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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
3.
Hautarzt ; 71(10): 741-751, 2020 Oct.
Artigo em Alemão | MEDLINE | ID: mdl-32880662

RESUMO

Melanocortins are peptides that share a common central pharmacophor. Melanin pigmentation of interfollicular epidermis and hair via MC1R remains the key physiologic function of the naturally occurring melanocortin peptides in skin. Moreover, the melanocortins are crucially involved in the ultraviolet light-induced tanning response. Under pathophysiologic conditions, melanocortin peptides induce cutaneous hyperpigmentation, likewise via the MC1R axis, e.g. in patients with Addison's disease, ectopic precursor pro-opiomelanocortin (POMC) syndrome and in those with abnormally elevated melanocortin blood levels. Translational research on α­MSH (melanocyte-stimulating hormones) and their antagonists has further revealed a variety of other biological activities beyond pigmentation. They include cytoprotection, antioxidative effects, regulation of collagen metabolism and fibrosis, sebum production, and cutaneous wound healing. These findings have also promoted the development of novel therapies in clinical dermatology including the exploitation of afamelanotide. In 2015, this agent became the first in-class synthetic α­MSH analogue to be approved in dermatology for the treatment of erythropoetic protoporphyria. In addition to afamelanotide, setmelanotide has recently emerged as a highly selective MC4R agonist useful for the treatment of distinct forms of genetically determined obesity, e.g., POMC deficiency. Future perspectives in dermatology reside in treatment of other difficult-to-treat skin diseases with α­MSH analogues, either with topical or systemic formulations. Moreover, synthetic melanocortin peptide derivatives lacking the central pharmacophor but with maintained anti-inflammatory effects could become a promising strategy for the design of new therapies in dermatology.


Assuntos
Dermatologia/tendências , Melanocortinas/química , Peptídeos/química , Pesquisa Médica Translacional , Humanos , Inflamação/metabolismo , Melanocortinas/farmacologia , Peptídeos/farmacologia , Pró-Opiomelanocortina , Pele/metabolismo , alfa-MSH
4.
PLoS One ; 15(9): e0238089, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32903266

RESUMO

A novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) is the source of a current pandemic (COVID-19) with devastating consequences in public health and economic stability. Using a peptide array to map the antibody response of plasma from healing patients (12) and heathy patients (6), we identified three immunodominant linear epitopes, two of which correspond to key proteolytic sites on the spike protein (S1/S2 and S2') known to be critical for cellular entry. We show biochemical evidence that plasma positive for the epitope adjacent to the S1/S2 cleavage site inhibits furin-mediated proteolysis of spike.


Assuntos
Infecções por Coronavirus/patologia , Epitopos/química , Pneumonia Viral/patologia , Sequência de Aminoácidos , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Mapeamento de Epitopos , Epitopos/sangue , Epitopos/imunologia , Furina/metabolismo , Humanos , Pandemias , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , Pneumonia Viral/virologia , Análise Serial de Proteínas , Estrutura Terciária de Proteína , Proteólise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
5.
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
6.
J Chromatogr A ; 1628: 461443, 2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32822982

RESUMO

Sodium dodecyl sulfate (SDS) in proteomics samples needs to be removed and estimated prior to mass spectrometry (MS)-based analysis and to avoid MS ion-source contamination. Here, we describe an organic solvent free method to remove SDS using a simple apparatus that mainly consists of an agarose gel inside a 1 mL plastic micropipette tip and a voltage power supply with electrodes. A small volume of sample (e.g., 50 µL) is loaded on top of the gel and then voltage (cathode at the sample side) is applied with an acidic solution at the other end of the micropipette tip. Within 25 min, SDS was removed (e.g., ≥99% SDS in 3.5 mM SDS) and the peptides were retained in the sample solution. The strategy was compared to the commercially available and expensive Pierce spin column for the removal of SDS and recovery of peptides from a digested bovine serum albumin sample.


Assuntos
Técnicas de Química Analítica/métodos , Técnicas Eletroquímicas , Proteômica/métodos , Dodecilsulfato de Sódio/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Peptídeos/química , Soroalbumina Bovina/química , Dodecilsulfato de Sódio/química
7.
Nat Commun ; 11(1): 4170, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32820174

RESUMO

Sulfur-sulfur motifs widely occur in vital function and drug design, which yearns for polysulfide construction in an efficient manner. However, it is a great challenge to install desired functional groups on both sides of sulfur-sulfur bonds at liberty. Herein, we designed a mesocyclic bilateral disulfurating reagent for sequential assembly and modular installation of polysulfides. Based on S-O bond dissociation energy imparity (mesocyclic compared to linear imparity is at least 5.34 kcal mol-1 higher), diverse types of functional molecules can be bridged via sulfur-sulfur bonds distinctly. With these stable reagents, excellent reactivities with nucleophiles including C, N and S are comprehensively demonstrated, sequentially installing on both sides of sulfur-sulfur motif with various substituents to afford six species of unsymmetrical polysulfides including di-, tri- and even tetra-sulfides. Life-related molecules, natural products and pharmaceuticals can be successively cross-linked with sulfur-sulfur bond. Remarkably, the cyclization of tri- and tetra-peptides affords 15- and 18-membered cyclic disulfide peptides with this reagent, respectively.


Assuntos
Dissulfetos/química , Indicadores e Reagentes/química , Peptídeos/química , Sulfetos/química , Enxofre/química , Produtos Biológicos/química , Técnicas de Química Sintética/métodos , Ciclização , Indicadores e Reagentes/síntese química , Modelos Químicos , Estrutura Molecular , Oxirredução , Preparações Farmacêuticas/química
8.
PLoS One ; 15(8): e0237328, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790707

RESUMO

α-Synuclein (αSyn) fibrils spread from one neuronal cell to another. This prion-like phenomenon is believed to contribute to the progression of the pathology in Parkinson's disease and other synucleinopathies. The binding of αSyn fibrils originating from affected cells to the plasma membrane of naïve cells is key in their prion-like propagation propensity. To interfere with this process, we designed polypeptides derived from proteins we previously showed to interact with αSyn fibrils, namely the molecular chaperone Hsc70 and the sodium/potassium pump NaK-ATPase and assessed their capacity to bind αSyn fibrils and/or interfere with their take-up by cells of neuronal origin. We demonstrate here that polypeptides that coat αSyn fibrils surfaces in such a way that they are changed affect αSyn fibrils binding to the plasma membrane components and/or their take-up by cells. Altogether our observations suggest that the rationale design of αSyn fibrils polypeptide binders that interfere with their propagation between neuronal cells holds therapeutic potential.


Assuntos
Neurônios/efeitos dos fármacos , Peptídeos/farmacologia , Agregação Patológica de Proteínas/tratamento farmacológico , alfa-Sinucleína/metabolismo , Sequência de Aminoácidos , Amiloide/antagonistas & inibidores , Amiloide/metabolismo , Animais , Linhagem Celular , Proteínas de Choque Térmico HSC70/química , Proteínas de Choque Térmico HSC70/metabolismo , Proteínas de Choque Térmico HSC70/farmacologia , Humanos , Camundongos , Modelos Moleculares , Neurônios/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Peptídeos/química , Príons/antagonistas & inibidores , Príons/metabolismo , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/farmacologia
9.
Protein Sci ; 29(10): 2038-2042, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32822073

RESUMO

The Envelope protein (E) is one of the four structural proteins encoded by the genome of SARS-CoV and SARS-CoV-2 Coronaviruses. It is an integral membrane protein, highly expressed in the host cell, which is known to have an important role in Coronaviruses maturation, assembly and virulence. The E protein presents a PDZ-binding motif at its C-terminus. One of the key interactors of the E protein in the intracellular environment is the PDZ containing protein PALS1. This interaction is known to play a key role in the SARS-CoV pathology and suspected to affect the integrity of the lung epithelia. In this paper we measured and compared the affinity of peptides mimicking the E protein from SARS-CoV and SARS-CoV-2 for the PDZ domain of PALS1, through equilibrium and kinetic binding experiments. Our results support the hypothesis that the increased virulence of SARS-CoV-2 compared to SARS-CoV may rely on the increased affinity of its Envelope protein for PALS1.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Proteínas de Membrana/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Pneumonia Viral/metabolismo , Vírus da SARS/metabolismo , Síndrome Respiratória Aguda Grave/metabolismo , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Betacoronavirus/química , Sítios de Ligação , Infecções por Coronavirus/virologia , Humanos , Proteínas de Membrana/química , Modelos Moleculares , Núcleosídeo-Fosfato Quinase/química , Domínios PDZ , Pandemias , Peptídeos/química , Peptídeos/metabolismo , Pneumonia Viral/virologia , Ligação Proteica , Vírus da SARS/química , Síndrome Respiratória Aguda Grave/virologia , Proteínas do Envelope Viral/química
10.
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
11.
SAR QSAR Environ Res ; 31(9): 643-654, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32847369

RESUMO

A quantitative structure-activity relationship (QSAR) model was built from a dataset of 54 peptide-type compounds as SARS-CoV inhibitors. The analysis was executed to identify prominent and hidden structural features that govern anti-SARS-CoV activity. The QSAR model was derived from the genetic algorithm-multi-linear regression (GA-MLR) methodology. This resulted in the generation of a statistically robust and highly predictive model. In addition, it satisfied the OECD principles for QSAR validation. The model was validated thoroughly and fulfilled the threshold values of a battery of statistical parameters (e.g. r 2 = 0.87, Q 2 loo = 0.82). The derived model is successful in identifying many atom-pairs as important structural features that govern the anti-SARS-CoV activity of peptide-type compounds. The newly developed model has a good balance of descriptive and statistical approaches. Consequently, the present work is useful for future modifications of peptide-type compounds for SARS-CoV and SARS-CoV-2 activity.


Assuntos
Antivirais , Betacoronavirus/efeitos dos fármacos , Peptídeos , Relação Quantitativa Estrutura-Atividade , Antivirais/química , Antivirais/farmacologia , Betacoronavirus/enzimologia , Cisteína Endopeptidases , Concentração Inibidora 50 , Modelos Lineares , Estrutura Molecular , Peptídeos/química , Peptídeos/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores
12.
Nat Commun ; 11(1): 4252, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843628

RESUMO

The 2019 novel respiratory virus (SARS-CoV-2) causes COVID-19 with rapid global socioeconomic disruptions and disease burden to healthcare. The COVID-19 and previous emerging virus outbreaks highlight the urgent need for broad-spectrum antivirals. Here, we show that a defensin-like peptide P9R exhibited potent antiviral activity against pH-dependent viruses that require endosomal acidification for virus infection, including the enveloped pandemic A(H1N1)pdm09 virus, avian influenza A(H7N9) virus, coronaviruses (SARS-CoV-2, MERS-CoV and SARS-CoV), and the non-enveloped rhinovirus. P9R can significantly protect mice from lethal challenge by A(H1N1)pdm09 virus and shows low possibility to cause drug-resistant virus. Mechanistic studies indicate that the antiviral activity of P9R depends on the direct binding to viruses and the inhibition of virus-host endosomal acidification, which provides a proof of concept that virus-binding alkaline peptides can broadly inhibit pH-dependent viruses. These results suggest that the dual-functional virus- and host-targeting P9R can be a promising candidate for combating pH-dependent respiratory viruses.


Assuntos
Antivirais/farmacologia , Coronavirus/efeitos dos fármacos , Vírus da Influenza A/efeitos dos fármacos , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , Linhagem Celular , Endossomos/química , Endossomos/efeitos dos fármacos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Vírus da Influenza A/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Ligação Proteica , Conformação Proteica , Rhinovirus/efeitos dos fármacos , Rhinovirus/metabolismo , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
13.
Sci Rep ; 10(1): 14179, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843695

RESUMO

A novel coronavirus (SARS-CoV-2) emerged from China in late 2019 and rapidly spread across the globe, infecting millions of people and generating societal disruption on a level not seen since the 1918 influenza pandemic. A safe and effective vaccine is desperately needed to prevent the continued spread of SARS-CoV-2; yet, rational vaccine design efforts are currently hampered by the lack of knowledge regarding viral epitopes targeted during an immune response, and the need for more in-depth knowledge on betacoronavirus immunology. To that end, we developed a computational workflow using a series of open-source algorithms and webtools to analyze the proteome of SARS-CoV-2 and identify putative T cell and B cell epitopes. Utilizing a set of stringent selection criteria to filter peptide epitopes, we identified 41 T cell epitopes (5 HLA class I, 36 HLA class II) and 6 B cell epitopes that could serve as promising targets for peptide-based vaccine development against this emerging global pathogen. To our knowledge, this is the first study to comprehensively analyze all 10 (structural, non-structural and accessory) proteins from SARS-CoV-2 using predictive algorithms to identify potential targets for vaccine development.


Assuntos
Betacoronavirus/imunologia , Biologia Computacional , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Interações Hospedeiro-Patógeno/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Betacoronavirus/classificação , Betacoronavirus/genética , Betacoronavirus/metabolismo , Biologia Computacional/métodos , Infecções por Coronavirus/metabolismo , Epitopos de Linfócito B/química , Epitopos de Linfócito T/química , Genoma Viral , Genômica/métodos , Humanos , Modelos Moleculares , Pandemias , Peptídeos/química , Peptídeos/imunologia , Filogenia , Pneumonia Viral/metabolismo , Relação Estrutura-Atividade , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vacinas de Subunidades/imunologia , Proteínas Virais/química , Vacinas Virais/imunologia
14.
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
15.
Nat Commun ; 11(1): 4065, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792501

RESUMO

Identification of post-translationally or chemically modified peptides in mass spectrometry-based proteomics experiments is a crucial yet challenging task. We have recently introduced a fragment ion indexing method and the MSFragger search engine to empower an open search strategy for comprehensive analysis of modified peptides. However, this strategy does not consider fragment ions shifted by unknown modifications, preventing modification localization and limiting the sensitivity of the search. Here we present a localization-aware open search method, in which both modification-containing (shifted) and regular fragment ions are indexed and used in scoring. We also implement a fast mass calibration and optimization method, allowing optimization of the mass tolerances and other key search parameters. We demonstrate that MSFragger with mass calibration and localization-aware open search identifies modified peptides with significantly higher sensitivity and accuracy. Comparing MSFragger to other modification-focused tools (pFind3, MetaMorpheus, and TagGraph) shows that MSFragger remains an excellent option for fast, comprehensive, and sensitive searches for modified peptides in shotgun proteomics data.


Assuntos
Peptídeos/química , Algoritmos , Animais , Bases de Dados de Proteínas , Humanos , Espectrometria de Massas , Proteômica/métodos
16.
Int J Mol Sci ; 21(15)2020 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-32751407

RESUMO

Enveloped viruses include the most dangerous human and animal pathogens, in particular coronavirus, influenza virus, and human immunodeficiency virus (HIV). For these viruses, receptor binding and entry are accomplished by a single viral envelope protein (termed the fusion protein), the structural changes of which trigger the remodeling and merger of the viral and target cellular membranes. The number of fusion proteins required for fusion activity is still under debate, and several studies report this value to range from 1 to 9 for type I fusion proteins. Here, we consider the earliest stage of viral fusion based on the continuum theory of membrane elasticity. We demonstrate that membrane deformations induced by the oblique insertion of amphipathic fusion peptides mediate the lateral interaction of these peptides and drive them to form into a symmetric fusion rosette. The pulling force produced by the structural rearrangements of the fusion protein ectodomains gives additional torque, which deforms the membrane and additionally stabilizes the symmetric fusion rosette, thus allowing a reduction in the number of fusion peptides needed for fusion. These findings can resolve the large range of published cooperativity indices for HIV, influenza, and other type I fusion proteins.


Assuntos
Infecções por HIV/virologia , HIV/fisiologia , Vírus da Influenza A/fisiologia , Influenza Humana/virologia , Peptídeos/química , Proteínas do Envelope Viral/química , Anisotropia , Membrana Celular/virologia , Humanos , Modelos Teóricos , Domínios Proteicos , Internalização do Vírus
17.
Nat Commun ; 11(1): 4278, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855388

RESUMO

Activation and migration of endogenous mesenchymal stromal cells (MSCs) are critical for bone regeneration. Here, we report a combinational peptide screening strategy for rapid discovery of ligands that not only bind strongly to osteogenic progenitor cells (OPCs) but also stimulate osteogenic cell Akt signaling in those OPCs. Two lead compounds are discovered, YLL3 and YLL8, both of which increase osteoprogenitor osteogenic differentiation in vitro. When given to normal or osteopenic mice, the compounds increase mineral apposition rate, bone formation, bone mass, and bone strength, as well as expedite fracture repair through stimulated endogenous osteogenesis. When covalently conjugated to alendronate, YLLs acquire an additional function resulting in a "tri-functional" compound that: (i) binds to OPCs, (ii) targets bone, and (iii) induces "pro-survival" signal. These bone-targeted, osteogenic peptides are well suited for current tissue-specific therapeutic paradigms to augment the endogenous osteogenic cells for bone regeneration and the treatment of bone loss.


Assuntos
Anabolizantes/farmacologia , Fraturas Ósseas/tratamento farmacológico , Osteogênese/efeitos dos fármacos , Peptídeos/farmacologia , Células-Tronco/efeitos dos fármacos , Anabolizantes/química , Animais , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Fraturas Ósseas/patologia , Humanos , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Orquiectomia , Osteogênese/fisiologia , Ovariectomia , Peptídeos/química , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Técnicas de Síntese em Fase Sólida , Células-Tronco/citologia
18.
PLoS Biol ; 18(7): e3000780, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32687489

RESUMO

Cells adjust to nutrient deprivation by reversible translational shutdown. This is accompanied by maintaining inactive ribosomes in a hibernation state, in which they are bound by proteins with inhibitory and protective functions. In eukaryotes, such a function was attributed to suppressor of target of Myb protein 1 (Stm1; SERPINE1 mRNA-binding protein 1 [SERBP1] in mammals), and recently, late-annotated short open reading frame 2 (Lso2; coiled-coil domain containing short open reading frame 124 [CCDC124] in mammals) was found to be involved in translational recovery after starvation from stationary phase. Here, we present cryo-electron microscopy (cryo-EM) structures of translationally inactive yeast and human ribosomes. We found Lso2/CCDC124 accumulating on idle ribosomes in the nonrotated state, in contrast to Stm1/SERBP1-bound ribosomes, which display a rotated state. Lso2/CCDC124 bridges the decoding sites of the small with the GTPase activating center (GAC) of the large subunit. This position allows accommodation of the duplication of multilocus region 34 protein (Dom34)-dependent ribosome recycling system, which splits Lso2-containing, but not Stm1-containing, ribosomes. We propose a model in which Lso2 facilitates rapid translation reactivation by stabilizing the recycling-competent state of inactive ribosomes.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Evolução Molecular , Células HEK293 , Humanos , Modelos Moleculares , Peptídeos/química , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/ultraestrutura , Saccharomyces cerevisiae/ultraestrutura , Relação Estrutura-Atividade
19.
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
20.
Int J Mol Sci ; 21(15)2020 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-32718020

RESUMO

The ongoing pandemic of coronavirus disease-2019 (COVID-19) is being caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The disease continues to present significant challenges to the health care systems around the world. This is primarily because of the lack of vaccines to protect against the infection and the lack of highly effective therapeutics to prevent and/or treat the illness. Nevertheless, researchers have swiftly responded to the pandemic by advancing old and new potential therapeutics into clinical trials. In this review, we summarize potential anti-COVID-19 therapeutics that block the early stage of the viral life cycle. The review presents the structures, mechanisms, and reported results of clinical trials of potential therapeutics that have been listed in clinicaltrials.gov. Given the fact that some of these therapeutics are multi-acting molecules, other relevant mechanisms will also be described. The reviewed therapeutics include small molecules and macromolecules of sulfated polysaccharides, polypeptides, and monoclonal antibodies. The potential therapeutics target viral and/or host proteins or processes that facilitate the early stage of the viral infection. Frequent targets are the viral spike protein, the host angiotensin converting enzyme 2, the host transmembrane protease serine 2, and clathrin-mediated endocytosis process. Overall, the review aims at presenting update-to-date details, so as to enhance awareness of potential therapeutics, and thus, to catalyze their appropriate use in combating the pandemic.


Assuntos
Antivirais/uso terapêutico , Betacoronavirus/fisiologia , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antivirais/química , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Ensaios Clínicos como Assunto , Humanos , Pandemias , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Polissacarídeos/química , Polissacarídeos/farmacologia , Polissacarídeos/uso terapêutico , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos
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