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1.
J Mater Chem B ; 12(12): 3006-3014, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38451210

RESUMO

Inorganic biomaterials are used in various orthopedic and dental implants. Nevertheless, they cause clinical issues such as loosening of implants and patient morbidity. Therefore, inspired by mussel adhesive proteins, we aimed to design an adhesive and dimer-forming highly active bone morphogenetic protein-2 (BMP-2) using bioorthogonal chemistry, in which recombinant DNA technology was combined with enzymatic modifications, to achieve long-term osseointegration with titanium. The prepared BMP-2 exhibited substantially higher binding activity than wild-type BMP-2, while the adhered BMP-2 was more active than soluble BMP-2. Therefore, the adhesive BMP-2 was immobilized onto titanium wires and screws and implanted into rat bones, and long-term osteogenesis was evaluated. Adhesive BMP-2 promoted the mechanical binding of titanium to bones, enabling efficient bone regeneration and effective stabilization of implants. Thus, such adhesive biosignaling proteins can be used in regenerative medicine.


Assuntos
Regeneração Óssea , Titânio , Ratos , Animais , Humanos , Titânio/farmacologia , Próteses e Implantes , Osteogênese , Osseointegração
2.
Int J Biol Macromol ; 264(Pt 2): 130568, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38447822

RESUMO

Polysaccharide based self-healing and injectable hydrogels with reversible characteristics have widespread potential in protein drug delivery. However, it is a challenge to design the dynamic hydrogel for sequential release of protein drugs. Herein, we developed a novel mussel inspired sequential protein delivery dynamic polysaccharide hydrogel. The nanocomposite hydrogel can be fabricated through doping polydopamine nanoparticles (PDA NPs) into reversible covalent bond (imine bonds) crosslinked polymer networks of oxidized hyaluronic acid (OHA) and carboxymethyl chitosan (CEC), named PDA NPs@OHA-l-CEC. Besides multiple capabilities (i.e., injection, self-healing, and biodegradability), the nanocomposite hydrogel can achieve sustained and sequential protein delivery of vascular endothelial growth factor (VEGF) and bovine serum albumin (BSA). PDA NPs doped in hydrogel matrix serve dual roles, acting as secondary protein release structures and form dynamic non-covalent interactions (i.e., hydrogen bonds) with polysaccharides. Moreover, by adjusting the oxidation degree of OHA, the hydrogels with different crosslinking density could control overall protein release rate. Analysis of different release kinetic models revealed that Fickian diffusion drove rapid VEGF release, while the slower BSA release followed a Super Case II transport mechanism. The novel biocompatible system achieved sequential release of protein drugs has potentials in multi-stage synergistic drug deliver based on dynamic hydrogel.


Assuntos
Quitosana , Fator A de Crescimento do Endotélio Vascular , Nanogéis , Fator A de Crescimento do Endotélio Vascular/química , Sistemas de Liberação de Medicamentos , Hidrogéis/química , Quitosana/química , Polissacarídeos/química , Ácido Hialurônico/química , Soroalbumina Bovina
3.
Carbohydr Polym ; 330: 121812, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38368083

RESUMO

Biomacromolecules based injectable and self-healing hydrogels possessing high mechanical properties have widespread potential in biomedical field. However, dynamic features are usually inversely proportional to toughness. It is challenging to simultaneously endow these properties to the dynamic hydrogels. Here, we fabricated an injectable nanocomposite hydrogel (CS-NPs@OSA-l-Gtn) stimultaneously possessing excellent autonomous self-healing performance and high mechanical strength by doping chitosan nanoparticles (CS-NPs) into dynamic polymer networks of oxidized sodium alginate (OSA) and gelatin (Gtn) in the presence of borax. The synergistic effect of the multiple reversible interactions combining dynamic covalent bonds (i.e., imine bond and borate ester bond) and noncovalent interactions (i.e., electrostatic interaction and hydrogen bond) provide effective energy dissipation to endure high fatigue resistance and cyclic loading. The dynamic hydrogel exhibited excellent mechanical properties like maximum 2.43 MPa compressive strength, 493.91 % fracture strain, and 89.54 kJ/m3 toughness. Moreover, the integrated hydrogel after injection and self-healing could withstand 150 successive compressive cycles. Besides, the bovine serum albumin embedded in CS-NPs could be sustainably released from the nanocomposite hydrogel for 12 days. This study proposes a novel strategy to synthesize an injectable and self-healing hydrogel combined with excellent mechanical properties for designing high-strength natural carriers with sustained protein delivery.


Assuntos
Alginatos , Quitosana , Alginatos/química , Nanogéis , Gelatina/química , Hidrogéis/química , Polímeros , Quitosana/química
4.
Anal Chem ; 95(19): 7503-7511, 2023 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-37130068

RESUMO

Accurate discrimination and classification of unknown species are the basis to predict its characteristics or applications to make correct decisions. However, for biogenic solutions that are ubiquitous in nature and our daily lives, direct determination of their similarities and disparities by their molecular compositions remains a scientific challenge. Here, we explore a standard and visualizable ontology, termed "biogenic solution map", that organizes multifarious classes of biogenic solutions into a map of hierarchical structures. To build the map, a novel 4-dimensional (4D) fingerprinting method based on data-independent acquisition data sets of untargeted metabolomics is developed, enabling accurate characterization of complex biogenic solutions. A generic parameter of metabolic correlation distance, calculated based on averaged similarities between 4D fingerprints of sample groups, is able to define "species", "genus", and "family" of each solution in the map. With the help of the "biogenic solution map", species of unknown biogenic solutions can be explicitly defined. Simultaneously, intrinsic correlations and subtle variations among biogenic solutions in the map are accurately illustrated. Moreover, it is worth mentioning that samples of the same analyte but prepared by alternative protocols may have significantly different metabolic compositions and could be classified into different "genera".


Assuntos
Metabolômica , Metabolômica/métodos
5.
Front Bioeng Biotechnol ; 11: 1169124, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37251573

RESUMO

The proper microenvironment is critical for the storage and transportation of embryonic stem cells (ESCs). To mimic a dynamic 3D microenvironment as it exists in vivo and consider "off-the-shelf" availability reaching the destination, we proposed an alternative approach that allows for facile storage and transportation of stem cells in the form of ESCs-dynamic hydrogel construct (CDHC) under ambient conditions. To form CDHC, mouse embryonic stem cells (mESCs) were in-situ encapsulated within a polysaccharide-based dynamic and self-biodegradable hydrogel. After storing CDHC in a sterile and hermetic environment for 3 days and then transferring to a sealed vessel with fresh medium for another 3 days, the large and compact colonies retained a 90% survival rate and pluripotency. Furthermore, after transporting and arriving at the destination, the encapsulated stem cell could be automatically released from the self-biodegradable hydrogel. After continuous cultivation of 15 generations of retrieved cells, automatically released from the CDHC, the mESCs underwent 3D encapsulation, storage, transportation, release, and continuous long-term subculture; resumed colony forming capacity and pluripotency were revealed by stem cell markers both in protein and mRNA levels. We believe that the dynamic and self-biodegradable hydrogel provides a simple, cost-effective, and valuable tool for storing and transporting "ready-to-use" CDHC under ambient conditions, facilitating "off-the-shelf" availability and widespread applications.

6.
ACS Omega ; 7(27): 23479-23486, 2022 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-35847293

RESUMO

In cancer, the mechanistic/mammalian target of rapamycin complex-1 (mTORC1) is hyperactivated to promote survival under adverse conditions. The kinase activity of mTORC1 is activated by small-GTPase RHEB-GTP. Therefore, a new modality to inhibit mTORC1 activity has emerged, through intercepting RHEB. However, due to the relatively large contact area involved in the interaction between RHEB and mTORC1, facilitating this inhibition through small molecules has been challenging. Here, we report the development of a peptide that can inhibit the RHEB-mTORC1 interaction. The peptide, P1_WT, was designed based on the α-helix (aa 101-115) of the N-heat domain of mTOR to interact with switch II of RHEB. P1_WT bound to RHEB (K D = 0.14 µM) and inhibited RHEB-mTORN-heat interaction (IC50 = 0.33 µM) in vitro. Consequently, P1_WT inhibited mTORC1 activity at a sub-micromolar level (IC50 ∼ 0.3 µM). P1_WT was predicted to be cell-permeable due to the rich content of arginine (23%), enhancing the intracellular translocation. These results show that P1_WT is a potential compound to further develop inhibitors for mTORC1 by intercepting RHEB from mTORC1.

7.
J Med Chem ; 65(2): 1329-1341, 2022 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-34191518

RESUMO

Cancer-specific metabolic alterations hyperactivate the kinase activity of the mammalian/mechanistic target of rapamycin (mTOR) for overcoming stressful environments. Rapalogs, which allosterically inhibit mTOR complex 1 (mTORC1), have been approved as anticancer agents. However, the immunosuppressive side effect of these compounds results in the promotion of tumor metastasis, thereby limiting their therapeutic efficacy. We first report a nonrapalog inhibitor, WRX606, identified by a hybrid strategy of in silico and in cell selections. Our studies showed that WRX606 formed a ternary complex with FK506-binding protein-12 (FKBP12) and FKBP-rapamycin-binding (FRB) domain of mTOR, resulting in the allosteric inhibition of mTORC1. WRX606 inhibited the phosphorylation of not only the ribosomal protein S6 kinase 1 (S6K1) but also eIF4E-binding protein-1 (4E-BP1). Hence, WRX606 efficiently suppressed tumor growth in mice without promotion of metastasis. These results suggest that WRX606 is a potent lead compound for developing anticancer drugs discovered by in silico and in cell methods.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias da Mama/tratamento farmacológico , Proteínas de Ciclo Celular/metabolismo , Simulação por Computador , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Regulação Alostérica , Animais , Apoptose , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular/genética , Proliferação de Células , Feminino , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Pharmacol Ther ; 232: 108012, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34624427

RESUMO

The mammalian/mechanistic target of rapamycin (mTOR) is a regulatory protein kinase involved in cell growth and proliferation. mTOR is usually assembled in two different complexes with different regulatory mechanisms, mTOR complex 1 (mTORC1) and mTORC2, which are involved in different functions such as cell proliferation and cytoskeleton assembly, respectively. In cancer cells, mTOR is hyperactivated in response to metabolic alterations and/or oncogenic signals to overcome the stressful microenvironments. Therefore, recent research progress for mTOR inhibition involves a variety of compounds that have been developed to disturb the metabolic processes of cancer cells through mTOR inhibition. In addition to competitive or allosteric inhibition, a new inhibition strategy that emerged mTOR complexes destabilization has recently been a concern. Here, we review the history of mTOR and its inhibition, along with the timeline of the mTOR inhibitors. We also introduce prospective drug targets to inhibit mTOR by disrupting the complexation of the components with peptides and small molecules.


Assuntos
Antineoplásicos , Sirolimo , Animais , Antineoplásicos/farmacologia , Proliferação de Células , Humanos , Mamíferos/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo
9.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445471

RESUMO

The mammalian/mechanistic target of rapamycin complex 1 (mTORC1) is activated by the small G-protein, Ras homolog enriched in brain (RHEB-GTPase). On lysosome, RHEB activates mTORC1 by binding the domains of N-heat, M-heat, and the focal adhesion targeting (FAT) domain, which allosterically regulates ATP binding in the active site for further phosphorylation. The crucial role of RHEB in regulating growth and survival through mTORC1 makes it a targetable site for anti-cancer therapeutics. However, the binding kinetics of RHEB to mTORC1 is still unknown at the molecular level. Therefore, we studied the kinetics by in vitro and in-cell protein-protein interaction (PPI) assays. To this end, we used the split-luciferase system (NanoBiT®) for in-cell studies and prepared proteins for the in vitro measurements. Consequently, we demonstrated that RHEB binds to the whole mTOR both in the presence or absence of GTPγS, with five-fold weaker affinity in the presence of GTPγS. In addition, RHEB bound to the truncated mTOR fragments of N-heat domain (∆N, aa 60-167) or M-heat domain (∆M, aa 967-1023) with the same affinity in the absence of GTP. The reconstructed binding site of RHEB, ∆N-FAT-M, however, bound to RHEB with the same affinity as ∆N-M, indicating that the FAT domain (∆FAT, aa 1240-1360) is dispensable for RHEB binding. Furthermore, RHEB bound to the truncated kinase domain (∆ATP, aa 2148-2300) with higher affinity than to ∆N-FAT-M. In conclusion, RHEB engages two different binding sites of mTOR, ∆N-FAT-M and ∆ATP, with higher affinity for ∆ATP, which likely regulates the kinase activity of mTOR through multiple different biding modes.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteína Enriquecida em Homólogo de Ras do Encéfalo/metabolismo , Sítios de Ligação , Células HEK293 , Humanos , Técnicas In Vitro , Cinética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Fosforilação , Proteína Enriquecida em Homólogo de Ras do Encéfalo/genética
10.
ACS Chem Biol ; 16(2): 316-323, 2021 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-33448787

RESUMO

Inhibiting the programmed death-1 (PD-1)/programmed death ligand 1 (PD-L1) axis by monoclonal antibodies (mAbs) is a successful cancer immunotherapy. However, mAb-based drugs have various disadvantages including high production costs and large molecular sizes, which motivated us to develop a smaller alternative drug. Since PD-L1 binds PD-1 with moderate affinity, a higher affinity PD-1 variant should serve as a competitive inhibitor of the wild-type PD-1/PD-L1 interaction. In this report, we conducted in silico point mutagenesis of PD-1 to identify potent PD-1 variants with a higher affinity toward PD-L1 and refined the in silico results using a luciferase-based in-cell protein-protein interaction (PPI) assay. As a result, a PD-1 variant was developed that had two mutated amino acids (T76Y, A132V), termed 2-PD-1. 2-PD-1 could bind with PD-L1 at a dissociation constant of 12.74 nM. Moreover, 2-PD-1 successfully inhibited the PD-1/PD-L1 interaction with a half maximal inhibitory concentration of 19.15 nM and reactivated the T cell with a half maximal effective concentration of 136.1 nM. These results show that in silico mutagenesis combined with an in-cell PPI assay verification strategy successfully prepared a non-IgG inhibitor of the PD-1/PD-L1 interaction.


Assuntos
Antígeno B7-H1/metabolismo , Proteínas de Checkpoint Imunológico/farmacologia , Receptor de Morte Celular Programada 1/metabolismo , Ligação Proteica/efeitos dos fármacos , Simulação por Computador , Células HeLa , Humanos , Proteínas de Checkpoint Imunológico/genética , Proteínas de Checkpoint Imunológico/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Mutagênese , Mutação Puntual , Receptor de Morte Celular Programada 1/genética , Engenharia de Proteínas , Linfócitos T/efeitos dos fármacos
11.
J Mater Chem B ; 8(44): 10162-10171, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33095222

RESUMO

Monoclonal antibodies have been developed as anticancer agents to block immune checkpoint pathways associated with programmed cell death 1 (PD-1) and its ligand PD-L1. However, the high cost of antibodies has encouraged researchers to develop other inhibitor types. Here, biphenyl compounds were conjugated with poly(ethylene glycol) (PEG) to enhance the activity of small molecular inhibitors. Immunoassay results revealed the decrease in the inhibition activity following conjugation with linear PEG, suggesting that the PEG moiety reduced the interaction between the biphenyl structure and PD-L1. However, the inhibitory effect on PD-1/PD-L1 interaction was further enhanced by using branched PEG conjugates. The increase in the number of conjugated biphenyl compounds resulted in increased inhibitory activity. The highest IC50 value was 0.33 µM, which was about 5 times higher than that observed for a non-conjugated monovalent compound. The inhibitory activity was more than 20 times the activity reported for the starting compound. Considering the increase in the inhibition activity, this multivalent strategy can be useful in the design of new immune checkpoint inhibitors.


Assuntos
Antígeno B7-H1/metabolismo , Compostos de Bifenilo/metabolismo , Inibidores de Checkpoint Imunológico/metabolismo , Polietilenoglicóis/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Antígeno B7-H1/antagonistas & inibidores , Compostos de Bifenilo/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Células Jurkat , Simulação de Acoplamento Molecular/métodos , Polietilenoglicóis/farmacologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores
12.
Int J Mol Sci ; 21(10)2020 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-32455628

RESUMO

Cancer immunotherapy has been revolutionized by the development of monoclonal antibodies (mAbs) that inhibit interactions between immune checkpoint molecules, such as programmed cell-death 1 (PD-1), and its ligand PD-L1. However, mAb-based drugs have some drawbacks, including poor tumor penetration and high production costs, which could potentially be overcome by small molecule drugs. BMS-8, one of the potent small molecule drugs, induces homodimerization of PD-L1, thereby inhibiting its binding to PD-1. Our assay system revealed that BMS-8 inhibited the PD-1/PD-L1 interaction with IC50 of 7.2 µM. To improve the IC50 value, we designed and synthesized a small molecule based on the molecular structure of BMS-8 by in silico simulation. As a result, we successfully prepared a biphenyl-conjugated bromotyrosine (X) with IC50 of 1.5 µM, which was about five times improved from BMS-8. We further prepared amino acid conjugates of X (amino-X), to elucidate a correlation between the docking modes of the amino-Xs and IC50 values. The results suggested that the displacement of amino-Xs from the BMS-8 in the pocket of PD-L1 homodimer correlated with IC50 values. This observation provides us a further insight how to derivatize X for better inhibitory effect.


Assuntos
Antígeno B7-H1/química , Compostos de Bifenilo/química , Inibidores de Checkpoint Imunológico/síntese química , Receptor de Morte Celular Programada 1/química , Tirosina/análogos & derivados , Antígeno B7-H1/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/química , Inibidores de Checkpoint Imunológico/farmacologia , Simulação de Acoplamento Molecular , Receptor de Morte Celular Programada 1/metabolismo , Ligação Proteica , Relação Quantitativa Estrutura-Atividade , Tirosina/química
13.
Int J Mol Sci ; 20(9)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052315

RESUMO

(1) Background: The folate receptor (FR) is a target for cancer treatment and detection. Expression of the FR is restricted in normal cells but overexpressed in many types of tumors. Folate was conjugated with peptides for enhancing binding affinity to the FR. (2) Materials and Methods: For conjugation, folate was coupled with propargyl or dibenzocyclooctyne, and 4-azidophenylalanine was introduced in peptides for "click" reactions. We measured binding kinetics including the rate constants of association (ka) and dissociation (kd) of folate-peptide conjugates with purified FR by biolayer interferometry. After optimization of the conditions for the click reaction, we successfully conjugated folate with designed peptides. (3) Results: The binding affinity, indicated by the equilibrium dissociation constant (KD), of folate toward the FR was enhanced by peptide conjugation. The enhanced FR binding affinity by peptide conjugation is a result of an increase in the number of interaction sites. (4) Conclusion: Such peptide-ligand conjugates will be important in the design of ligands with higher affinity. These high affinity ligands can be useful for targeted drug delivery system.


Assuntos
Receptores de Folato com Âncoras de GPI/metabolismo , Ácido Fólico/análogos & derivados , Alcinos/química , Azidas/química , Química Click/métodos , Ciclo-Octanos/química , Receptores de Folato com Âncoras de GPI/química , Ácido Fólico/metabolismo , Simulação de Acoplamento Molecular , Peptídeos/química , Fenilalanina/análogos & derivados , Fenilalanina/química , Propanóis/química , Ligação Proteica
14.
Biochem Biophys Res Commun ; 513(4): 952-957, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31010685

RESUMO

Phytoplasmas are plant pathogenic bacteria that often induce unique phyllody symptoms in which the floral organs are transformed into leaf-like structures. Recently, a novel family of bacterial effector genes, called phyllody-inducing genes (phyllogens), was identified as being involved in the induction of phyllody by degrading floral MADS-domain transcription factors (MTFs). However, the structural characteristics of phyllogens are unknown. In this study, we elucidated the crystal structure of PHYL1OY, a phyllogen of 'Candidatus Phytoplasma asteris' onion yellows strain, at a resolution of 2.4 Å. The structure of PHYL1 consisted of two α-helices connected by a random loop in a coiled-coil manner. In both α-helices, the distributions of hydrophobic residues were conserved among phyllogens. Amino acid insertion mutations into either α-helix resulted in the loss of phyllody-inducing activity and the ability of the phyllogen to degrade floral MTF. In contrast, the same insertion in the loop region did not affect either activity, indicating that both conserved α-helices are important for the function of phyllogens. This is the first report on the crystal structure of an effector protein of phytoplasmas.


Assuntos
Proteínas de Bactérias/química , Phytoplasma/química , Cristalografia por Raios X , Estrutura Molecular , Doenças das Plantas/microbiologia , Conformação Proteica em alfa-Hélice
15.
Anal Chem ; 90(19): 11179-11182, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30175583

RESUMO

A new type of turn-on electrochemical protein detection is developed using an electropolymerizable molecular probe. To detect trypsin, a benzamidine ligand is conjugated with a thiophene moiety. Encapsulation of the probe in the trypsin pocket prevents electropolymerization, leading to efficient electron transfer from the electrolyte to the electrode. In contrast, unbound probes can become electropolymerized, yielding a polythiophene layer on the electrode. The polythiophene formed this way suppressed electron transfer. The detection limit of trypsin using this electrochemical strategy is 50 nM. The method is shown to be useful for nonenzymatic turn-on electrochemical detection.


Assuntos
Sondas Moleculares/química , Polímeros/química , Tiofenos/química , Tripsina/análise , Eletroquímica , Eletrodos , Ligantes , Polimerização , Tripsina/química
16.
Protein Expr Purif ; 149: 17-22, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29654824

RESUMO

Human folate receptors (hFRα and hFRß) are membrane proteins anchored to the cell surface by glycosylphosphatidylinositol. They play an important role in cell growth by taking up folate for de novo synthesis of purines and methylation of DNA, lipids, and proteins. Thus, controlling folate uptake through hFRs may lead to the development of anti-cancer drugs. Development of hFRs-targeting drug requires a large amount of hFRs. However, it is difficult to prepare active forms of hFRs from prokaryotic cells because of their high content of cysteine residues that form disulfide bonds. Here, we prepared active forms of hFRα and hFRß from inclusion bodies of Escherichia coli. The crucial steps in our preparation were intensive washing of the inclusion bodies to remove impurities derived from E. coli and gradual dropping of solubilized hFRs into refolding buffers to correctly reform disulfide bonds. The binding activity of prepared hFRs to folate was confirmed by biolayer interferometry measurements. Finally, we successfully prepared the active form of 2.52 mg hFRα and 2.4 mg hFRß from 10 g of E. coli cell bodies.


Assuntos
Receptor 1 de Folato/biossíntese , Receptor 2 de Folato/biossíntese , Dobramento de Proteína , Escherichia coli , Receptor 1 de Folato/genética , Receptor 2 de Folato/genética , Expressão Gênica , Humanos , Corpos de Inclusão/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
17.
Proc Natl Acad Sci U S A ; 114(18): 4661-4666, 2017 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-28416699

RESUMO

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is an IP3-gated ion channel that releases calcium ions (Ca2+) from the endoplasmic reticulum. The IP3-binding sites in the large cytosolic domain are distant from the Ca2+ conducting pore, and the allosteric mechanism of how IP3 opens the Ca2+ channel remains elusive. Here, we identify a long-range gating mechanism uncovered by channel mutagenesis and X-ray crystallography of the large cytosolic domain of mouse type 1 IP3R in the absence and presence of IP3 Analyses of two distinct space group crystals uncovered an IP3-dependent global translocation of the curvature α-helical domain interfacing with the cytosolic and channel domains. Mutagenesis of the IP3R channel revealed an essential role of a leaflet structure in the α-helical domain. These results suggest that the curvature α-helical domain relays IP3-controlled global conformational dynamics to the channel through the leaflet, conferring long-range allosteric coupling from IP3 binding to the Ca2+ channel.


Assuntos
Receptores de Inositol 1,4,5-Trifosfato/química , Inositol 1,4,5-Trifosfato/química , Ativação do Canal Iônico , Regulação Alostérica , Animais , Cristalografia por Raios X , Humanos , Inositol 1,4,5-Trifosfato/genética , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Domínios Proteicos , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
18.
FASEB J ; 31(4): 1301-1322, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27492925

RESUMO

We identified a novel, nontoxic mushroom protein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM- and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K-Ras. The use of nakanori as a lipid-domain-specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.-Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C.-G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., Kobayashi, T. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.


Assuntos
Colesterol/metabolismo , Proteínas Fúngicas/farmacologia , Grifola/química , Microdomínios da Membrana/efeitos dos fármacos , Doença de Niemann-Pick Tipo C/metabolismo , Esfingomielinas/metabolismo , Sítios de Ligação , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Células HeLa , Humanos , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/virologia , Ligação Proteica , Liberação de Vírus
19.
Angew Chem Int Ed Engl ; 55(38): 11447-51, 2016 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-27383212

RESUMO

The generation of metal surfaces with biological properties, such as cell-growth-enhancing and differentiation-inducing abilities, could be potentially exciting for the development of functional materials for use in humans, including artificial dental implants and joint replacements. However, currently the immobilization of proteins on the surfaces of the metals are limited. In this study, we have used a mussel-inspired bioorthogonal approach to design a 3,4-hydroxyphenalyalanine-containing recombinant insulin-like growth-factor-1 using a combination of recombinant DNA technology and tyrosinase treatment for the surface modification of titanium. The modified growth factor prepared in this study exhibited strong binding affinity to titanium, and significantly enhanced the growth of NIH3T3 cells on the surface of titanium.


Assuntos
Fator de Crescimento Insulin-Like I/química , Monofenol Mono-Oxigenase/metabolismo , Titânio/química , Sequência de Aminoácidos , Animais , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Proteínas Imobilizadas/análise , Proteínas Imobilizadas/química , Proteínas Imobilizadas/farmacologia , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Células NIH 3T3 , Técnicas de Microbalança de Cristal de Quartzo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Propriedades de Superfície , Espectrometria de Massas em Tandem , Titânio/metabolismo
20.
J Mol Biol ; 428(13): 2744-57, 2016 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-27181198

RESUMO

Viral protein R (Vpr) is an accessory gene product of human immunodeficiency virus type 1 (HIV-1) that plays multiple important roles associated with viral replication. Structural studies using NMR have revealed that Vpr consists of three α-helices and contains flexible N- and C-termini. However, the molecular mechanisms associated with Vpr function have not been elucidated. To investigate Vpr multifunctionality, we performed an X-ray crystallographic study of Vpr complexes containing importin-α, a known Vpr binding partner present in host cells. Elucidation of the crystal structure revealed that the flexible C-terminus changes its conformation to a twisted ß-turn via an induced-fit mechanism, enabling binding to a minor nuclear localization signal (NLS) site of importin-α. The Vpr C-terminus can also bind with major NLS sites of importin-α in an extended conformation in different ways. These results, which represent the first reported crystallographic analysis of Vpr, demonstrate the multifunctional aspects that enable Vpr interaction with a variety of cellular proteins.


Assuntos
Produtos do Gene vpr/metabolismo , HIV-1/metabolismo , Ligação Proteica/fisiologia , alfa Carioferinas/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Humanos , Sinais de Localização Nuclear/metabolismo , Replicação Viral/fisiologia
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