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1.
Viruses ; 12(9)2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32971895

RESUMO

Coronaviruses (CoVs) are enveloped, positive sense, single strand RNA viruses that cause respiratory, intestinal and neurological diseases in mammals and birds. Following replication, CoVs assemble on intracellular membranes including the endoplasmic reticulum Golgi intermediate compartment (ERGIC) where the envelope protein (E) functions in virus assembly and release. In consequence, E potentially contains membrane-modifying peptides. To search for such peptides, the E coding sequence of Mouse Hepatitis Virus (MHV) was inspected for its amino acid conservation, proximity to the membrane and/or predicted amphipathic helices. Peptides identified in silico were synthesized and tested for membrane-modifying activity in the presence of giant unilamellar vesicles (GUVs) consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), sphingomyelin and cholesterol. To confirm the presence of membrane binding peptides identified in the context of a full-length E protein, the wild type and a number of mutants in the putative membrane binding peptide were expressed in Lenti-X-293T mammalian and insect cells, and the distribution of E antigen within the expressing cell was assessed. Our data identify a role for the post-transmembrane region of MHV E in membrane binding.


Assuntos
Vírus da Hepatite Murina/química , Peptídeos/química , Proteínas do Envelope Viral/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Infecções por Coronavirus , Humanos , Membranas Intracelulares/metabolismo , Camundongos , Vírus da Hepatite Murina/genética , Vírus da Hepatite Murina/metabolismo , Mutação , Peptídeos/síntese química , Peptídeos/metabolismo , Células Sf9 , Spodoptera , Lipossomas Unilamelares/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
2.
Molecules ; 25(19)2020 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-32987757

RESUMO

There is a vast practice of using antimalarial drugs, RAS inhibitors, serine protease inhibitors, inhibitors of the RNA-dependent RNA polymerase of the virus and immunosuppressants for the treatment of the severe form of COVID-19, which often occurs in patients with chronic diseases and older persons. Currently, the clinical efficacy of these drugs for COVID-19 has not been proven yet. Side effects of antimalarial drugs can worsen the condition of patients and increase the likelihood of death. Peptides, given their physiological mechanism of action, have virtually no side effects. Many of them are geroprotectors and can be used in patients with chronic diseases. Peptides may be able to prevent the development of the pathological process during COVID-19 by inhibiting SARS-CoV-2 virus proteins, thereby having immuno- and bronchoprotective effects on lung cells, and normalizing the state of the hemostasis system. Immunomodulators (RKDVY, EW, KE, AEDG), possessing a physiological mechanism of action at low concentrations, appear to be the most promising group among the peptides. They normalize the cytokines' synthesis and have an anti-inflammatory effect, thereby preventing the development of disseminated intravascular coagulation, acute respiratory distress syndrome and multiple organ failure.


Assuntos
Anti-Inflamatórios/uso terapêutico , Antivirais/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Fatores Imunológicos/uso terapêutico , Peptídeos/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Fármacos do Sistema Respiratório/uso terapêutico , Doença Aguda , Anti-Inflamatórios/síntese química , Antivirais/síntese química , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/crescimento & desenvolvimento , Infecções por Coronavirus/complicações , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Síndrome da Liberação de Citocina/complicações , Síndrome da Liberação de Citocina/diagnóstico , Síndrome da Liberação de Citocina/tratamento farmacológico , Síndrome da Liberação de Citocina/virologia , Coagulação Intravascular Disseminada/complicações , Coagulação Intravascular Disseminada/diagnóstico , Coagulação Intravascular Disseminada/tratamento farmacológico , Coagulação Intravascular Disseminada/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Fatores Imunológicos/síntese química , Pulmão/irrigação sanguínea , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/virologia , Pandemias , Peptídeos/síntese química , Pneumonia Viral/complicações , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , Insuficiência Respiratória/complicações , Insuficiência Respiratória/diagnóstico , Insuficiência Respiratória/prevenção & controle , Insuficiência Respiratória/virologia , Fármacos do Sistema Respiratório/síntese química , Relação Estrutura-Atividade
3.
Nat Commun ; 11(1): 3895, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753588

RESUMO

The mussel byssus has long been a source of inspiration for the adhesion community. Recently, adhesive synergy between flanking lysine (Lys, K) and 3,4-Dihydroxyphenylalanine (DOPA, Y) residues in the mussel foot proteins (Mfps) has been highlighted. However, the complex topological relationship of DOPA and Lys as well as the interfacial adhesive roles of other amino acids have been understudied. Herein, we study adhesion of Lys and DOPA-containing peptides to organic and inorganic substrates using single-molecule force spectroscopy (SMFS). We show that a modest increase in peptide length, from KY to (KY)3, increases adhesion strength to TiO2. Surprisingly, further increase in peptide length offers no additional benefit. Additionally, comparison of adhesion of dipeptides containing Lys and either DOPA (KY) or phenylalanine (KF) shows that DOPA is stronger and more versatile. We furthermore demonstrate that incorporating a nonadhesive spacer between (KY) repeats can mimic the hidden length in the Mfp and act as an effective strategy to dissipate energy.


Assuntos
Adesivos/química , Di-Hidroxifenilalanina/química , Lisina/química , Sequência de Aminoácidos , Animais , Bivalves , Dipeptídeos , Peptídeos/síntese química , Propriedades de Superfície , Titânio/química
4.
PLoS One ; 15(8): e0237473, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813720

RESUMO

Solid phase peptide synthesis (SPPS) has enabled widespread use of synthetic peptides in applications ranging from pharmaceuticals to materials science. The demand for synthetic peptides has driven recent efforts to produce automated SPPS synthesizers which utilize fluid-handling components common to chemistry laboratories to drive costs down to several thousand dollars. Herein, we describe the design and validation of a more 'frugal' SPPS synthesizer that uses inexpensive, consumer-grade fluid-handling components to achieve a prototype price point between US$300 and $600. We demonstrated functionality by preparing and characterizing peptides with a variety of distinct properties including binding functionality, nanoscale self-assembly, and oxidation-induced fluorescence. This system yielded micromoles of peptide at a cost of approximately $1/residue, a cost which may be further reduced by optimization and bulk purchasing.


Assuntos
Peptídeos/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Sequência de Aminoácidos , Automação , Peptídeos Penetradores de Células/síntese química , Peptídeos Penetradores de Células/química , Desenho de Equipamento , Fluorometria , Nanoestruturas/química , Oxirredução , Peptídeos/química , Técnicas de Síntese em Fase Sólida/economia , Técnicas de Síntese em Fase Sólida/instrumentação
5.
Nat Commun ; 11(1): 3818, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732937

RESUMO

The formation of peptide bonds by energetic processing of amino acids is an important step towards the formation of biologically relevant molecules. As amino acids are present in space, scenarios have been developed to identify the roots of life on Earth, either by processes occurring in outer space or on Earth itself. We study the formation of peptide bonds in single collisions of low-energy He2+ ions (α-particles) with loosely bound clusters of ß-alanine molecules at impact energies typical for solar wind. Experimental fragmentation mass spectra produced by collisions are compared with results of molecular dynamics simulations and an exhaustive exploration of potential energy surfaces. We show that peptide bonds are efficiently formed by water molecule emission, leading to the formation of up to tetrapeptide. The present results show that a plausible route to polypeptides formation in space is the collision of energetic ions with small clusters of amino acids.


Assuntos
Aminoácidos/química , Simulação de Dinâmica Molecular , Peptídeos/química , Termodinâmica , beta-Alanina/química , Dipeptídeos/síntese química , Dipeptídeos/química , Íons/química , Oligopeptídeos/síntese química , Oligopeptídeos/química , Peptídeos/síntese química , Espectrometria de Massas por Ionização por Electrospray/métodos , Água/química
6.
Proc Natl Acad Sci U S A ; 117(28): 16127-16137, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601214

RESUMO

Thrombogenic reaction, aggressive smooth muscle cell (SMC) proliferation, and sluggish endothelial cell (EC) migration onto bioinert metal vascular stents make poststenting reendothelialization a dilemma. Here, we report an easy to perform, biomimetic surface engineering strategy for multiple functionalization of metal vascular stents. We first design and graft a clickable mussel-inspired peptide onto the stent surface via mussel-inspired adhesion. Then, two vasoactive moieties [i.e., the nitric-oxide (NO)-generating organoselenium (SeCA) and the endothelial progenitor cell (EPC)-targeting peptide (TPS)] are clicked onto the grafted surfaces via bioorthogonal conjugation. We optimize the blood and vascular cell compatibilities of the grafted surfaces through changing the SeCA/TPS feeding ratios. At the optimal ratio of 2:2, the surface-engineered stents demonstrate superior inhibition of thrombosis and SMC migration and proliferation, promotion of EPC recruitment, adhesion, and proliferation, as well as prevention of in-stent restenosis (ISR). Overall, our biomimetic surface engineering strategy represents a promising solution to address clinical complications of cardiovascular stents and other blood-contacting metal materials.


Assuntos
Adesivos/química , Materiais Revestidos Biocompatíveis/química , Peptídeos/química , Stents , Adesivos/síntese química , Animais , Materiais Biomiméticos/síntese química , Materiais Biomiméticos/química , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Química Click , Células Progenitoras Endoteliais/citologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Humanos , Miócitos de Músculo Liso/citologia , Óxido Nítrico/química , Compostos Organosselênicos/química , Peptídeos/síntese química , Proteínas/química , Coelhos , Stents/efeitos adversos , Trombose/etiologia , Trombose/prevenção & controle
7.
J Chromatogr A ; 1625: 461237, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709313

RESUMO

The quest for ligands alternative to Protein A for the purification of monoclonal antibodies (mAbs) has been pursued for almost three decades. Yet, the IgG-binding peptides known to date still fall short of the host cell protein (HCP) logarithmic removal value (LRV) set by Protein A media (2.5-3.1). In this study, we present an integrated computational-experimental approach leading to the discovery of peptide ligands that provide HCP LRVs on par with Protein A. First, the screening of 60,000 peptide variants was performed using a high-throughput search algorithm to identify sequences that ensure IgG affinity binding. Select sequences WQRHGI, MWRGWQ, RHLGWF, and GWLHQR were then negatively screened in silico against a panel of model HCPs to ensure the selection of peptides with high binding selectivity. Candidate ligands WQRHGI and MWRGWQ were conjugated to chromatographic resins and characterized by isothermal binding and breakthrough assays to quantify static and dynamic binding capacity (Qmax and DBC10%), respectively. The resulting Qmax were 52.6 mg of IgG per mL of adsorbent for WQRHGI and 57.48 mg/mL for MWRGWQ, while the DBC10% (2 minutes residence time) were 30.1 mg/mL for WQRHGI and 36.4 mg/mL for MWRGWQ. Evaluation of the peptides by isothermal titration calorimetry (ITC) confirmed the binding energy predicted in silico, and an amino acid scanning study corroborated the affinity-like binding activity of the peptides. WQRHGI-WorkBeads resin was finally characterized by purification of a monoclonal antibody from a Chinese Hamster Ovary (CHO) cell culture harvest, affording a remarkable HCP LRV of 2.7, and consistent product yield and purity over 100 chromatographic cycles. These results demonstrate the potential of WQRHGI as an effective alternative to Protein A for antibody purification.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade/métodos , Peptídeos/química , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Cricetulus , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Ligantes , Peptídeos/síntese química , Peptídeos/metabolismo , Ligação Proteica , Proteína Estafilocócica A/química , Proteína Estafilocócica A/metabolismo
8.
Nat Commun ; 11(1): 2793, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32493905

RESUMO

Biology utilizes multiple strategies, including sequestration in lipid vesicles, to raise the rate and specificity of chemical reactions through increases in effective molarity of reactants. We show that micelle-assisted reaction can facilitate native chemical ligations (NCLs) between a peptide-thioester - in which the thioester leaving group contains a lipid-like alkyl chain - and a Cys-peptide modified by a lipid-like moiety. Hydrophobic lipid modification of each peptide segment promotes the formation of mixed micelles, bringing the reacting peptides into close proximity and increasing the reaction rate. The approach enables the rapid synthesis of polypeptides using low concentrations of reactants without the need for thiol catalysts. After NCL, the lipid moiety is removed to yield an unmodified ligation product. This micelle-based methodology facilitates the generation of natural peptides, like Magainin 2, and the derivatization of the protein Ubiquitin. Formation of mixed micelles from lipid-modified reactants shows promise for accelerating chemical reactions in a traceless manner.


Assuntos
Lipídeos/química , Micelas , Peptídeos/química , Tensoativos/química , Sequência de Aminoácidos , Cinética , Luz , Magaininas/síntese química , Magaininas/química , Peptídeos/síntese química , Ubiquitina/metabolismo
9.
PLoS One ; 15(6): e0234901, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579565

RESUMO

Lasso peptides are unique in that the tail of the lasso peptide threads through its macrolactam ring. The unusual structure and biological activity of lasso peptides have generated increased interest from the scientific community in recent years. Because of this, many new types of lasso peptides have been discovered. These peptides can be synthesized by microorganisms efficiently, and yet, their chemical assembly is challenging. Herein, we investigated the possibility of high pressure inducing the cyclization of linear precursors of lasso peptides. Unlike other molecules like rotaxanes which mechanically interlock at high pressure, the threaded lasso peptides did not form, even at pressures the high pressure up to 14 000 kbar.


Assuntos
Peptídeos/química , Peptídeos/síntese química , Sequência de Aminoácidos , Ciclização , Dissulfetos/química , Oxirredução , Pressão , Conformação Proteica , Soluções
10.
Aging (Albany NY) ; 12(12): 11263-11276, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32544884

RESUMO

The outbreak of COVID-19 has now become a global pandemic that has severely impacted lives and economic stability. There is, however, no effective antiviral drug that can be used to treat COVID-19 to date. Built on the fact that SARS-CoV-2 initiates its entry into human cells by the receptor binding domain (RBD) of its spike protein binding to the angiotensin-converting enzyme 2 (hACE2), we extended a recently developed approach, EvoDesign, to design multiple peptide sequences that can competitively bind to the SARS-CoV-2 RBD to inhibit the virus from entering human cells. The protocol starts with the construction of a hybrid peptidic scaffold by linking two fragments grafted from the interface of the hACE2 protein (a.a. 22-44 and 351-357) with a linker glycine, which is followed by the redesign and refinement simulations of the peptide sequence to optimize its binding affinity to the interface of the SARS-CoV-2 RBD. The binding experiment analyses showed that the designed peptides exhibited a significantly stronger binding potency to hACE2 than the wild-type hACE2 receptor (with -53.35 vs. -46.46 EvoEF2 energy unit scores for the top designed and wild-type peptides, respectively). This study demonstrates a new avenue to utilize computationally designed peptide motifs to treat the COVID-19 disease by blocking the critical spike-RBD and hACE2 interactions.


Assuntos
Infecções por Coronavirus/tratamento farmacológico , Peptídeos/síntese química , Peptídeos/farmacologia , Peptidil Dipeptidase A/fisiologia , Pneumonia Viral/tratamento farmacológico , Glicoproteína da Espícula de Coronavírus/fisiologia , Sequência de Aminoácidos , Antivirais , Sítios de Ligação , Desenho de Fármacos , Evolução Molecular , Humanos , Modelos Moleculares , Pandemias , Ligação Proteica , Conformação Proteica , Internalização do Vírus/efeitos dos fármacos
11.
Science ; 368(6494): 980-987, 2020 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-32467387

RESUMO

Ribosomes can produce proteins in minutes and are largely constrained to proteinogenic amino acids. Here, we report highly efficient chemistry matched with an automated fast-flow instrument for the direct manufacturing of peptide chains up to 164 amino acids long over 327 consecutive reactions. The machine is rapid: Peptide chain elongation is complete in hours. We demonstrate the utility of this approach by the chemical synthesis of nine different protein chains that represent enzymes, structural units, and regulatory factors. After purification and folding, the synthetic materials display biophysical and enzymatic properties comparable to the biologically expressed proteins. High-fidelity automated flow chemistry is an alternative for producing single-domain proteins without the ribosome.


Assuntos
Peptídeos/síntese química , Proteínas/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Peptídeos/química , Peptídeos/isolamento & purificação , Domínios Proteicos , Dobramento de Proteína , Proteínas/química , Proteínas/isolamento & purificação
12.
Org Biomol Chem ; 18(15): 2823-2827, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32232252

RESUMO

Here, we report peptide probes with either single or cyclic double stranded collagen-like sequences that spontaneously acquire collagen-hybridizing ability at physiological pH. These peptides have ester bonds derived from O-acyl isopeptide units that are converted to amide bonds via intramolecular O-to-N acyl migration by a pH shift. The peptides that do not require pre-treatment for disassembly will be useful as prodrugs in theranostic treatments targeting unfolded collagen.


Assuntos
Colágeno/química , Peptídeos/química , Concentração de Íons de Hidrogênio , Conformação Molecular , Peptídeos/síntese química
13.
Chem Commun (Camb) ; 56(16): 2507-2510, 2020 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-32003763

RESUMO

Radiolabeling of peptides with fluorine-18 is hurdled by their chemical sensitivity and complicated processes. Original triflyl-pyridine intermediates afforded ammonium precursors that were radiolabeled at low temperature. From that study, a generic tag has been designed to allow a simple one-step/late-stage radiolabelling of peptides. The strategy has been transposed to an automated "on-resin" radiolabelling.


Assuntos
Peptídeos/síntese química , Compostos Radiofarmacêuticos/síntese química , Resinas Sintéticas/síntese química , Radioisótopos de Flúor , Halogenação , Estrutura Molecular , Peptídeos/química , Compostos Radiofarmacêuticos/química , Resinas Sintéticas/química , Temperatura
14.
Chem Commun (Camb) ; 56(18): 2767-2770, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32022009

RESUMO

We synthesized "glyco-arylopeptides", whose folding structure significantly changes depending on the kind of saccharide in their side chain. The saccharide moiety interacts with the main chain via hydrogen bonding, and the non-natural polypeptides form two well-defined architectures-(P)-31- and (M)-41-helices-depending on the length of the saccharide chains and even the configuration of a single stereo-genic center in the epimers.


Assuntos
Glicopeptídeos/química , Oligossacarídeos/química , Peptídeos/síntese química , Teoria da Densidade Funcional , Estrutura Molecular , Peptídeos/química , Dobramento de Proteína
15.
Int J Nanomedicine ; 15: 1117-1128, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32110011

RESUMO

Introduction: Antibiotic-resistant bacteria kill 25,000 people every year in the EU. Patients subject to recurrent lung infections are the most vulnerable to severe or even lethal infections. For these patients, pulmonary delivery of antibiotics would be advantageous, since inhalation can achieve higher concentration in the lungs than iv administration and can provide a faster onset of action. This would allow for the delivery of higher doses and hence reduce the number of treatments required. We report here about a new nanosystem (M33-NS) obtained by capturing SET-M33 peptide on single-chain dextran nanoparticles. SET-M33 is a non-natural antimicrobial peptide synthesized in branched form. This form gives the peptide resistance to degradation in biological fluids. SET-M33 has previously shown efficacy in vitro against about one hundred of Gram-negative multidrug and extensively drug-resistant clinical isolates and was also active in preclinical infection models of pneumonia, sepsis and skin infections. Methods: The new nanosystem was evaluated for its efficacy in bacteria cells and in a mouse model of pneumonia. Toxicity and genotoxicity were also tested in vitro. Biodistribution and pharmacokinetic studies in healthy rats were carried out using a radiolabeled derivative of the nanosystem. Results: The M33-nanosystem, studied here, showed to be effective against Pseudomonas aeruginosa in time-kill kinetic experiments. Cytotoxicity towards different animal cell lines was acceptable. Lung residence time of the antimicrobial peptide, administered via aerosol in healthy rats, was markedly improved by capturing SET-M33 on dextran nanoparticles. M33-NS was also efficient in eradicating pulmonary infection in a BALB/c mouse model of pneumonia caused by P. aeruginosa. Discussion: This study revealed that the encapsulation of the antimicrobial peptide in dextran nanoparticles markedly improved lung residence time of the peptide administered via aerosol. The result has to be considered among the aims of the development of a new therapeutic option for patients suffering recurrent infections, that will benefit from high local doses of persistent antimicrobials.


Assuntos
Antibacterianos/administração & dosagem , Nanopartículas/administração & dosagem , Peptídeos/administração & dosagem , Infecções por Pseudomonas/tratamento farmacológico , Administração por Inalação , Animais , Antibacterianos/farmacologia , Dextranos , Sistemas de Liberação de Medicamentos/métodos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Nanopartículas/química , Peptídeos/síntese química , Peptídeos/farmacologia , Pneumonia Bacteriana/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Ratos , Terapia Respiratória , Distribuição Tecidual
16.
Biochem Biophys Res Commun ; 524(4): 923-928, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32057360

RESUMO

Amyloid ß (Aß) oligomers may be a real culprit in the pathogenesis of Alzheimer's disease (AD); therefore, the elimination of these toxic oligomers may be of great significance for AD therapy. Autophagy is the catabolic process by which lysosomes degrade cytosolic components, and heat shock cognate 70 kDa protein (Hsc70) binds to proteins with their KFERQ-like motifs [also known as chaperone-mediated autophagy (CMA) motifs] and carries them to lysosomes through CMA or late endosomes through endosomal microautophagy (eMI) for degradation. In this study, our strategy is to make the pathological Aß become one selective and suitable substrate for CMA and eMI (termed as Hsc70-based autophagy) by tagging its oligomers with multiple CMA motifs. First, we design and synthesize Aß oligomer binding peptides with three CMA motifs. Second, we determine that the peptide can help Aß oligomers enter endosomes and lysosomes, which can be further enhanced by ketone. More importantly, we find that the peptide can dramatically reduce Aß oligomers in induced pluripotent stem cell (iPSC) cortical neurons derived from AD patient fibroblasts and protect primary cultured cortical neurons against the Aß oligomer-induced neurotoxicity. In conclusion, we demonstrate that the peptide targeting Hsc70-based autophagy can effectively eliminate Aß oligomers and have superior neuroprotective activity.


Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Autofagia Mediada por Chaperonas/efeitos dos fármacos , Proteínas de Choque Térmico HSC70/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Peptídeos/farmacologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Motivos de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Diferenciação Celular , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteínas de Choque Térmico HSC70/genética , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Terapia de Alvo Molecular , Neurônios/metabolismo , Neurônios/patologia , Fármacos Neuroprotetores/síntese química , Peptídeos/síntese química , Cultura Primária de Células , Ligação Proteica , Proteólise , Ratos , Ratos Long-Evans
17.
Biochim Biophys Acta Mol Cell Res ; 1867(6): 118674, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32035967

RESUMO

Increased Pur-alpha (Pura) protein levels in animal models alleviate certain cellular symptoms of the disease spectrum amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). Pura is a member of the Pur family of evolutionarily conserved guanine-rich polynucleotide binding proteins containing a repeated signature PUR domain of 60-80 amino acids. Here we have employed a synthetic peptide, TZIP, similar to a Pur domain, but with sequence alterations based on a consensus of evolutionarily conserved Pur family binding domains and having an added transporter sequence. A major familial form of ALS/FTD, C9orf72 (C9), is due to a hexanucleotide repeat expansion (HRE) of (GGGGCC), a Pur binding element. We show by circular dichroism that RNA oligonucleotides containing this purine-rich sequence consist largely of parallel G-quadruplexes. TZIP peptide binds this repeat sequence in both DNA and RNA. It binds the RNA element, including the G-quadruplexes, with a high degree of specificity versus a random oligonucleotide. In addition, TZIP binds both linear and G-quadruplex repeat RNA to form higher order G-quadruplex secondary structures. This change in conformational form by Pur-based peptide represents a new mechanism for regulating G quadruplex secondary structure within the C9 repeat. TZIP modulation of C9 RNA structural configuration may alter interaction of the complex with other proteins. This Pur-based mechanism provides new targets for therapy, and it may help to explain Pura alleviation of certain cellular pathological aspects of ALS/FTD.


Assuntos
Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Proteínas de Ligação a DNA/química , Peptídeos/farmacologia , Fatores de Transcrição/química , Proteína C9orf72/química , Dicroísmo Circular , Expansão das Repetições de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Quadruplex G/efeitos dos fármacos , Humanos , Modelos Moleculares , Mimetismo Molecular , Peptídeos/síntese química , RNA/química , RNA/metabolismo , Termodinâmica , Fatores de Transcrição/metabolismo
18.
Nat Commun ; 11(1): 982, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080186

RESUMO

Although peptide chemistry has made great progress, the frequent occurrence of aspartimide formation during peptide synthesis remains a formidable challenge. Aspartimide formation leads to low yields in addition to costly purification or even inaccessible peptide sequences. Here, we report an alternative approach to address this longstanding challenge of peptide synthesis by utilizing cyanosulfurylides to mask carboxylic acids by a stable C-C bond. These functional groups-formally zwitterionic species-are exceptionally stable to all common manipulations and impart improved solubility during synthesis. Deprotection is readily and rapidly achieved under aqueous conditions with electrophilic halogenating agents via a highly selective C-C bond cleavage reaction. This protecting group is employed for the synthesis of a range of peptides and proteins including teduglutide, ubiquitin, and the low-density lipoprotein class A. This protecting group strategy has the potential to overcome one of the most difficult aspects of modern peptide chemistry.


Assuntos
Peptídeos/química , Peptídeos/síntese química , Técnicas de Síntese em Fase Sólida/métodos , Ácido Aspártico/análogos & derivados , Ácido Aspártico/síntese química , Ácido Aspártico/química , Ácidos Carboxílicos/química , Cianetos/química , Lipoproteínas LDL/síntese química , Lipoproteínas LDL/química , Dobramento de Proteína , Ubiquitina/química
20.
Cancer Sci ; 111(4): 1357-1366, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31991041

RESUMO

Survivin belongs to the inhibitor of apoptosis protein family, which is consistently overexpressed in most cancer cells but rarely expressed in normal adult tissues. Therefore, the detection and inhibition of survivin are regarded as attractive strategies for cancer-specific treatment. In this study, we designed and synthesized 7-19 residues of inner centromere protein (INCENP)-derived small peptides (INC peptides) as novel survivin-targeting agents. The INC peptides showed binding affinity for the human survivin protein (Kd  = 91.4-255 nmol L-1 ); INC16-22 , which contains residues 16-22 of INCENP, showed the highest affinity (91.4 nmol L-1 ). Confocal fluorescence imaging showed consistent colocalization of FITC-INC16-22 and survivin in cell lines. Nona-arginine-linked INC16-22 (r9-INC16-22 ) rendered INC16-22 cells penetrable and strongly inhibited cell growth of MIA PaCa-2 cells (52% inhibition at 1.0 µmol L-1 ) and MDA-MB-231 cells (60% inhibition at 10 µmol L-1 ) as determined by MTT assays. The exposure of MIA PaCa-2 cells to 40 µmol L-1 r9-INC16-22 apparently reduced the intracellular protein expression levels of survivin. However, cleaved caspase-3 was significantly increased in cells treated with r9-INC16-22 , even at 10 µmol L-1 , compared to untreated cells. Flow cytometry revealed that r9-INC16-22 strongly induced apoptosis in MIA PaCa-2 cells. These results indicate that the cytotoxic effects of r9-INC16-22 could be mediated mainly through the disruption of survivin-dependent antiapoptotic functions and partly because of the direct degradation of the survivin protein. Our findings suggest that INC peptides can act as useful scaffolds for novel cancer imaging and anticancer agents.


Assuntos
Neoplasias da Mama/diagnóstico por imagem , Proteínas Cromossômicas não Histona/genética , Peptídeos/farmacologia , Survivina/isolamento & purificação , Apoptose/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caspases/química , Caspases/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Proteínas Cromossômicas não Histona/química , Feminino , Humanos , Proteínas Inibidoras de Apoptose/química , Proteínas Inibidoras de Apoptose/isolamento & purificação , Imagem Molecular/métodos , Peptídeos/síntese química , Peptídeos/química , Survivina/química , Survivina/genética
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