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1.
Nat Commun ; 11(1): 4554, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917865

RESUMO

Non-ribosomal peptide synthetase (NRPS) enzymes form modular assembly-lines, wherein each module governs the incorporation of a specific monomer into a short peptide product. Modules are comprised of one or more key domains, including adenylation (A) domains, which recognise and activate the monomer substrate; condensation (C) domains, which catalyse amide bond formation; and thiolation (T) domains, which shuttle reaction intermediates between catalytic domains. This arrangement offers prospects for rational peptide modification via substitution of substrate-specifying domains. For over 20 years, it has been considered that C domains play key roles in proof-reading the substrate; a presumption that has greatly complicated rational NRPS redesign. Here we present evidence from both directed and natural evolution studies that any substrate-specifying role for C domains is likely to be the exception rather than the rule, and that novel non-ribosomal peptides can be generated by substitution of A domains alone. We identify permissive A domain recombination boundaries and show that these allow us to efficiently generate modified pyoverdine peptides at high yields. We further demonstrate the transferability of our approach in the PheATE-ProCAT model system originally used to infer C domain substrate specificity, generating modified dipeptide products at yields that are inconsistent with the prevailing dogma.


Assuntos
Monofosfato de Adenosina/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Domínios Proteicos , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Embaralhamento de DNA , Modelos Moleculares , Família Multigênica , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Conformação Proteica , Pseudomonas , Especificidade por Substrato
2.
Nat Commun ; 11(1): 4414, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887877

RESUMO

CD4+ helper T cells contribute important functions to the immune response during pathogen infection and tumor formation by recognizing antigenic peptides presented by class II major histocompatibility complexes (MHC-II). While many computational algorithms for predicting peptide binding to MHC-II proteins have been reported, their performance varies greatly. Here we present a yeast-display-based platform that allows the identification of over an order of magnitude more unique MHC-II binders than comparable approaches. These peptides contain previously identified motifs, but also reveal new motifs that are validated by in vitro binding assays. Training of prediction algorithms with yeast-display library data improves the prediction of peptide-binding affinity and the identification of pathogen-associated and tumor-associated peptides. In summary, our yeast-display-based platform yields high-quality MHC-II-binding peptide datasets that can be used to improve the accuracy of MHC-II binding prediction algorithms, and potentially enhance our understanding of CD4+ T cell recognition.


Assuntos
Epitopos de Linfócito T/genética , Oligopeptídeos , Sítios de Ligação , Linfócitos T CD4-Positivos/imunologia , Técnicas de Visualização da Superfície Celular , Bases de Dados de Proteínas , Epitopos de Linfócito T/química , Epitopos de Linfócito T/metabolismo , Genes MHC da Classe II , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Ligação Proteica/genética , Receptores de Antígenos de Linfócitos T , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo
3.
Nat Commun ; 11(1): 3922, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764664

RESUMO

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a key contributor to multidrug resistance and is also essential for the survival of the malaria parasite, yet its natural function remains unresolved. We identify host-derived peptides of 4-11 residues, varying in both charge and composition, as the substrates of PfCRT in vitro and in situ, and show that PfCRT does not mediate the non-specific transport of other metabolites and/or ions. We find that drug-resistance-conferring mutations reduce both the peptide transport capacity and substrate range of PfCRT, explaining the impaired fitness of drug-resistant parasites. Our results indicate that PfCRT transports peptides from the lumen of the parasite's digestive vacuole to the cytosol, thereby providing a source of amino acids for parasite metabolism and preventing osmotic stress of this organelle. The resolution of PfCRT's native substrates will aid the development of drugs that target PfCRT and/or restore the efficacy of existing antimalarials.


Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Transporte Biológico Ativo , Resistência a Medicamentos/genética , Feminino , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/fisiologia , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oligopeptídeos/metabolismo , Oócitos/metabolismo , Plasmodium falciparum/genética , Transporte Proteico , Proteínas de Protozoários/genética , Xenopus laevis
4.
Endocr Res ; 45(3): 210-215, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32628899

RESUMO

BACKGROUND: Uptake of coronaviruses by target cells involves binding of the virus by cell ectoenzymes. For the etiologic agent of COVID-19 (SARS-CoV-2), a receptor has been identified as angiotensin-converting enzyme-2 (ACE2). Recently it has been suggested that plasma membrane integrins may be involved in the internalization and replication of clinically important coronaviruses. For example, integrin αvß3 is involved in the cell uptake of a model porcine enteric α-coronavirus that causes human epidemics. ACE2 modulates the intracellular signaling generated by integrins. OBJECTIVE: We propose that the cellular internalization of αvß3 applies to uptake of coronaviruses bound to the integrin, and we evaluate the possibility that clinical host T4 may contribute to target cell uptake of coronavirus and to the consequence of cell uptake of the virus. DISCUSSION AND CONCLUSIONS: The viral binding domain of the integrin is near the Arg-Gly-Asp (RGD) peptide-binding site and RGD molecules can affect virus binding. In this same locale on integrin αvß3 is the receptor for thyroid hormone analogues, particularly, L-thyroxine (T4). By binding to the integrin, T4 has been shown to modulate the affinity of the integrin for other proteins, to control internalization of αvß3 and to regulate the expression of a panel of cytokine genes, some of which are components of the 'cytokine storm' of viral infections. If T4 does influence coronavirus uptake by target cells, other thyroid hormone analogues, such as deaminated T4 and deaminated 3,5,3'-triiodo-L-thyronine (T3), are candidate agents to block the virus-relevant actions of T4 at integrin αvß3 and possibly restrict virus uptake.


Assuntos
Infecções por Coronavirus/virologia , Integrina alfaVbeta3/metabolismo , Vírus da Diarreia Epidêmica Suína/metabolismo , Receptores Virais/efeitos dos fármacos , Hormônios Tireóideos/farmacologia , Animais , Betacoronavirus/metabolismo , Sítios de Ligação , Citocinas/fisiologia , Células Epiteliais/virologia , Humanos , Oligopeptídeos/metabolismo , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/virologia , Receptores Virais/química , Receptores Virais/metabolismo , Suínos , Hormônios Tireóideos/fisiologia , Tiroxina/fisiologia , Internalização do Vírus
5.
Proc Natl Acad Sci U S A ; 117(26): 15363-15373, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554501

RESUMO

Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin-protein interacting regions. These results offer a glimpse of the protein interaction landscape of SS-31 and provide mechanistic insight relevant to SS-31 mitochondrial therapy.


Assuntos
Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Oligopeptídeos/farmacologia , Envelhecimento , Animais , Masculino , Camundongos , Modelos Químicos , Simulação de Dinâmica Molecular , Oligopeptídeos/metabolismo , Ligação Proteica
6.
Proc Natl Acad Sci U S A ; 117(21): 11432-11443, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32381732

RESUMO

The structure and mechanics of many connective tissues are dictated by a collagen-rich extracellular matrix (ECM), where collagen fibers provide topological cues that direct cell migration. However, comparatively little is known about how cells navigate the hyaluronic acid (HA)-rich, nanoporous ECM of the brain, a problem with fundamental implications for development, inflammation, and tumor invasion. Here, we demonstrate that glioblastoma cells adhere to and invade HA-rich matrix using microtentacles (McTNs), which extend tens of micrometers from the cell body and are distinct from filopodia. We observe these structures in continuous culture models and primary patient-derived tumor cells, as well as in synthetic HA matrix and organotypic brain slices. High-magnification and superresolution imaging reveals McTNs are dynamic, CD44-coated tubular protrusions containing microtubules and actin filaments, which respectively drive McTN extension and retraction. Molecular mechanistic studies reveal that McTNs are stabilized by an interplay between microtubule-driven protrusion, actomyosin-driven retraction, and CD44-mediated adhesion, where adhesive and cytoskeletal components are mechanistically coupled by an IQGAP1-CLIP170 complex. McTNs represent a previously unappreciated mechanism through which cells engage nanoporous HA matrix and may represent an important molecular target in physiology and disease.


Assuntos
Glioblastoma/patologia , Receptores de Hialuronatos/metabolismo , Actinas/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Feminino , Técnicas de Inativação de Genes , Glioblastoma/metabolismo , Humanos , Ácido Hialurônico/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismo , Proteínas de Neoplasias/metabolismo , Oligopeptídeos/metabolismo , Técnicas de Cultura de Órgãos , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo
7.
Clin Nucl Med ; 45(7): 561-562, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32433166

RESUMO

We present a 78-year-old man with suspicion of prostate cancer due to a PSA of 200 ng/mL, who underwent F-PSMA-1007 (prostate specific membrane antigen) PET/CT for primary staging. Besides heterogeneous uptake to the prostate, an increased PSMA uptake in the cecum was observed, located in the thickened cecal wall with suspicion of a secondary malignancy. Colonoscopic biopsy followed by hemicolectomy confirmed the diagnosis of colon adenocarcinoma. This case demonstrates the importance of bioptic workup of suspicious findings on PSMA PET/CT, which are unlikely to be related to prostate cancer as PSMA ligand uptake is not exclusively prostate cancer specific.


Assuntos
Adenocarcinoma/diagnóstico por imagem , Adenocarcinoma/metabolismo , Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/metabolismo , Achados Incidentais , Niacinamida/análogos & derivados , Oligopeptídeos/metabolismo , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Idoso , Transporte Biológico , Humanos , Masculino , Niacinamida/metabolismo
8.
Pharm Res ; 37(6): 98, 2020 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-32419062

RESUMO

PURPOSE: A Na+-coupled transport system in mammalian cells is responsible for the uptake of oligopeptides consisting of 5 or more amino acids. Here we investigated if this transport system is expressed in brain cells and transports the 42-amino-acid ß-amyloid peptide (Aß1-42). METHODS: The human and mouse neuronal cell lines SK-N-SH and HT22, human microglial cell line HMC-3, and human blood-brain barrier endothelial cell line hCMEC/D3 were used to monitor the uptake of [3H]-deltorphin II (a heptapeptide) and fluorescence-labeled Aß1-42. RESULTS: All four cell lines exhibited Na+-coupled uptake of deltorphin II. Aß1-42 competed with deltorphin II for the uptake. Uptake of fluorescence-labeled Aß1-42 was detectable in these cell lines, and the uptake was Na+-dependent and inhibitable by deltorphin II. The Na+-coupled uptake disappeared at high concentrations of Aß1-42 due to oligomerization of the peptide. Exposure of the cells to excess iron abolished the uptake. In hCMEC/D3 cells cultured on Transwell filters, the uptake was localized preferentially to the abluminal membrane. CONCLUSION: A Na+-coupled transport system mediates the uptake of Aß1-42 monomers in neuronal and microglial cells. The same system is also responsible for the uptake of Aß1-42 from brain into blood-brain barrier endothelial cells. These findings have relevance to Alzheimer's disease.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Oligopeptídeos/metabolismo , Fragmentos de Peptídeos/metabolismo , Sódio/metabolismo , Animais , Transporte Biológico , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Células Endoteliais/metabolismo , Humanos , Cinética , Moduladores de Transporte de Membrana/metabolismo , Camundongos , Modelos Biológicos
9.
Chemistry ; 26(33): 7492-7496, 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32227540

RESUMO

The use of multimeric ligands is considered as a promising strategy to improve tumor targeting for diagnosis and therapy. Herein, tetrameric RGD (Arg-Gly-Asp) peptidomimetics were designed to target αv ß3 integrin-expressing tumor cells. These compounds were prepared by an oxime chemoselective assembly of cyclo(DKP-RGD) ligands and a cyclodecapeptide scaffold, which allows a tetrameric presentation. The resulting tetrameric RGD peptidomimetics were shown to improve αv ß3 integrin binding compared with the monomeric form. Interestingly, these compounds were also able to enhance tumor cell endocytosis in the same way as tetrameric RGD peptides. Altogether, the results show the potential of the tetrameric cyclo(DKP-RGD) ligands for in vivo imaging and drug delivery.


Assuntos
Integrina alfaVbeta3/metabolismo , Oligopeptídeos/química , Peptidomiméticos/química , Transporte Biológico , Humanos , Integrina alfaVbeta3/química , Ligantes , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacocinética , Oligopeptídeos/farmacologia
10.
Clin Nucl Med ; 45(6): e276-e278, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32332302

RESUMO

A 72-year-old man, who is known with a case of metastatic carcinoma of the breast, was referred for F-PMSA 1007 PET/CT with clinical suspicion of synchronous prostate cancer. F-PSMA 1007 PET/CT scan detected no abnormal tracer concentrating lesion in the prostate gland; however, abnormal tracer concentration was noted in soft tissue lesions in left breast, metastatic lymph nodes, and skeletal lesions. Compared with F-FDG PET/CT, more bone lesions were detected on F-PSMA 1007 imaging. The findings of our case open the possibility of imaging metastatic breast cancer with F-PSMA 1007 in men.


Assuntos
Neoplasias da Mama Masculina/metabolismo , Neoplasias da Mama Masculina/patologia , Radioisótopos de Flúor , Niacinamida/análogos & derivados , Oligopeptídeos/metabolismo , Idoso , Neoplasias da Mama Masculina/diagnóstico por imagem , Humanos , Masculino , Metástase Neoplásica , Niacinamida/metabolismo , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons
11.
Artigo em Inglês | MEDLINE | ID: mdl-32331227

RESUMO

Blooms of the cyanobacterium Planktothrix agardhii are common in shallow, eutrophic freshwaters. P. agardhii may produce hepatotoxic microcystins (MCs) and many other bioactive secondary metabolites belonging mostly to non-ribosomal oligopeptides. The aim of this work was to study the effects of two extracts (Pa-A and Pa-B) of P. agardhii-predominated bloom samples with different oligopeptide profiles and high concentration of biogenic compounds on another natural P. agardhii population. We hypothesised that the P. agardhii biomass and content of oligopeptides in P. agardhii is shaped in a different manner by diverse mixtures of metabolites of different P. agardhii-dominated cyanobacterial assemblages. For this purpose, the biomass, chlorophyll a and oligopeptides content in the treated P. agardhii were measured. Seven-day microcosm experiments with four concentrations of the extracts Pa-A and Pa-B were carried out. Generally, aeruginosins (AERs), cyanopeptolins (CPs) and anabaenopeptins (APs) were the most numerous peptides; however, only 16% of them were common for both extracts. The addition of the extracts resulted in similar effects on P. agardhii: an increase in biomass, Chl-a and MC content in the exposed P. agardhii as well as changes in its oligopeptide profile were observed. MCs present in the extracts did not inhibit accumulation of P. agardhii biomass, and did not have any negative effect on MC and Chl-a content. No evidence for bioaccumulation of dissolved peptides in the P. agardhii exposed was found. As the two tested extracts differed considerably in oligopeptide composition, but contained similar high concentrations of nutrients, it seems that biogenic compounds, not oligopeptides themselves, positively influenced the mixed natural P. agardhii population.


Assuntos
Clorofila A , Cianobactérias , Eutrofização , Microcistinas , Oligopeptídeos , Biomassa , Cianobactérias/metabolismo , Oligopeptídeos/metabolismo , Extratos Vegetais
12.
Nature ; 579(7799): 421-426, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32188939

RESUMO

Bioorthogonal chemistry capable of operating in live animals is needed to investigate biological processes such as cell death and immunity. Recent studies have identified a gasdermin family of pore-forming proteins that executes inflammasome-dependent and -independent pyroptosis1-5. Pyroptosis is proinflammatory, but its effect on antitumour immunity is unknown. Here we establish a bioorthogonal chemical system, in which a cancer-imaging probe phenylalanine trifluoroborate (Phe-BF3) that can enter cells desilylates and 'cleaves' a designed linker that contains a silyl ether. This system enabled the controlled release of a drug from an antibody-drug conjugate in mice. When combined with nanoparticle-mediated delivery, desilylation catalysed by Phe-BF3 could release a client protein-including an active gasdermin-from a nanoparticle conjugate, selectively into tumour cells in mice. We applied this bioorthogonal system to gasdermin, which revealed that pyroptosis of less than 15% of tumour cells was sufficient to clear the entire 4T1 mammary tumour graft. The tumour regression was absent in immune-deficient mice or upon T cell depletion, and was correlated with augmented antitumour immune responses. The injection of a reduced, ineffective dose of nanoparticle-conjugated gasdermin along with Phe-BF3 sensitized 4T1 tumours to anti-PD1 therapy. Our bioorthogonal system based on Phe-BF3 desilylation is therefore a powerful tool for chemical biology; our application of this system suggests that pyroptosis-induced inflammation triggers robust antitumour immunity and can synergize with checkpoint blockade.


Assuntos
Preparações de Ação Retardada/administração & dosagem , Neoplasias Mamárias Experimentais/imunologia , Piroptose/imunologia , Animais , Cumarínicos/administração & dosagem , Cumarínicos/química , Cumarínicos/metabolismo , Cumarínicos/farmacocinética , Preparações de Ação Retardada/química , Preparações de Ação Retardada/metabolismo , Preparações de Ação Retardada/farmacocinética , Feminino , Proteínas de Fluorescência Verde/administração & dosagem , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/farmacocinética , Células HeLa , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/química , Imunoconjugados/metabolismo , Imunoconjugados/farmacocinética , Inflamassomos/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/administração & dosagem , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacocinética , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Proteínas/administração & dosagem , Proteínas/química , Proteínas/metabolismo , Proteínas/farmacocinética , Silanos/administração & dosagem , Silanos/química , Silanos/metabolismo , Silanos/farmacocinética , Linfócitos T/imunologia , Trastuzumab/administração & dosagem , Trastuzumab/química , Trastuzumab/metabolismo , Trastuzumab/farmacocinética , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Biochim Biophys Acta Biomembr ; 1862(6): 183260, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32142822

RESUMO

Because of their potential as novel antibiotic agents, antimicrobial peptides (AMPs) have generated considerable interest. The mechanism of bacterial toxicity of AMPs often involves the disruption and/or permeabilization of the bacterial membrane; even those that act intracellularly first have to traverse the membrane. In this work we have explored the incorporation of the fluorinated aromatic amino acids fluoro-Phe and fluoro-Tyr into the Trp- and Arg-rich AMP tritrpticin, and investigated their role in the membrane binding properties and the antimicrobial activity of the peptide. Fluorinated peptides were obtained with good yield by recombinant expression of tritrpticin as a calmodulin-fusion protein in Escherichia coli. Cells were grown in the presence of glyphosate, an inhibitor of aromatic amino acid biosynthesis, and the peptides were released by proteolysis from the purified fusion protein. By using SDS micelles, as a simplified model of the bacterial cytoplasmic membrane, we could study the peptide-membrane interactions and the preferred location of individual fluorinated residues in the micelles by 19F NMR spectroscopy. Solvent-perturbation 19F NMR measurements revealed that para-fluoro-Phe residues are embedded deeply in the hydrophobic region of the micelles. On the other hand, 3-fluoro-Tyr residues introduced in tritrpticin were located near the surface of the micelles with high solvent exposure, while 2-fluoro-Tyr sidechains were less solvent exposed. In combination with the outcome of determinations of their antimicrobial activity, our 19F NMR results indicate that the higher solvent exposure of Tyr residues correlates with a decrease of the antimicrobial potency. This different role of Tyr can likely be extended from tritrpticin to other cationic AMPs.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Flúor , Espectroscopia de Ressonância Magnética/métodos , Oligopeptídeos/química , Tirosina/fisiologia , Peptídeos Catiônicos Antimicrobianos/toxicidade , Membrana Celular/metabolismo , Micelas , Oligopeptídeos/metabolismo , Dodecilsulfato de Sódio
14.
PLoS One ; 15(3): e0230282, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32160243

RESUMO

Cloning and expression of a desired gene is indispensable in molecular biology studies. Expression vectors, in this regard, should offer much needed flexibility and choice of cloning strategies for both in vivo and in vitro protein expression experiments. Furthermore, availability of option to choose from various reporter tags allows one to be flexible during designing of an experiment in a more relevant manner. Thus, the need of a versatile expression system cannot be ignored. Although several different expression vectors are available for gene expression in mycobacteria, they lack the required versatility of expression and the inclusion of reporter tags. We here present the construction of a set of nine E. coli-Mycobacterium shuttle plasmids, which offer a combination of three mycobacterial promoter systems (heat shock inducible-hsp60, tetracycline-, and acetamide-inducible) along with three polypeptide tags (Green Fluorescent Protein (GFP), Glutathione S-transferase (GST) and hexa-histidine tag). These vectors offer the cloning of a target gene in all the nine given vectors in parallel, thus allowing the generation of recombinant plasmids that will express the target gene from different promoters with different tags. Here, while the hexa-histidine and GST tags can be used for protein purification and pull-down experiments, the GFP-tag can be used for protein localization within the cell. Additionally, the vectors also offer the choice of positioning of the reporter tag either at the N-terminus or at the C-terminus of the expressed protein, which is achieved by cloning of the gene at any of the two blunt-end restriction enzyme sites available in the vector. We believe that these plasmids will be extremely useful in the gene expression studies in mycobacteria by offering the choices of promoters and reporters. Our work also paves the way to developing more such plasmids with other tags and promoters that may find use in mycobacterial biology.


Assuntos
Engenharia Genética/métodos , Vetores Genéticos/genética , Mycobacterium/genética , Escherichia coli/genética , Genes Reporter , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina/genética , Histidina/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Regiões Promotoras Genéticas , Ativação Transcricional
15.
Org Biomol Chem ; 18(10): 1978-1986, 2020 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-32104826

RESUMO

Development of an intracellular delivery method for functional peptides via cell-penetrating peptides (CPPs) expands peptide use in basic research and therapeutic applications. Although direct conjugation of a functional peptide with a CPP is the simplest method for delivery, this method has not always been reliable. CPPs usually contain several positively charged amino acids that potentially interact non-specifically with negatively charged molecules in cells and subsequently interfere with conjugated functional peptide function. Here we demonstrate a new intracellular delivery method for peptides in which a functional peptide is released from a positively charged CPP via peptide nucleic acids (PNAs). We prepared an 8-mer PNA conjugated to octa-arginine in tandem (PNA1-CPP) and linked its complementary PNA to an autophagy inducing peptide (PNA2-AIP) by solid-phase peptide synthesis. PNA1-CPP and PNA2-AIP formed a 1 : 1 hybrid via PNA1/PNA2 interaction, thereby indirectly but stably connecting the AIP to the CPP. PNA2-AIP was successfully delivered into cells in a hybrid formation-dependent manner and at least some portion of the PNA1-CPP/PNA2-AIP hybrids dissociated into PNA2-AIP and PNA1-CPP inside the cells. Notably, PNA2-AIP delivered to cells induced more autophagy than AIP directly conjugated to CPP (CPP-AIP). Further, the PNA hybrid did not induce significant cell death. These findings indicate that the PNA1/PNA2 hybrid can function as a molecular glue enabling the delivery of functional peptides into cells.


Assuntos
Proteína Beclina-1/farmacologia , Peptídeos Penetradores de Células/metabolismo , Portadores de Fármacos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ácidos Nucleicos Peptídicos/metabolismo , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Proteína Beclina-1/toxicidade , Peptídeos Penetradores de Células/toxicidade , Portadores de Fármacos/toxicidade , Liberação Controlada de Fármacos , Células HeLa , Humanos , Zíper de Leucina , Oligopeptídeos/metabolismo , Oligopeptídeos/toxicidade , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Ácidos Nucleicos Peptídicos/toxicidade , Ligação Proteica
16.
J Mater Chem B ; 8(11): 2350-2362, 2020 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-32104824

RESUMO

Mineralised collagen fibrils constitute the basic building blocks of bone, dentin and cementum. Noncollagenous proteins (NCPs) that are indispensable for collagen biomineralisation are not commercially available, and the mechanism of intrafibrillar mineralisation remains debatable. Herein, synthetic biomimetic molecules are regarded as alternative candidates for NCPs, and more convenient in revealing the mechanism of intrafibrillar mineralisation in vitro. Here, we fabricated a novel amphiphilic oligopeptide imitating a natural NCP. We aimed to investigate the effectiveness of the oligopeptide in intrafibrillar mineralisation and partially reveal the corresponding mechanism in vitro. The effectiveness of the oligopeptide in intrafibrillar mineralisation was characterised from the following aspects: (1) mineral interaction, (2) collagen binding and (3) induction of intrafibrillar mineralisation. Results indicated that the self-assembled oligopeptide could attract calcium ions inducing the formation of amorphous precursors; and bind onto the surface of collagen fibrils. These processes were mainly driven by the electrostatic attraction and hydrogen bonds. The self-assembled oligopeptide induced the intrafibrillar mineralisation of reconstituted collagen fibrils, in which the c-axis of apatite crystallites was roughly parallel to the long axis of the fibrils. The collagen mineralisation was achieved by binding with the self-assembled oligopeptide to increase the pool of mineralization precursors available for intrafibrillar mineralisation. In addition, the self-assembled oligopeptide induced dentin collagen remineralisation and formed a 30 µm-thick remineralised layer within 96 h. Our work sheds light on the fabrication of a novel biomimetic molecule for collagen mineralisation. The results should serve as a reference for understanding the mechanism of intrafibrillar mineralisation.


Assuntos
Biomineralização/efeitos dos fármacos , Colágeno/metabolismo , Minerais/metabolismo , Oligopeptídeos/farmacologia , Animais , Biomimética , Cálcio/metabolismo , Humanos , Oligopeptídeos/metabolismo
17.
Infect Immun ; 88(5)2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32094256

RESUMO

Peptidoglycan, the sugar-amino acid polymer that composes the bacterial cell wall, requires a significant expenditure of energy to synthesize and is highly immunogenic. To minimize the loss of an energetically expensive metabolite and avoid host detection, bacteria often recycle their peptidoglycan, transporting its components back into the cytoplasm, where they can be used for subsequent rounds of new synthesis. The peptidoglycan-recycling substrate binding protein (SBP) MppA, which is responsible for recycling peptidoglycan fragments in Escherichia coli, has not been annotated for most intracellular pathogens. One such pathogen, Chlamydia trachomatis, has a limited capacity to synthesize amino acids de novo and therefore must obtain oligopeptides from its host cell for growth. Bioinformatics analysis suggests that the putative C. trachomatis oligopeptide transporter OppABCDF (OppABCDF Ct ) encodes multiple SBPs (OppA1 Ct , OppA2 Ct , and OppA3 Ct ). Intracellular pathogens often encode multiple SBPs, while only one, OppA, is encoded in the E. coli opp operon. We hypothesized that the putative OppABCDF transporter of C. trachomatis functions in both oligopeptide transport and peptidoglycan recycling. We coexpressed the putative SBP genes (oppA1Ct , oppA2Ct , oppA3Ct ) along with oppBCDFCt in an E. coli mutant lacking the Opp transporter and determined that all three chlamydial OppA subunits supported oligopeptide transport. We also demonstrated the in vivo functionality of the chlamydial Opp transporter in C. trachomatis Importantly, we found that one chlamydial SBP, OppA3 Ct , possessed dual substrate recognition properties and is capable of transporting peptidoglycan fragments (tri-diaminopimelic acid) in E. coli and in C. trachomatis These findings suggest that Chlamydia evolved an oligopeptide transporter to facilitate the acquisition of oligopeptides for growth while simultaneously reducing the accumulation of immunostimulatory peptidoglycan fragments in the host cell cytosol. The latter property reflects bacterial pathoadaptation that dampens the host innate immune response to Chlamydia infection.


Assuntos
Chlamydia trachomatis/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Oligopeptídeos/metabolismo , Peptidoglicano/metabolismo , Aminoácidos/genética , Aminoácidos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico/genética , Transporte Biológico/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Parede Celular/genética , Parede Celular/metabolismo , Infecções por Chlamydia/metabolismo , Chlamydia trachomatis/genética , Citosol/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos/genética , Células HeLa , Humanos , Imunidade Inata/genética , Proteínas de Membrana Transportadoras/genética , Oligopeptídeos/genética , Óperon/genética , Peptidoglicano/genética
18.
Artigo em Inglês | MEDLINE | ID: mdl-31931329

RESUMO

The silkworm, Bombyx mori, is a promising expression system for the production of recombinant proteins, but the purification of these proteins is not easy because of the large amount of host proteins present. To investigate purity, recovery and scale-up ability of the purification of recombinant proteins expressed in silkworm larval hemolymph without any affinity tags, we used mCherry, a red fluorescence protein, as a model. The host cell proteins could be greatly reduced using a three-step chromatography protocol consisting of hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) and heparin chromatography after heat pretreatment. The thermal treatment had the greatest impact on the removal of host cell extracellular proteins and increasing purity. There were still some minor traces of host cell proteins in the purified sample, which showed that the purification of recombinant proteins from the silkworm hemolymph was still challenging. The proposed protocol and affinity tag purification reduced the overall protein content by 99.84% and 99.95%, respectively, while the amount of DNA was reduced by 98.41% and 99.53%, respectively. Purities of our proposed protocol based on SDS-PAGE and capillary electrophoresis (CE) analyses were 85.45% and 43.60%, respectively, while those of Strep-tag affinity purification were 100% or 63.69%, respectively. Using densitometry, the overall recovery was calculated was 5.78%, which was higher than 4.09% using Strep-tag affinity purification. This proposed protocol, mainly based on thermal treatment, HIC, SEC and HiTrap Heparin HP column chromatography, is applicable to an upscalable purification for the silkworm expression system without employing affinity tag chromatography process.


Assuntos
Bombyx/química , Cromatografia de Afinidade/métodos , Hemolinfa/química , Larva/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Animais , Bombyx/metabolismo , Eletroforese em Gel de Poliacrilamida , Larva/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética
19.
J Exp Biol ; 223(Pt 2)2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31900346

RESUMO

Social isolation strongly modulates behavior across the animal kingdom. We utilized the fruit fly Drosophila melanogaster to study social isolation-driven changes in animal behavior and gene expression in the brain. RNA-seq identified several head-expressed genes strongly responding to social isolation or enrichment. Of particular interest, social isolation downregulated expression of the gene encoding the neuropeptide Drosulfakinin (Dsk), the homologue of vertebrate cholecystokinin (CCK), which is critical for many mammalian social behaviors. Dsk knockdown significantly increased social isolation-induced aggression. Genetic activation or silencing of Dsk neurons each similarly increased isolation-driven aggression. Our results suggest a U-shaped dependence of social isolation-induced aggressive behavior on Dsk signaling, similar to the actions of many neuromodulators in other contexts.


Assuntos
Agressão , Drosophila melanogaster/fisiologia , Neuropeptídeos/metabolismo , Oligopeptídeos/metabolismo , Isolamento Social , Animais , Comportamento Animal/fisiologia , Encéfalo/metabolismo , Drosophila melanogaster/genética , Masculino , Neuropeptídeos/genética , Oligopeptídeos/genética , Análise de Sequência de RNA
20.
Mol Pharmacol ; 97(3): 191-201, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31924695

RESUMO

We have previously shown that the retinoid-related orphan receptor alpha (RORα) phosphorylation plays a pivotal role in sulfotransferase 1E1 gene regulation within mouse liver. Here, we found serine 100-phosphorylated RORα orchestrates constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4 alpha (HNF4α) to induce CYP2B6 by phenobarbital (PB) in human primary hepatocytes (HPHs). RORα knockdown using small interfering RNAs suppressed CYP2B6 mRNAs in HPH, whereas transient expression of RORα in COS-1 cells activated CYP2B6 promoter activity in reporter assays. Through chromatin immunoprecipitation (IP) and gel shift assays, we found that RORα in the form of phosphorylated (p-) S100 directly bound to a newly identified RORα response element (RORα response element on CYP2B6 promoter, -660/-649) within the CYP2B6 promoter in untreated or treated HPH. In PB-treated HPH, p-Ser100 RORα was both enriched in the distal phenobarbital response element module (PBREM) and the proximal okadaic acid response element (OARE), a known HNF4α binding site. Chromatin conformation capture assay revealed direct contact between the PBREM and OARE only in PB-treated HPH. Moreover, CAR preferably interacted with phosphomimetically mutated RORα at Ser100 residue in co-IP assay. A gel shift assay with a radiolabeled OARE module and nuclear extracts prepared from PB-treated mouse liver confirmed that HNF4α formed a complex with Ser 100-phosphorylated RORα, as shown by supershifted complexes with anti-p-Ser100 RORα and anti-HNF4α antibodies. Altogether, the results established that p-Ser100 RORα bridging the PBREM and OARE orchestrates CAR and HNF4α to form active chromatin complex during PB-induced CYP2B6 expression in human primary hepatocytes. SIGNIFICANCE STATEMENT: CYP2B6 is a vital enzyme for the metabolic elimination of xenobiotics, and it is prone to induction by xenobiotics, including phenobarbital via constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4 alpha (HNF4α). Here, we show that retinoid-related orphan receptor alpha (RORα), through phosphorylated S100 residue, orchestrated CAR-HNF4α interaction on the CYP2B6 promoter in human primary hepatocyte cultures. These results signify not only the role of RORα in the molecular process of CYP2B6 induction, but it also reveals the importance of conserved phosphorylation sites within the DNA-binding domain of the receptor.


Assuntos
Cromatina/metabolismo , Indutores do Citocromo P-450 CYP2B6/farmacologia , Fator 4 Nuclear de Hepatócito/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Oligopeptídeos/metabolismo , Fenobarbital/farmacologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Células Cultivadas , Citocromo P-450 CYP2B6/biossíntese , Citocromo P-450 CYP2B6/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia
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