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1.
Mediators Inflamm ; 2020: 8198963, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33029105

RESUMO

The novel coronavirus is not only causing respiratory problems, but it may also damage the heart, kidneys, liver, and other organs; in Wuhan, 14 to 30% of COVID-19 patients have lost their kidney function and now require either dialysis or kidney transplants. The novel coronavirus gains entry into humans by targeting the ACE2 receptor that found on lung cells, which destroy human lungs through cytokine storms, and this leads to hyperinflammation, forcing the immune cells to destroy healthy cells. This is why some COVID-19 patients need intensive care. The inflammatory chemicals released during COVID-19 infection cause the liver to produce proteins that defend the body from infections. However, these proteins can cause blood clotting, which can clog blood vessels in the heart and other organs; as a result, the organs are deprived of oxygen and nutrients which could ultimately lead to multiorgan failure and consequent progression to acute lung injury, acute respiratory distress syndrome, and often death. However, there are novel protein modification tools called the QTY code, which are similar in their structure to antibodies, which could provide a solution to excess cytokines. These synthetic proteins can be injected into the body to bind the excess cytokines created by the cytokine storm; this will eventually remove the excessive cytokines and inhibit the severe symptoms caused by the COVID-19 infection. In this review, we will focus on cytokine storm in COVID-19 patients, their impact on the body organs, and the potential treatment by QTY code-designed detergent-free chemokine receptors.


Assuntos
Infecções por Coronavirus/complicações , Infecções por Coronavirus/imunologia , Síndrome da Liberação de Citocina/etiologia , Síndrome da Liberação de Citocina/terapia , Pneumonia Viral/complicações , Pneumonia Viral/imunologia , Receptores de Quimiocinas/uso terapêutico , Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/terapia , Síndrome da Liberação de Citocina/imunologia , Citocinas/antagonistas & inibidores , Desenho de Fármacos , Humanos , Mediadores da Inflamação/sangue , Mediadores da Inflamação/imunologia , Modelos Moleculares , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/imunologia , Insuficiência de Múltiplos Órgãos/terapia , Pandemias , Pneumonia Viral/terapia , Engenharia de Proteínas , Modificação Traducional de Proteínas , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo
2.
Nat Commun ; 11(1): 4476, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32900995

RESUMO

Mechanically stable specific heterodimerization between small protein domains have a wide scope of applications, from using as a molecular anchorage in single-molecule force spectroscopy studies of protein mechanics, to serving as force-bearing protein linker for modulation of mechanotransduction of cells, and potentially acting as a molecular crosslinker for functional materials. Here, we explore the possibility to develop heterodimerization system with a range of mechanical stability from a set of recently engineered helix-heterotetramers whose mechanical properties have yet to be characterized. We demonstrate this possibility using two randomly chosen helix-heterotetramers, showing that their mechanical properties can be modulated by changing the stretching geometry and the number of interacting helices. These helix-heterotetramers and their derivatives are sufficiently stable over physiological temperature range. Using it as mechanically stable anchorage, we demonstrate the applications in single-molecule manipulation studies of the temperature dependent unfolding and refolding of a titin immunoglobulin domain and α-actinin spectrin repeats.


Assuntos
Engenharia de Proteínas , Multimerização Proteica , Estabilidade Proteica , Actinina/química , Fenômenos Biomecânicos , Conectina/química , Modelos Moleculares , Simulação de Dinâmica Molecular , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Dobramento de Proteína , Estrutura Quaternária de Proteína , Desdobramento de Proteína , Imagem Individual de Molécula , Temperatura
3.
Nature ; 585(7826): 614-619, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32879484

RESUMO

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.


Assuntos
Alcaloides/biossíntese , Alcaloides/provisão & distribução , Hiosciamina/biossíntese , Saccharomyces cerevisiae/metabolismo , Escopolamina/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Atropa belladonna/enzimologia , Derivados da Atropina/metabolismo , Transporte Biológico , Datura/enzimologia , Glucosídeos/biossíntese , Glucosídeos/metabolismo , Hiosciamina/provisão & distribução , Lactatos/metabolismo , Ligases/genética , Ligases/metabolismo , Modelos Moleculares , Doenças do Sistema Nervoso/tratamento farmacológico , Oxirredutases/genética , Oxirredutases/metabolismo , Engenharia de Proteínas , Saccharomyces cerevisiae/genética , Escopolamina/provisão & distribução , Vacúolos/metabolismo
4.
Nat Commun ; 11(1): 4808, 2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32968058

RESUMO

The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M-1s-1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M-1s-1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.


Assuntos
Simulação por Computador , Evolução Molecular Direcionada/métodos , Enzimas/química , Evolução Química , Liases/química , Catálise , Domínio Catalítico , Cristalografia por Raios X , Estabilidade Enzimática , Enzimas/genética , Enzimas/metabolismo , Cinética , Liases/genética , Liases/metabolismo , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Engenharia de Proteínas
5.
J Theor Biol ; 505: 110425, 2020 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-32735992

RESUMO

The interaction between the angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the spike protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a pivotal role in virus entry into the host cells. Since recombinant ACE2 protein has been suggested as an anti-SARS-CoV-2 therapeutic agent, this study was conducted to design an ACE2 protein with more desirable properties. In this regard, the amino acids with central roles in enzymatic activity of the ACE2 were substituted. Moreover, saturation mutagenesis at the interaction interface between the ACE2 and RBD was performed to increase their interaction affinity. The best mutations to increase the structural and thermal stability of the ACE2 were also selected based on B factors and mutation effects. The obtained resulted revealed that the Arg273Gln and Thr445Gly mutation have drastically reduced the binding affinity of the angiotensin-II into the active site of ACE2. The Thr27Arg mutation was determined to be the most potent mutation to increase the binding affinity. The Asp427Arg mutation was done to decrease the flexibility of the region with high B factor. The Pro451Met mutation along with the Gly448Trp mutation was predicted to increase the thermodynamic stability and thermostability of the ACE2. The designed therapeutic ACE2 would have no enzymatic activity while it could bear stronger interaction with Spike glycoprotein of the SARS-CoV-2. Moreover, decreased in vivo enzymatic degradation would be anticipated due to increased thermostability. This engineered ACE2 could be exploited as a novel therapeutic agent against COVID-19 after necessary evaluations.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/tratamento farmacológico , Desenho de Fármacos , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/tratamento farmacológico , Engenharia de Proteínas/métodos , Substituição de Aminoácidos , Betacoronavirus/genética , Sítios de Ligação , Evolução Molecular Direcionada , Humanos , Pandemias , Peptidil Dipeptidase A/uso terapêutico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Estabilidade Proteica , Glicoproteína da Espícula de Coronavírus/metabolismo
6.
Nature ; 585(7823): 129-134, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32848250

RESUMO

Transmembrane channels and pores have key roles in fundamental biological processes1 and in biotechnological applications such as DNA nanopore sequencing2-4, resulting in considerable interest in the design of pore-containing proteins. Synthetic amphiphilic peptides have been found to form ion channels5,6, and there have been recent advances in de novo membrane protein design7,8 and in redesigning naturally occurring channel-containing proteins9,10. However, the de novo design of stable, well-defined transmembrane protein pores that are capable of conducting ions selectively or are large enough to enable the passage of small-molecule fluorophores remains an outstanding challenge11,12. Here we report the computational design of protein pores formed by two concentric rings of α-helices that are stable and monodisperse in both their water-soluble and their transmembrane forms. Crystal structures of the water-soluble forms of a 12-helical pore and a 16-helical pore closely match the computational design models. Patch-clamp electrophysiology experiments show that, when expressed in insect cells, the transmembrane form of the 12-helix pore enables the passage of ions across the membrane with high selectivity for potassium over sodium; ion passage is blocked by specific chemical modification at the pore entrance. When incorporated into liposomes using in vitro protein synthesis, the transmembrane form of the 16-helix pore-but not the 12-helix pore-enables the passage of biotinylated Alexa Fluor 488. A cryo-electron microscopy structure of the 16-helix transmembrane pore closely matches the design model. The ability to produce structurally and functionally well-defined transmembrane pores opens the door to the creation of designer channels and pores for a wide variety of applications.


Assuntos
Simulação por Computador , Genes Sintéticos/genética , Canais Iônicos/química , Canais Iônicos/genética , Modelos Moleculares , Biologia Sintética , Linhagem Celular , Microscopia Crioeletrônica , Cristalografia por Raios X , Condutividade Elétrica , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrazinas , Canais Iônicos/metabolismo , Transporte de Íons , Lipossomos/metabolismo , Técnicas de Patch-Clamp , Porinas/química , Porinas/genética , Porinas/metabolismo , Engenharia de Proteínas , Estrutura Secundária de Proteína , Solubilidade , Água/química
7.
Science ; 369(6507): 1132-1136, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32855341

RESUMO

Naturally occurring proteins vary the precise geometries of structural elements to create distinct shapes optimal for function. We present a computational design method, loop-helix-loop unit combinatorial sampling (LUCS), that mimics nature's ability to create families of proteins with the same overall fold but precisely tunable geometries. Through near-exhaustive sampling of loop-helix-loop elements, LUCS generates highly diverse geometries encompassing those found in nature but also surpassing known structure space. Biophysical characterization showed that 17 (38%) of 45 tested LUCS designs encompassing two different structural topologies were well folded, including 16 with designed non-native geometries. Four experimentally solved structures closely matched the designs. LUCS greatly expands the designable structure space and offers a new paradigm for designing proteins with tunable geometries that may be customizable for novel functions.


Assuntos
Desenho Assistido por Computador , Engenharia de Proteínas/métodos , Dobramento de Proteína , Estrutura Secundária de Proteína
8.
Science ; 369(6508): 1261-1265, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753553

RESUMO

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds angiotensin-converting enzyme 2 (ACE2) on host cells to initiate entry, and soluble ACE2 is a therapeutic candidate that neutralizes infection by acting as a decoy. By using deep mutagenesis, mutations in ACE2 that increase S binding are found across the interaction surface, in the asparagine 90-glycosylation motif and at buried sites. The mutational landscape provides a blueprint for understanding the specificity of the interaction between ACE2 and S and for engineering high-affinity decoy receptors. Combining mutations gives ACE2 variants with affinities that rival those of monoclonal antibodies. A stable dimeric variant shows potent SARS-CoV-2 and -1 neutralization in vitro. The engineered receptor is catalytically active, and its close similarity with the native receptor may limit the potential for viral escape.


Assuntos
Betacoronavirus/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Engenharia de Proteínas , Receptores Virais/genética , Receptores Virais/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Humanos , Modelos Moleculares , Mutagênese , Mutação , Peptidil Dipeptidase A/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores Virais/química , Glicoproteína da Espícula de Coronavírus/química
9.
BMC Bioinformatics ; 21(1): 275, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32611389

RESUMO

BACKGROUND: Protein engineering has many applications for industry, such as the development of new drugs, vaccines, treatment therapies, food, and biofuel production. A common way to engineer a protein is to perform mutations in functionally essential residues to optimize their function. However, the discovery of beneficial mutations for proteins is a complex task, with a time-consuming and high cost for experimental validation. Hence, computational approaches have been used to propose new insights for experiments narrowing the search space and reducing the costs. RESULTS: In this study, we developed Proteus (an acronym for Protein Engineering Supporter), a new algorithm for proposing mutation pairs in a target 3D structure. These suggestions are based on contacts observed in other known structures from Protein Data Bank (PDB). Proteus' basic assumption is that if a non-interacting pair of amino acid residues in the target structure is exchanged to an interacting pair, this could enhance protein stability. This trade is only allowed if the main-chain conformation of the residues involved in the contact is conserved. Furthermore, no steric impediment is expected between the proposed mutations and the surrounding protein atoms. To evaluate Proteus, we performed two case studies with proteins of industrial interests. In the first case study, we evaluated if the mutations suggested by Proteus for four protein structures enhance the number of inter-residue contacts. Our results suggest that most mutations proposed by Proteus increase the number of interactions into the protein. In the second case study, we used Proteus to suggest mutations for a lysozyme protein. Then, we compared Proteus' outcomes to mutations with available experimental evidence reported in the ProTherm database. Four mutations, in which our results agree with the experimental data, were found. This could be initial evidence that changes in the side-chain of some residues do not cause disturbances that harm protein structure stability. CONCLUSION: We believe that Proteus could be used combined with other methods to give new insights into the rational development of engineered proteins. Proteus user-friendly web-based tool is available at < http://proteus.dcc.ufmg.br >.


Assuntos
Proteínas/química , Interface Usuário-Computador , Algoritmos , Bases de Dados de Proteínas , Muramidase/química , Muramidase/genética , Muramidase/metabolismo , Mutagênese , Engenharia de Proteínas/métodos , Estrutura Terciária de Proteína , Proteínas/genética , Proteínas/metabolismo
11.
Nat Commun ; 11(1): 3314, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620861

RESUMO

The amyloid conformation can be adopted by a variety of sequences, but the precise boundaries of amyloid sequence space are still unclear. The currently charted amyloid sequence space is strongly biased towards hydrophobic, beta-sheet prone sequences that form the core of globular proteins and by Q/N/Y rich yeast prions. Here, we took advantage of the increasing amount of high-resolution structural information on amyloid cores currently available in the protein databank to implement a machine learning approach, named Cordax (https://cordax.switchlab.org), that explores amyloid sequence beyond its current boundaries. Clustering by t-Distributed Stochastic Neighbour Embedding (t-SNE) shows how our approach resulted in an expansion away from hydrophobic amyloid sequences towards clusters of lower aliphatic content and higher charge, or regions of helical and disordered propensities. These clusters uncouple amyloid propensity from solubility representing sequence flavours compatible with surface-exposed patches in globular proteins, functional amyloids or sequences associated to liquid-liquid phase transitions.


Assuntos
Algoritmos , Amiloide/química , Proteínas Amiloidogênicas/química , Modelos Químicos , Peptídeos/química , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Amiloidose/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Aprendizado de Máquina , Peptídeos/metabolismo , Conformação Proteica , Engenharia de Proteínas/métodos , Solubilidade
12.
Nat Commun ; 11(1): 3746, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32719315

RESUMO

Recently, the clinical proof of concept for the first ultra-long oral insulin was reported, showing efficacy and safety similar to subcutaneously administered insulin glargine. Here, we report the molecular engineering as well as biological and pharmacological properties of these insulin analogues. Molecules were designed to have ultra-long pharmacokinetic profile to minimize variability in plasma exposure. Elimination plasma half-life of ~20 h in dogs and ~70 h in man is achieved by a strong albumin binding, and by lowering the insulin receptor affinity 500-fold to slow down receptor mediated clearance. These insulin analogues still stimulate efficient glucose disposal in rats, pigs and dogs during constant intravenous infusion and euglycemic clamp conditions. The albumin binding facilitates initial high plasma exposure with a concomitant delay in distribution to peripheral tissues. This slow appearance in the periphery mediates an early transient hepato-centric insulin action and blunts hypoglycaemia in dogs in response to overdosing.


Assuntos
Insulina/administração & dosagem , Engenharia de Proteínas , Administração Oral , Sequência de Aminoácidos , Animais , Glicemia/metabolismo , Simulação por Computador , Cães , Relação Dose-Resposta a Droga , Overdose de Drogas/sangue , Técnica Clamp de Glucose , Meia-Vida , Humanos , Hiperinsulinismo/tratamento farmacológico , Hipoglicemia/diagnóstico , Insulina/análogos & derivados , Insulina/química , Insulina/farmacocinética , Masculino , Estabilidade Proteica , Proteólise , Ratos Sprague-Dawley , Suínos , Resultado do Tratamento
13.
Proc Natl Acad Sci U S A ; 117(28): 16537-16545, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601191

RESUMO

Engineering biological nitrogen fixation in eukaryotic cells by direct introduction of nif genes requires elegant synthetic biology approaches to ensure that components required for the biosynthesis of active nitrogenase are stable and expressed in the appropriate stoichiometry. Previously, the NifD subunits of nitrogenase MoFe protein from Azotobacter vinelandii and Klebsiella oxytoca were found to be unstable in yeast and plant mitochondria, respectively, presenting a bottleneck to the assembly of active MoFe protein in eukaryotic cells. In this study, we have delineated the region and subsequently a key residue, NifD-R98, from K. oxytoca that confers susceptibility to protease-mediated degradation in mitochondria. The effect observed is pervasive, as R98 is conserved among all NifD proteins analyzed. NifD proteins from four representative diazotrophs, but not their R98 variants, were observed to be unstable in yeast mitochondria. Furthermore, by reconstituting mitochondrial-processing peptidases (MPPs) from yeast, Oryza sativa, Nicotiana tabacum, and Arabidopsis thaliana in Escherichia coli, we demonstrated that MPPs are responsible for cleavage of NifD. These results indicate a pervasive effect on the stability of NifD proteins in mitochondria resulting from cleavage by MPPs. NifD-R98 variants that retained high levels of nitrogenase activity were obtained, with the potential to stably target active MoFe protein to mitochondria. This reconstitution approach could help preevaluate the stability of Nif proteins for plant expression and paves the way for engineering active nitrogenase in plant organelles.


Assuntos
Proteínas de Bactérias/genética , Expressão Gênica , Klebsiella oxytoca/enzimologia , Nitrogenase/genética , Engenharia de Proteínas/métodos , Biologia Sintética/métodos , Proteínas de Bactérias/metabolismo , Klebsiella oxytoca/genética , Mitocôndrias/enzimologia , Mitocôndrias/genética , Nitrogenase/metabolismo , Plantas/genética , Plantas/metabolismo , Processamento de Proteína Pós-Traducional
14.
Nature ; 583(7817): 631-637, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32641830

RESUMO

Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Such proteins include a group of predicted interbacterial toxins of the deaminase superfamily, members of which have found application in gene-editing techniques1,2. Because previously described cytidine deaminases operate on single-stranded nucleic acids3, their use in base editing requires the unwinding of double-stranded DNA (dsDNA)-for example by a CRISPR-Cas9 system. Base editing within mitochondrial DNA (mtDNA), however, has thus far been hindered by challenges associated with the delivery of guide RNA into the mitochondria4. As a consequence, manipulation of mtDNA to date has been limited to the targeted destruction of the mitochondrial genome by designer nucleases9,10.Here we describe an interbacterial toxin, which we name DddA, that catalyses the deamination of cytidines within dsDNA. We engineered split-DddA halves that are non-toxic and inactive until brought together on target DNA by adjacently bound programmable DNA-binding proteins. Fusions of the split-DddA halves, transcription activator-like effector array proteins, and a uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that catalyse C•G-to-T•A conversions in human mtDNA with high target specificity and product purity. We used DdCBEs to model a disease-associated mtDNA mutation in human cells, resulting in changes in respiration rates and oxidative phosphorylation. CRISPR-free DdCBEs enable the precise manipulation of mtDNA, rather than the elimination of mtDNA copies that results from its cleavage by targeted nucleases, with broad implications for the study and potential treatment of mitochondrial disorders.


Assuntos
Toxinas Bacterianas/metabolismo , Citidina Desaminase/metabolismo , DNA Mitocondrial/genética , Edição de Genes/métodos , Genes Mitocondriais/genética , Mitocôndrias/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Sequência de Bases , Burkholderia cenocepacia/enzimologia , Burkholderia cenocepacia/genética , Respiração Celular/genética , Citidina/metabolismo , Citidina Desaminase/química , Citidina Desaminase/genética , Genoma Mitocondrial/genética , Células HEK293 , Humanos , Doenças Mitocondriais/genética , Doenças Mitocondriais/terapia , Mutação , Fosforilação Oxidativa , Engenharia de Proteínas , RNA Guia/genética , Especificidade por Substrato , Sistemas de Secreção Tipo VI/metabolismo
15.
Life Sci ; 257: 118052, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32634431

RESUMO

AIMS: Granulocyte colony-stimulating factor (G-CSF) is a cytokine that induces proliferation and differentiation of hematopoietic precursor cells and activation of mature neutrophils. G-CSF is overexpressed in several malignant tumors and blocking its binding to the receptor can lead to significant decrease in tumor growth, vascularization and metastasis. Furthermore, targeting G-CSF receptor has shown therapeutic benefit in other diseases such as rheumatoid arthritis, progressive neurodegenerative disorder and uveitis. Camelid single-chain antibodies (nanobodies) have exceptional properties making them appropriate for tumor imaging and therapeutic application. In this study we aim to use the rational design approach to engineer a previously described G-CSF-R targeting nanobody (VHH1), to improve its affinity toward G-CSF-R. MAIN METHODS: We redesigned the complementary determining region 3 (CDR3) domain of the VHH1 nanobody to mimic G-CSF interaction to its receptor and developed five new engineered nanobodies. Binding affinity of the engineered nanobodies was evaluated by ELISA (Enzyme-linked immunosorbent assay) on NFS60 cells. KEY FINDINGS: Enzyme-linked immunosorbent assay (ELISA) confirmed the specificity of the engineered nanobodies and ELISA-based determination of affinity revealed that two of the engineered nanobodies (1c and 5a) bind to G-CSF-R on the surface of NFS60 cells in a dose-dependent manner and with a higher potency compared to the parental nanobody. SIGNIFICANCE: Additional studies are required to better characterize these nanobodies and assess their interaction with G-CSF-R in vitro and in vivo. These newly developed nanobodies could be beneficial in tumor imaging and therapy and make a basis for development of additional engineered nanobodies.


Assuntos
Fator Estimulador de Colônias de Granulócitos/ultraestrutura , Receptores de Fator Estimulador de Colônias de Granulócitos/imunologia , Anticorpos de Domínio Único/imunologia , Anticorpos , Anticorpos Monoclonais/imunologia , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia de Afinidade/métodos , Ensaio de Imunoadsorção Enzimática , Fator Estimulador de Colônias de Granulócitos/imunologia , Fator Estimulador de Colônias de Granulócitos/metabolismo , Humanos , Neovascularização Patológica/tratamento farmacológico , Engenharia de Proteínas/métodos , Anticorpos de Cadeia Única
16.
Nat Commun ; 11(1): 3583, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32681005

RESUMO

The phosphatases PP1 and PP2A are responsible for the majority of dephosphorylation reactions on phosphoserine (pSer) and phosphothreonine (pThr), and are involved in virtually all cellular processes and numerous diseases. The catalytic subunits exist in cells in form of holoenzymes, which impart substrate specificity. The contribution of the catalytic subunits to the recognition of substrates is unclear. By developing a phosphopeptide library approach and a phosphoproteomic assay, we demonstrate that the specificity of PP1 and PP2A holoenzymes towards pThr and of PP1 for basic motifs adjacent to the phosphorylation site are due to intrinsic properties of the catalytic subunits. Thus, we dissect this amino acid specificity of the catalytic subunits from the contribution of regulatory proteins. Furthermore, our approach enables discovering a role for PP1 as regulator of the GRB-associated-binding protein 2 (GAB2)/14-3-3 complex. Beyond this, we expect that this approach is broadly applicable to detect enzyme-substrate recognition preferences.


Assuntos
Proteína Fosfatase 1/química , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Domínio Catalítico , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Humanos , Fosforilação , Ligação Proteica , Engenharia de Proteínas , Proteína Fosfatase 1/genética , Proteína Fosfatase 2/genética , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Especificidade por Substrato
17.
Nat Biotechnol ; 38(7): 845-855, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32601435

RESUMO

Genome editing has the potential to treat an extensive range of incurable monogenic and complex diseases. In particular, advances in sequence-specific nuclease technologies have dramatically accelerated the development of therapeutic genome editing strategies that are based on either the knockout of disease-causing genes or the repair of endogenous mutated genes. These technologies are progressing into human clinical trials. However, challenges remain before the therapeutic potential of genome editing can be fully realized. Delivery technologies that have serendipitously been developed over the past couple decades in the protein and nucleic acid delivery fields have been crucial to genome editing success to date, including adeno-associated viral and lentiviral vectors for gene therapy and lipid nanoparticle and other non-viral vectors for nucleic acid and protein delivery. However, the efficiency and tissue targeting capabilities of these vehicles must be further improved. In addition, the genome editing enzymes themselves need to be optimized, and challenges regarding their editing efficiency, specificity and immunogenicity must be addressed. Emerging protein engineering and synthetic chemistry approaches can offer solutions and enable the development of safe and efficacious clinical genome editing.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/tendências , Doenças Genéticas Inatas/terapia , Terapia Genética , Doenças Genéticas Inatas/genética , Vetores Genéticos/genética , Vetores Genéticos/uso terapêutico , Humanos , Nanopartículas/uso terapêutico , Engenharia de Proteínas
18.
Med Hypotheses ; 143: 110108, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32721804

RESUMO

The present hypothesis suggests an innovative therapeutic strategy to cease Covid 19 infection. It is based on the inhibition of Spike glycoprotein and ACE-2 receptor interaction that provides the entry of virus in human host cells, by targeting the S protein with a recombinant molecule made of the ACE-2 receptor ectodomain and an opsonin, the formed complex would enhance its phagocytosis.


Assuntos
Betacoronavirus , Infecções por Coronavirus/tratamento farmacológico , Proteínas Opsonizantes/química , Fagocitose , Pneumonia Viral/tratamento farmacológico , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Desenho de Fármacos , Humanos , Pandemias , Peptidil Dipeptidase A/química , Domínios Proteicos , Engenharia de Proteínas , Proteínas Recombinantes/química , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus
19.
Proc Natl Acad Sci U S A ; 117(25): 14110-14118, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32522868

RESUMO

Interleukin-6 (IL-6) family cytokines signal through multimeric receptor complexes, providing unique opportunities to create novel ligand-based therapeutics. The cardiotrophin-like cytokine factor 1 (CLCF1) ligand has been shown to play a role in cancer, osteoporosis, and atherosclerosis. Once bound to ciliary neurotrophic factor receptor (CNTFR), CLCF1 mediates interactions to coreceptors glycoprotein 130 (gp130) and leukemia inhibitory factor receptor (LIFR). By increasing CNTFR-mediated binding to these coreceptors we generated a receptor superagonist which surpassed the potency of natural CNTFR ligands in neuronal signaling. Through additional mutations, we generated a receptor antagonist with increased binding to CNTFR but lack of binding to the coreceptors that inhibited tumor progression in murine xenograft models of nonsmall cell lung cancer. These studies further validate the CLCF1-CNTFR signaling axis as a therapeutic target and highlight an approach of engineering cytokine activity through a small number of mutations.


Assuntos
Subunidade alfa do Receptor do Fator Neutrófico Ciliar/agonistas , Citocinas/metabolismo , Engenharia de Proteínas/métodos , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Células Cultivadas , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/antagonistas & inibidores , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Receptor gp130 de Citocina/metabolismo , Citocinas/química , Citocinas/genética , Humanos , Subunidade alfa de Receptor de Fator Inibidor de Leucemia/metabolismo , Ligantes , Neurônios/metabolismo , Ligação Proteica , Ratos , Transdução de Sinais
20.
Proc Natl Acad Sci U S A ; 117(25): 14602-14608, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32522869

RESUMO

Bioinspired actuators with stimuli-responsive and deformable properties are being pursued in fields such as artificial tissues, medical devices and diagnostics, and intelligent biosensors. These applications require that actuator systems have biocompatibility, controlled deformability, biodegradability, mechanical durability, and stable reversibility. Herein, we report a bionic actuator system consisting of stimuli-responsive genetically engineered silk-elastin-like protein (SELP) hydrogels and wood-derived cellulose nanofibers (CNFs), which respond to temperature and ionic strength underwater by ecofriendly methods. Programmed site-selective actuation can be predicted and folded into three-dimensional (3D) origami-like shapes. The reversible deformation performance of the SELP/CNF actuators was quantified, and complex spatial transformations of multilayer actuators were demonstrated, including a biomimetic flower design with selective petal movements. Such actuators consisting entirely of biocompatible and biodegradable materials will offer an option toward constructing stimuli-responsive systems for in vivo biomedicine soft robotics and bionic research.


Assuntos
Materiais Biocompatíveis/química , Materiais Biomiméticos/química , Biônica/métodos , Celulose/química , Elastina/química , Elastina/genética , Hidrogéis/química , Conformação Molecular , Nanofibras/química , Engenharia de Proteínas , Robótica/métodos , Seda/química , Seda/genética
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