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1.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198626

RESUMO

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a') tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.


Assuntos
Fator de Células-Tronco/biossíntese , Sequência de Aminoácidos , Ciclo Celular , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Solubilidade , Fator de Células-Tronco/química
2.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204592

RESUMO

NADH dehydrogenase (ubiquinone) Fe-S protein 8 (NDUFS8) is a nuclear-encoded core subunit of human mitochondrial complex I. Defects in NDUFS8 are associated with Leigh syndrome and encephalomyopathy. Cell-penetrating peptide derived from the HIV-1 transactivator of transcription protein (TAT) has been successfully applied as a carrier to bring fusion proteins into cells without compromising the biological function of the cargoes. In this study, we developed a TAT-mediated protein transduction system to rescue complex I deficiency caused by NDUFS8 defects. Two fusion proteins (TAT-NDUFS8 and NDUFS8-TAT) were exogenously expressed and purified from Escherichia coli for transduction of human cells. In addition, similar constructs were generated and used in transfection studies for comparison. The results showed that both exogenous TAT-NDUFS8 and NDUFS8-TAT were delivered into mitochondria and correctly processed. Interestingly, the mitochondrial import of TAT-containing NDUFS8 was independent of mitochondrial membrane potential. Treatment with TAT-NDUFS8 not only significantly improved the assembly of complex I in an NDUFS8-deficient cell line, but also partially rescued complex I functions both in the in-gel activity assay and the oxygen consumption assay. Our current findings suggest the considerable potential of applying the TAT-mediated protein transduction system for treatment of complex I deficiency.


Assuntos
Complexo I de Transporte de Elétrons/deficiência , Potencial da Membrana Mitocondrial , Mitocôndrias/genética , Mitocôndrias/metabolismo , NADH Desidrogenase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Sobrevivência Celular , Células Cultivadas , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Mitocôndrias/efeitos dos fármacos , NADH Desidrogenase/genética , Transporte Proteico , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
3.
Molecules ; 26(11)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34205205

RESUMO

Total body irradiation is a standard procedure of bone marrow transplantation (BMT) which causes a rapid increase in reactive oxygen species (ROS) in the bone marrow microenvironment during BMT. The increase in ROS reduces the engraftment ability of donor cells, thereby affecting the bone marrow recovery of recipients after BMT. In the early weeks following transplantation, recipients are at high risk of severe infection due to weakened hematopoiesis. Thus, it is imperative to improve engraftment capacity and accelerate bone marrow recovery in BMT recipients. In this study, we constructed recombinant copper/zinc superoxide dismutase 1 (SOD1) fused with the cell-penetrating peptide (CPP), the trans-activator of transcription (Tat), and showed that this fusion protein has penetrating ability and antioxidant activity in both RAW264.7 cells and bone marrow cells in vitro. Furthermore, irradiated mice transplanted with SOD1-Tat-treated total bone marrow donor cells showed an increase in total bone marrow engraftment capacity two weeks after transplantation. This study explored an innovative method for enhancing engraftment efficiency and highlights the potential of CPP-SOD1 in ROS manipulation during BMT.


Assuntos
Antioxidantes/farmacologia , Células da Medula Óssea/citologia , Peptídeos Penetradores de Células/genética , Produtos do Gene tat/genética , Proteínas Recombinantes de Fusão/farmacologia , Superóxido Dismutase-1/genética , Animais , Antioxidantes/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Transplante de Medula Óssea , Peptídeos Penetradores de Células/metabolismo , Células Cultivadas , Produtos do Gene tat/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espécies Reativas de Oxigênio , Proteínas Recombinantes de Fusão/metabolismo , Superóxido Dismutase-1/metabolismo , Irradiação Corporal Total
4.
FASEB J ; 35(8): e21681, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34196428

RESUMO

The sodium/iodide symporter (NIS) expresses at the basolateral plasma membrane of the thyroid follicular cell and mediates iodide accumulation required for normal thyroid hormonogenesis. Loss-of-function NIS variants cause congenital hypothyroidism due to impaired iodide accumulation in thyroid follicular cells underscoring the significance of NIS for thyroid physiology. Here we report novel findings derived from the thorough characterization of the nonsense NIS mutant p.R636* NIS-leading to a truncated protein missing the last eight amino acids-identified in twins with congenital hypothyroidism. R636* NIS is severely mislocalized into intracellular vesicular compartments due to the lack of a conserved carboxy-terminal type 1 PDZ-binding motif. As a result, R636* NIS is barely targeted to the plasma membrane and therefore iodide transport is reduced. Deletion of the PDZ-binding motif causes NIS accumulation into late endosomes and lysosomes. Using PDZ domain arrays, we revealed that the PDZ-domain containing protein SCRIB binds to the carboxy-terminus of NIS by a PDZ-PDZ interaction. Furthermore, in CRISPR/Cas9-based SCRIB deficient cells, NIS expression at the basolateral plasma membrane is compromised, leading to NIS localization into intracellular vesicular compartments. We conclude that the PDZ-binding motif is a plasma membrane retention signal that participates in the polarized expression of NIS by selectively interacting with the PDZ-domain containing protein SCRIB, thus retaining the transporter at the basolateral plasma membrane. Our data provide insights into the molecular mechanisms that regulate NIS expression at the plasma membrane, a topic of great interest in the thyroid cancer field considering the relevance of NIS-mediated radioactive iodide therapy for differentiated thyroid carcinoma.


Assuntos
Proteínas de Membrana/metabolismo , Simportadores/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/metabolismo , Códon sem Sentido , Hipotireoidismo Congênito/genética , Hipotireoidismo Congênito/metabolismo , Sequência Conservada , Cães , Endossomos/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Células Madin Darby de Rim Canino , Proteínas de Membrana/química , Proteínas de Membrana/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios PDZ/genética , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Simportadores/química , Simportadores/genética , Glândula Tireoide/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética
5.
Nat Commun ; 12(1): 3388, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099676

RESUMO

Wearable smart electronic devices, such as smart watches, are generally equipped with green-light-emitting diodes, which are used for photoplethysmography to monitor a panoply of physical health parameters. Here, we present a traceless, green-light-operated, smart-watch-controlled mammalian gene switch (Glow Control), composed of an engineered membrane-tethered green-light-sensitive cobalamin-binding domain of Thermus thermophilus (TtCBD) CarH protein in combination with a synthetic cytosolic TtCBD-transactivator fusion protein, which manage translocation of TtCBD-transactivator into the nucleus to trigger expression of transgenes upon illumination. We show that Apple-Watch-programmed percutaneous remote control of implanted Glow-controlled engineered human cells can effectively treat experimental type-2 diabetes by producing and releasing human glucagon-like peptide-1 on demand. Directly interfacing wearable smart electronic devices with therapeutic gene expression will advance next-generation personalized therapies by linking biopharmaceutical interventions to the internet of things.


Assuntos
Proteínas de Bactérias/efeitos da radiação , Diabetes Mellitus Tipo 2/terapia , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Optogenética/métodos , Transativadores/efeitos da radiação , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Engenharia Celular , Diabetes Mellitus Tipo 2/genética , Feminino , Engenharia Genética , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células HEK293 , Humanos , Luz , Masculino , Células-Tronco Mesenquimais , Camundongos , Camundongos Obesos , Optogenética/instrumentação , Fotopletismografia/instrumentação , Domínios Proteicos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/efeitos da radiação , Thermus thermophilus/genética , Transativadores/genética , Transativadores/metabolismo , Transgenes , Dispositivos Eletrônicos Vestíveis
6.
Int J Mol Sci ; 22(9)2021 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-34066779

RESUMO

The mechanisms underlying the transport of leptin into the brain are still largely unclear. While the leptin receptor has been implicated in the transport process, recent evidence has suggested an additional role of LRP2 (megalin). To evaluate the function of LRP2 for leptin transport across the blood-brain barrier (BBB), we developed a novel leptin-luciferase fusion protein (pLG), which stimulated leptin signaling and was transported in an in vitro BBB model based on porcine endothelial cells. The LRP inhibitor RAP did not affect leptin transport, arguing against a role of LRP2. In line with this, the selective deletion of LRP2 in brain endothelial cells and epithelial cells of the choroid plexus did not influence bodyweight, body composition, food intake, or energy expenditure of mice. These findings suggest that LRP2 at the BBB is not involved in the transport of leptin into the brain, nor in the development of obesity as has previously been described.


Assuntos
Barreira Hematoencefálica/metabolismo , Leptina/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Obesidade/metabolismo , Obesidade/patologia , Animais , Sítios de Ligação , Composição Corporal , Peso Corporal , Células CHO , Plexo Corióideo/metabolismo , Cricetulus , Células Endoteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Luciferases/metabolismo , Masculino , Modelos Biológicos , Fosforilação , Transporte Proteico , Receptores para Leptina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Suínos
7.
Methods Mol Biol ; 2268: 61-76, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085261

RESUMO

G protein-coupled receptors (GPCR) are integral membrane proteins that regulate multiple cellular processes. To obtain insights into structural properties of GPCR and mechanism of activity, these proteins should be isolated in significant (milligram) quantities, in a pure, homogenous, and stable form. Here we describe the expression and purification of type II human cannabinoid receptor CB2, a class A GPCR, in two different types of expression hosts: in Escherichia coli and in mammalian suspension cell culture Expi293. Our method allows preparation of milligram quantities of the purified receptors suitable for a wide array of downstream applications including high-resolution structural studies and functional assays.


Assuntos
Cromatografia de Afinidade/métodos , Cristalografia por Raios X/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Receptor CB2 de Canabinoide/isolamento & purificação , Receptor CB2 de Canabinoide/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Detergentes/química , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Receptor CB2 de Canabinoide/genética , Proteínas Recombinantes de Fusão/genética
8.
Nat Commun ; 12(1): 3310, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083531

RESUMO

FtsZ is a key component in bacterial cell division, being the primary protein of the presumably contractile Z ring. In vivo and in vitro, it shows two distinctive features that could so far, however, not be mechanistically linked: self-organization into directionally treadmilling vortices on solid supported membranes, and shape deformation of flexible liposomes. In cells, circumferential treadmilling of FtsZ was shown to recruit septum-building enzymes, but an active force production remains elusive. To gain mechanistic understanding of FtsZ dependent membrane deformations and constriction, we design an in vitro assay based on soft lipid tubes pulled from FtsZ decorated giant lipid vesicles (GUVs) by optical tweezers. FtsZ filaments actively transform these tubes into spring-like structures, where GTPase activity promotes spring compression. Operating the optical tweezers in lateral vibration mode and assigning spring constants to FtsZ coated tubes, the directional forces that FtsZ-YFP-mts rings exert upon GTP hydrolysis can be estimated to be in the pN range. They are sufficient to induce membrane budding with constricting necks on both, giant vesicles and E.coli cells devoid of their cell walls. We hypothesize that these forces result from torsional stress in a GTPase activity dependent manner.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas do Citoesqueleto/metabolismo , Guanosina Trifosfato/metabolismo , Fenômenos Biomecânicos , Divisão Celular/fisiologia , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Hidrólise , Lipossomos/metabolismo , Proteínas Luminescentes/metabolismo , Membranas/metabolismo , Modelos Biológicos , Pinças Ópticas , Proteínas Recombinantes de Fusão/metabolismo , Torção Mecânica
9.
Molecules ; 26(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066245

RESUMO

Natural backbone-cyclized proteins have an increased thermostability and resistance towards proteases, characteristics that have sparked interest in head-to-tail cyclization as a method to stability-enhance proteins used in diagnostics and therapeutic applications, for example. In this proof-of principle study, we have produced and investigated a head-to-tail cyclized and HER2-specific ZHER2:342 Affibody dimer. The sortase A-mediated cyclization reaction is highly efficient (>95%) under optimized conditions, and renders a cyclic ZHER3:342-dimer with an apparent melting temperature, Tm, of 68 °C, which is 3 °C higher than that of its linear counterpart. Circular dichroism spectra of the linear and cyclic dimers looked very similar in the far-UV range, both before and after thermal unfolding to 90 °C, which suggests that cyclization does not negatively impact the helicity or folding of the cyclic protein. The cyclic dimer had an apparent sub-nanomolar affinity (Kd ~750 pM) to the HER2-receptor, which is a ~150-fold reduction in affinity relative to the linear dimer (Kd ~5 pM), but the anti-HER2 Affibody dimer remained a high-affinity binder even after cyclization. No apparent difference in proteolytic stability was detected in an endopeptidase degradation assay for the cyclic and linear dimers. In contrast, in an exopeptidase degradation assay, the linear dimer was shown to be completely degraded after 5 min, while the cyclic dimer showed no detectable degradation even after 60 min. We further demonstrate that a site-specifically DyLight 594-labeled cyclic dimer shows specific binding to HER2-overexpressing cells. Taken together, the results presented here demonstrate that head-to-tail cyclization can be an effective strategy to increase the stability of an Affibody dimer.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Biocatálise , Neoplasias da Mama/metabolismo , Cisteína Endopeptidases/metabolismo , Multimerização Proteica , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Neoplasias da Mama/patologia , Dicroísmo Circular , Ciclização , Feminino , Humanos , Cinética , Células MCF-7 , Microscopia de Fluorescência , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Ressonância de Plasmônio de Superfície
10.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33947115

RESUMO

Cortisol, a stress hormone, plays key roles in mediating stress and anti-inflammatory responses. As abnormal cortisol levels can induce various adverse effects, screening cortisol and cortisol analogues is important for monitoring stress levels and for identifying drug candidates. A novel cell-based sensing system was adopted for rapid screening of cortisol and its functional analogues under complex cellular regulation. We used glucocorticoid receptor (GR) fused to a split intein which reconstituted with the counterpart to trigger conditional protein splicing (CPS) in the presence of targets. CPS generates functional signal peptides which promptly translocate the fluorescent cargo. The sensor cells exhibited exceptional performance in discriminating between the functional and structural analogues of cortisol with improved sensitivity. Essential oil extracts with stress relief activity were screened using the sensor cells to identify GR effectors. The sensor cells responded to peppermint oil, and L-limonene and L-menthol were identified as potential GR effectors from the major components of peppermint oil. Further analysis indicated L-limonene as a selective GR agonist (SEGRA) which is a potential anti-inflammatory agent as it attenuates proinflammatory responses without causing notable adverse effects of GR agonists.


Assuntos
Técnicas Biossensoriais , Avaliação Pré-Clínica de Medicamentos/métodos , Polarização de Fluorescência/métodos , Hidrocortisona/análise , Óleos Voláteis/farmacologia , Receptores de Glucocorticoides/agonistas , Atrofia , Acetato de Ciproterona/farmacologia , Dexametasona/farmacologia , Estradiol/farmacologia , Fluorometria , Células HeLa , Humanos , Inteínas , Limoneno/farmacologia , Proteínas Luminescentes/análise , Mentol/farmacologia , Mifepristona/farmacologia , Estrutura Molecular , Músculo Esquelético/patologia , Mioblastos/efeitos dos fármacos , Óleos Vegetais/farmacologia , Processamento de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
11.
EMBO J ; 40(11): e102277, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: covidwho-1194823

RESUMO

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS-CoV-2 and SARS-CoV share an otherwise non-conserved part of non-structural protein 3 (Nsp3), therefore named as "SARS-unique domain" (SUD). We previously found a yeast-2-hybrid screen interaction of the SARS-CoV SUD with human poly(A)-binding protein (PABP)-interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS-CoV SUD:Paip1 interaction by size-exclusion chromatography, split-yellow fluorescent protein, and co-immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS-CoV-2 and Paip1. The three-dimensional structure of the N-terminal domain of SARS-CoV SUD ("macrodomain II", Mac2) in complex with the middle domain of Paip1, determined by X-ray crystallography and small-angle X-ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC-SARS-CoV replicon-transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS-CoV and SARS-CoV-2.


Assuntos
Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Regulação Viral da Expressão Gênica , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Vírus da SARS/fisiologia , SARS-CoV-2/fisiologia , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias , Cromatografia em Gel , Proteases Semelhantes à Papaína de Coronavírus/química , Cristalografia por Raios X , Genes Reporter , Células HEK293 , Humanos , Imunoprecipitação , Proteínas Luminescentes , Modelos Moleculares , Fatores de Iniciação de Peptídeos/química , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , RNA Viral/genética , Proteínas de Ligação a RNA/química , RNA Polimerase Dependente de RNA/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Subunidades Ribossômicas/metabolismo , Vírus da SARS/genética , SARS-CoV-2/genética , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas não Estruturais Virais/química , Difração de Raios X
12.
EMBO J ; 40(11): e102277, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33876849

RESUMO

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS-CoV-2 and SARS-CoV share an otherwise non-conserved part of non-structural protein 3 (Nsp3), therefore named as "SARS-unique domain" (SUD). We previously found a yeast-2-hybrid screen interaction of the SARS-CoV SUD with human poly(A)-binding protein (PABP)-interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS-CoV SUD:Paip1 interaction by size-exclusion chromatography, split-yellow fluorescent protein, and co-immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS-CoV-2 and Paip1. The three-dimensional structure of the N-terminal domain of SARS-CoV SUD ("macrodomain II", Mac2) in complex with the middle domain of Paip1, determined by X-ray crystallography and small-angle X-ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC-SARS-CoV replicon-transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS-CoV and SARS-CoV-2.


Assuntos
Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Regulação Viral da Expressão Gênica , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Vírus da SARS/fisiologia , SARS-CoV-2/fisiologia , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias , Cromatografia em Gel , Proteases Semelhantes à Papaína de Coronavírus/química , Cristalografia por Raios X , Genes Reporter , Células HEK293 , Humanos , Imunoprecipitação , Proteínas Luminescentes , Modelos Moleculares , Fatores de Iniciação de Peptídeos/química , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , RNA Viral/genética , Proteínas de Ligação a RNA/química , RNA Polimerase Dependente de RNA/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Subunidades Ribossômicas/metabolismo , Vírus da SARS/genética , SARS-CoV-2/genética , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas não Estruturais Virais/química , Difração de Raios X
13.
Nat Commun ; 12(1): 2287, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863894

RESUMO

Both adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.


Assuntos
Adenosina Desaminase/genética , Proteína 9 Associada à CRISPR/genética , DNA/genética , Proteínas de Escherichia coli/genética , Edição de Genes/métodos , Adenina/metabolismo , Adenosina Desaminase/metabolismo , Toxinas Bacterianas/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Citosina/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Genes Reporter , Células HEK293 , Humanos , Inosina/genética , Inosina/metabolismo , Engenharia de Proteínas , Edição de RNA/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA-Seq , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
14.
SLAS Discov ; 26(6): 749-756, 2021 07.
Artigo em Inglês | MEDLINE | ID: covidwho-1136206

RESUMO

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5' end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3'-5' exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.


Assuntos
Antivirais/farmacologia , Exorribonucleases/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Nitrocompostos/farmacologia , Capuzes de RNA/antagonistas & inibidores , RNA Viral/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Tiazóis/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Antiparasitários/química , Antiparasitários/farmacologia , Antivirais/química , COVID-19/virologia , Clonagem Molecular , Reposicionamento de Medicamentos , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Espectrometria de Massas/métodos , Metilação , Nitrocompostos/química , Medicamentos sob Prescrição/química , Medicamentos sob Prescrição/farmacologia , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tiazóis/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
15.
Biol Cell ; 113(7): 311-328, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33666950

RESUMO

BACKGROUND INFORMATION: Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. RESULTS: We present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localisation of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria. CONCLUSIONS AND SIGNIFICANCE: This library should facilitate further cellular investigations, notably by imaging techniques.


Assuntos
COVID-19/virologia , Biblioteca de Peptídeos , SARS-CoV-2/metabolismo , Proteínas Virais/metabolismo , Células A549 , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , SARS-CoV-2/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Imagem com Lapso de Tempo , Proteínas Virais/genética
16.
J Mol Biol ; 433(10): 166948, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33744316

RESUMO

Liquid-liquid phase separation (LLPS) of proteins is involved in a growing number of cellular processes. Most proteins with LLPS harbor intrinsically disordered regions (IDR), which serve as a guideline to search for cellular proteins that potentially phase separate. Herein, we reveal that oligomerization lowers the barriers for LLPS and could act as a general mechanism to enhance LLPS of proteins domains independent of IDR. Using TDP43 as a model system, we found that deleting its IDR resulted in LLPS that was dependent on the oligomerization of the N-terminal domain (NTD). Replacing TDP43's NTD with other oligomerization domains enhanced the LLPS proportionately to the state of oligomerization. In addition to TDP43, fusing NTD to other globular proteins without known LLPS behavior also drove their phase separation in a manner dependent on oligomerization. Finally, we demonstrate that heterooligomers composed of NTD-fused proteins can be driven into droplets through NTD interactions. Our results potentiate a new paradigm for using oligomerization domains as a signature to systematically identify cellular proteins with LLPS behavior, thus broadening the scope of this exciting research field.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas Intrinsicamente Desordenadas/química , Coloração e Rotulagem/métodos , Sítios de Ligação , Cumarínicos/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fluoresceínas/química , Corantes Fluorescentes/química , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Rodaminas/química
17.
Methods Mol Biol ; 2297: 105-113, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33656674

RESUMO

Cryptochromes (CRYs) belong to an ancient and conserved class of blue-light receptor regulating circadian clock and development in animals and plants. Arabidopsis CRY2 form physiologically active homodimers in response to blue light treatment and further oligomerize into photobodies, which are expected to be the foci harboring protein interaction, phosphorylation, and ubiquitination. Here we describe two efficient methods developed to test the formation of blue-light-dependent photobodies of CRY-GFP fusing proteins using the mesophyll protoplasts of Arabidopsis or soybean, respectively.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Criptocromos/metabolismo , Protoplastos/metabolismo , Soja/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relógios Circadianos , Criptocromos/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Luz , Fosforilação , Multimerização Proteica , Proteínas Recombinantes de Fusão/metabolismo , Soja/genética , Soja/metabolismo , Ubiquitinação
18.
Cancer Sci ; 112(6): 2299-2313, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33735501

RESUMO

Podoplanin is a key molecule for enhancing tumor-induced platelet aggregation. Podoplanin interacts with CLEC-2 on platelets via PLatelet Aggregation-inducing domains (PLAGs). Among our generated antibodies, those targeting the fourth PLAG domain (PLAG4) strongly suppress podoplanin-CLEC-2 binding and podoplanin-expressing tumor growth and metastasis. We previously performed a single-dose toxicity study of PLAG4-targeting anti-podoplanin-neutralizing antibodies and found no acute toxicity in cynomolgus monkeys. To confirm the therapeutic efficacy and toxicity of podoplanin-targeting antibodies, a syngeneic mouse model that enables repeated dose toxicity tests is needed. Replacement of mouse PLAG1-PLAG4 domains with human homologous domains drastically decreased the platelet-aggregating activity. Therefore, we searched the critical domain of the platelet-aggregating activity in mouse podoplanin and found that the mouse PLAG4 domain played a critical role in platelet aggregation, similar to the human PLAG4 domain. Human/mouse chimeric podoplanin, in which a limited region containing mouse PLAG4 was replaced with human homologous region, exhibited a similar platelet-aggregating activity to wild-type mouse podoplanin. Thus, we generated knock-in mice with human/mouse chimeric podoplanin expression (PdpnKI/KI mice). Our previously established PLAG4-targeting antibodies could suppress human/mouse chimeric podoplanin-mediated platelet aggregation and tumor growth in PdpnKI/KI mice. Repeated treatment of PdpnKI/KI mice with antibody-dependent cell-mediated cytotoxicity activity-possessing PG4D2 antibody did not result in toxicity or changes in hematological and biochemical parameters. Our results suggest that anti-podoplanin-neutralizing antibodies could be used safely as novel anti-tumor agents. Our generated PdpnKI/KI mice are useful for investigating the efficacy and toxicity of human podoplanin-targeting drugs.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Antineoplásicos/uso terapêutico , Glicoproteínas de Membrana/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ativação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Am J Hematol ; 96(6): 698-707, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33761144

RESUMO

Myeloproliferative neoplasms (MPNs) are characterized by a pathologic expansion of myeloid lineages. Mutations in JAK2, CALR and MPL genes are known to be three prominent MPN disease drivers. Mutant CALR (mutCALR) is an oncoprotein that interacts with and activates the thrombopoietin receptor (MPL) and represents an attractive target for targeted therapy of CALR mutated MPN. We generated a transgenic murine model with conditional expression of the human mutant exon 9 (del52) from the murine endogenous Calr locus. These mice develop essential thrombocythemia like phenotype with marked thrombocytosis and megakaryocytosis. The disease exacerbates with age showing prominent signs of splenomegaly and anemia. The disease is transplantable and mutCALR stem cells show proliferative advantage when compared to wild type stem cells. Transcriptome profiling of hematopoietic stem cells revealed oncogenic and inflammatory gene expression signatures. To demonstrate the applicability of the transgenic animals for immunotherapy, we treated mice with monoclonal antibody raised against the human mutCALR. The antibody treatment lowered platelet and stem cell counts in mutant mice. Secretion of mutCALR did not constitute a significant antibody sink. This animal model not only recapitulates human MPN but also serves as a relevant model for testing immunotherapeutic strategies targeting epitopes of the human mutCALR.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Calreticulina/antagonistas & inibidores , Modelos Animais de Doenças , Células-Tronco Hematopoéticas/metabolismo , Terapia de Alvo Molecular , Trombocitemia Essencial/terapia , Animais , Anticorpos Monoclonais/imunologia , Plaquetas/imunologia , Plaquetas/metabolismo , Calreticulina/genética , Calreticulina/imunologia , Calreticulina/fisiologia , Éxons/genética , Mutação da Fase de Leitura , Técnicas de Introdução de Genes , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Quimera por Radiação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Esplenomegalia/etiologia , Trombocitemia Essencial/sangue , Trombocitemia Essencial/complicações , Trombocitemia Essencial/genética , Transcriptoma
20.
Nat Chem Biol ; 17(5): 593-600, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33686291

RESUMO

O-linked N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification that is presented on thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a single target protein is crucial, yet challenging to perform in cells. Herein, we developed a nanobody-fused split O-GlcNAcase (OGA) as an O-GlcNAc eraser for selective deglycosylation of a target protein in cells. After systematic cellular optimization, we identified a split OGA with reduced inherent deglycosidase activity that selectively removed O-GlcNAc from the desired target protein when directed by a nanobody. We demonstrate the generality of the nanobody-fused split OGA using four nanobodies against five target proteins and use the system to study the impact of O-GlcNAc on the transcription factors c-Jun and c-Fos. The nanobody-directed O-GlcNAc eraser provides a new strategy for the functional evaluation and engineering of O-GlcNAc via the selective removal of O-GlcNAc from individual proteins directly in cells.


Assuntos
Antígenos de Neoplasias/metabolismo , Histona Acetiltransferases/metabolismo , Hialuronoglucosaminidase/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Glicoproteínas de Membrana/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Domínio Único/química , Fator de Transcrição Sp1/metabolismo , Fatores de Transcrição/metabolismo , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Bioensaio , Domínio Catalítico , Sistemas de Liberação de Medicamentos/métodos , Expressão Gênica , Glicosilação , Células HEK293 , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Humanos , Hialuronoglucosaminidase/química , Hialuronoglucosaminidase/genética , Hidrólise , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Glicoproteínas de Membrana/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Anticorpos de Domínio Único/metabolismo , Fator de Transcrição Sp1/genética , Fatores de Transcrição/genética , Transfecção/métodos
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