with the W-box DNA by NMR. A four-stranded ß-sheet enters the major groove of DNA in an atypical mode termed the ß-wedge, where the sheet is nearly perpendicular to the DNA helical axis. Residues in the conserved WRKYGQK motif contact DNA bases mainly through extensive apolar contacts with thymine methyl groups. The importance of these contacts was verified by substituting the relevant T bases with U and by surface plasmon resonance analyses of DNA binding.


Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , ADN de Plantas/química , Factores de Transcripción/química , Secuencias de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , ADN de Plantas/metabolismo , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie , Factores de Transcripción/metabolismo
12.
Anal Chem ; 85(20): 9439-43, 2013 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-24028433

RESUMEN

An NMR method was developed that allows for real-time monitoring of reactions (on the order of seconds) induced by a temperature jump. In a recycle flow system, heating and cooling baths were integrated, with the latter inside the NMR probe. A refolding reaction of ribonuclease A was triggered by rapid cooling and monitored by a series of NMR measurements over 12 s. Data were processed by principal component analysis, in which a factor related to the structural change with an exponential rate constant of 0.2-0.7 s(-1) was successfully separated from factors related to baseline instability and/or noise. Temperature dependency of the rate constant revealed the entropy-driven formation of the transition state of the refolding reaction.


Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Pliegue de Proteína , Temperatura , Animales , Bovinos , Cinética , Análisis de Componente Principal , Ribonucleasa Pancreática/química , Factores de Tiempo
13.
Chem Biol Drug Des ; 101(6): 1241-1251, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-36869438

RESUMEN

Malaria remains a threat to global public health and the available antimalarial drugs are undermined by side effects and parasite resistance, suggesting an emphasis on new potential targets. Among the novel targets, Plasmodium falciparum autophagy-related proteins (PfAtg) remain a priority. In this paper, we reviewed the existing knowledge on the functions and structural biology of PfAtg including the compounds with inhibitory activity toward P. falciparum Atg8-Atg3 protein-protein interaction (PfAtg8-PfAtg3 PPI). A total of five PfAtg (PfAtg5, PfAtg8, PfAtg12, PfAtg18, and Rab7) were observed to have autophagic and/or non-autophagic roles. Available data showed that PfAtg8 has conserved hydrophobic pockets, which allows it to interact with PfAtg3 to form PfAtg8-PfAtg3 PPI. Additionally, 2-bromo-N-(4-pyridin-2-yl-1,3-thiazol-2-yl) benzamide was identified as the most powerful inhibitor of PfAtg8-PfAtg3 PPI. Due to the dearth of knowledge in this field, we hope that the article would open an avenue to further research on the remaining PfAtg as possible drug candidates.


Asunto(s)
Antimaláricos , Malaria Falciparum , Humanos , Antimaláricos/química , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/farmacología , Plasmodium falciparum , Proteínas Protozoarias/metabolismo , Biología
14.
FEBS J ; 290(2): 412-427, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36007953

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S protein) is highly N-glycosylated, and a "glycan shield" is formed to limit the access of other molecules; however, a small open area coincides with the interface to the host's receptor and also neutralising antibodies. Most of the variants of concern have mutations in this area, which could reduce the efficacy of existing antibodies. In contrast, N-glycosylation sites are relatively invariant, and some are essential for infection. Here, we observed that the S proteins of the ancestral (Wuhan) and Omicron strains bind with Pholiota squarrosa lectin (PhoSL), a 40-amino-acid chemically synthesised peptide specific to core-fucosylated N-glycans. The affinities were at a low nanomolar level, which were ~ 1000-fold stronger than those between PhoSL and the core-fucosylated N-glycans at the micromolar level. We demonstrated that PhoSL inhibited infection by both strains at similar submicromolar levels, suggesting its broad-spectrum effect on SARS-CoV-2 variants. Cryogenic electron microscopy revealed that PhoSL caused an aggregation of the S protein, which was likely due to the multivalence of both the trimeric PhoSL and S protein. This characteristic is likely relevant to the inhibitory mechanism. Structural modelling of the PhoSL-S protein complex indicated that PhoSL was in contact with the amino acids of the S protein, which explains the enhanced affinity with S protein and also indicates the significant potential for developing specific binders by the engineering of PhoSL.


Asunto(s)
Antivirales , Lectinas , SARS-CoV-2 , Humanos , COVID-19 , Fucosa/química , Lectinas/farmacología , Polisacáridos/química , SARS-CoV-2/efectos de los fármacos , Antivirales/farmacología , Pholiota/química
15.
FEBS J ; 289(18): 5527-5530, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-35587530

RESUMEN

Old yellow enzymes (OYEs) are flavoproteins that catalyze stereoselective reduction of a wide variety of small molecules including xenobiotic toxins, and are considered as synthetic tools in industrial and pharmaceutical applications. Despite their broad specificity, differences in the enzyme structures influence the yield and stereochemistry of the products. Singh et al. present the three-dimensional structure and biochemical properties of an OYE of a necrotrophic fungus, Ascochyta rabiei, which belongs to a recently identified fungi-specific class. Observations of distinct structural features and arrangements of the catalytic-site residues should contribute to understanding the catalytic mechanism of OYEs of this class. Comment on: https://doi.org/10.1111/febs.16445.


Asunto(s)
NADPH Deshidrogenasa , Xenobióticos , Secuencia de Aminoácidos , Dominio Catalítico , Flavoproteínas/química , NADPH Deshidrogenasa/química
16.
J Biochem ; 171(6): 619-629, 2022 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-35191956

RESUMEN

Glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) of Trypanosoma brucei, the causative protozoan parasite of African trypanosomiasis, is a membrane-bound enzyme essential for antigenic variation, because it catalyses the release of the membrane-bound form of variable surface glycoproteins. Here, we performed a fragment-based drug discovery of TbGPI-PLC inhibitors using a combination of enzymatic inhibition assay and water ligand observed via gradient spectroscopy (WaterLOGSY) NMR experiment. The TbGPI-PLC was cloned and overexpressed using an Escherichia coli expression system followed by purification using three-phase partitioning and gel filtration. Subsequently, the inhibitory activity of 873 fragment compounds against the recombinant TbGPI-PLC led to the identification of 66 primary hits. These primary hits were subjected to the WaterLOGSY NMR experiment where 10 fragment hits were confirmed to directly bind to the TbGPI-PLC. These included benzothiazole, chlorobenzene, imidazole, indole, pyrazol and quinolinone derivatives. Molecular docking simulation indicated that six of them share a common binding site, which corresponds to the catalytic pocket. The present study identified chemically diverse fragment hits that could directly bind and inhibit the TbGPI-PLC activity, which constructed a framework for fragment optimization or linking towards the design of novel drugs for African trypanosomiasis.


Asunto(s)
Trypanosoma brucei brucei , Tripanosomiasis Africana , Animales , Descubrimiento de Drogas , Glicosilfosfatidilinositol Diacilglicerol-Liasa/metabolismo , Ligandos , Simulación del Acoplamiento Molecular , Análisis Espectral , Trypanosoma brucei brucei/metabolismo , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitología , Fosfolipasas de Tipo C/metabolismo , Agua
17.
J Biochem ; 170(3): 363-368, 2021 Oct 12.
Artículo en Inglés | MEDLINE | ID: mdl-33831188

RESUMEN

NMR spectroscopy permits real-time monitoring of reactions that involve changes in the spectra of reactants. MICCS (MIcro Channelled Cell for Synthesis monitoring) is a microfluidic chip for such purposes, which is used to rapidly activate reactions by mixing the reactant solutions in the chip inserted into the typical NMR tube. Although it allows monitoring of chemical reactions of small compounds, its simple mixing system dependent on diffusion in the microchannel was not suitable for macromolecules such as proteins with low diffusion rates. Here, we developed a new microfluidic chip based on MICCS by incorporating a mixer of split-and-recombination type within the microchannel. We applied it to monitoring of the protein-folding reaction in a stopped-flow mode. A solution of denaturant-unfolded RNase A was injected from a syringe pump into the microchip set inside the NMR magnet and mixed with a buffer for dilution to reach the folding condition. Immediately after dilution, the reaction was initiated and detected by a series of NMR measurements that were synchronized with activation and inactivation of the pump. The process was repeated for accumulation of the data. By analysing the change of the spectra by factor analysis, a kinetic constant of 0.57 min-1 was obtained.


Asunto(s)
Sustancias Macromoleculares/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Microfluídica/métodos , Cinética , Sustancias Macromoleculares/química , Pliegue de Proteína , Proteínas/metabolismo
18.
PLoS One ; 16(1): e0241157, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33406123

RESUMEN

We previously reported that IF7 peptide, which binds to the annexin A1 (ANXA1) N-terminus, functions as a tumor vasculature-targeted drug delivery vehicle after intravenous injection. To enhance IF7 stability in vivo, we undertook mirror-image peptide phage display using a synthetic D-peptide representing the ANXA1 N-terminus as target. We then identified peptide sequences, synthesized them as D-amino acids, and designated the resulting peptide dTIT7, which we showed bound to the ANXA1 N-terminus. Whole body imaging of mouse brain tumor models injected with near infrared fluorescent IRDye-conjugated dTIT7 showed fluorescent signals in brain and kidney. Furthermore, orally-administered dTIT7/geldanamycin (GA) conjugates suppressed brain tumor growth. Ours is a proof-of-concept experiment showing that ANXA1-binding D-peptide can be developed as an orally-administrable tumor vasculature-targeted therapeutic.


Asunto(s)
Anexina A1/antagonistas & inhibidores , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/tratamiento farmacológico , Sistemas de Liberación de Medicamentos , Proteínas de Neoplasias/antagonistas & inhibidores , Neovascularización Patológica/tratamiento farmacológico , Péptidos , Administración Oral , Animales , Anexina A1/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Neoplasias/metabolismo , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Péptidos/síntesis química , Péptidos/química , Péptidos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto
19.
J Am Chem Soc ; 132(16): 5548-9, 2010 Apr 28.
Artículo en Inglés | MEDLINE | ID: mdl-20355726

RESUMEN

The hinge region of human immunoglobulin A1 (IgA1), connecting the Fab and Fc regions, is mostly composed of Ser, Thr, and Pro (VPSTPPTPSPSTPPTPSPS); hinge peptide (HP). O-Glycans are naturally attached on only particular five Ser/Thr residues in this region. NMR was employed for analysis of the structural changes in HP upon the glycosylation, especially focusing on the cis/trans isomerization of Pro residues. A series of HP containing (13)C,(15)N-labeled Pro residues were chemically synthesized and enzymatically glycosylated. The signals from cis and trans forms of the labeled Pro were identified by two-dimensional NMR spectroscopy. Cis/trans ratios of the Pro residues at the C-terminal side of the glycosylated Ser/Thr were reduced from 9-10% to 2-3% by the glycosylation. Thermodynamic analyses indicated that the decrease in the cis/trans ratio was enthalpy-driven. Hydrogen-deuterium exchange experiments and NOE-based structure determination revealed that the intraresidue hydrogen bonds between the amide group of GalNAc and carbonyl oxygen of the peptide backbone of GalNAc-Thr are formed in the major trans conformers, which is consistent with the thermodynamic parameters. These hydrogen bonds largely restrict the psi angle of the peptide backbone and, thereby, should make the trans conformation of the C-terminal Pro residue more stable than the cis conformation. Namely, it is predicted that the restricted psi angle causes interresidue steric hindrance for the cis conformation. The appropriate glycosylation of HP probably contributes to the decrease in the unfavorable variety of relative orientations between Fab and Fc in IgA1, through stabilizing the conformation of HP.


Asunto(s)
Inmunoglobulina A/química , Fragmentos de Péptidos/química , Prolina/química , Secuencia de Aminoácidos , Glicosilación , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estereoisomerismo , Termodinámica
20.
Biosci Rep ; 40(4)2020 04 30.
Artículo en Inglés | MEDLINE | ID: mdl-32266936

RESUMEN

Hydroxyoctadecadienoic acids (HODEs) are produced by oxidation and reduction of linoleates. There are several regio- and stereo-isomers of HODE, and their concentrations in vivo are higher than those of other lipids. Although conformational isomers may have different biological activities, comparative analysis of intracellular function of HODE isomers has not yet been performed. We evaluated the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ), a therapeutic target for diabetes, and analyzed PPARγ agonist activity of HODE isomers. The lowest scores for docking poses of 12 types of HODE isomers (9-, 10-, 12-, and 13-HODEs) were almost similar in docking simulation of HODEs into PPARγ ligand-binding domain (LBD). Direct binding of HODE isomers to PPARγ LBD was determined by water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR experiments. In contrast, there were differences in PPARγ agonist activities among 9- and 13-HODE stereo-isomers and 12- and 13-HODE enantio-isomers in a dual-luciferase reporter assay. Interestingly, the activity of 9-HODEs was less than that of other regio-isomers, and 9-(E,E)-HODE tended to decrease PPARγ-target gene expression during the maturation of 3T3-L1 cells. In addition, 10- and 12-(Z,E)-HODEs, which we previously proposed as biomarkers for early-stage diabetes, exerted PPARγ agonist activity. These results indicate that all HODE isomers have PPARγ-binding affinity; however, they have different PPARγ agonist activity. Our findings may help to understand the biological function of lipid peroxidation products.


Asunto(s)
Ácidos Linoleicos/farmacología , PPAR gamma/agonistas , Células 3T3-L1 , Animales , Ácidos Linoleicos/química , Peroxidación de Lípido , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , PPAR gamma/química , PPAR gamma/metabolismo , Estereoisomerismo , Relación Estructura-Actividad
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