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1.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2915-2923, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472308

RESUMO

Antimicrobial peptides are the most promising alternatives to antibiotics. However, the strategy of producing antimicrobial peptides by recombinant technology is complicated and expensive, which is not conducive to the large-scale production. Oxysterlin 1 is a novel type of cecropin antimicrobial peptide mainly targeting on Gram-negative bacteria and is of low cytotoxicity. In this study, a simple and cost-effective method was developed to produce Oxysterlin 1 in Escherichia coli. The Oxysterlin 1 gene was cloned into a plasmid containing elastin-like polypeptide (ELP) and protein splicing elements (intein) to construct the recombinant expression plasmid (pET-ELP-I-Oxysterlin 1). The recombinant protein was mainly expressed in soluble form in E. coli, and then the target peptide can be purified with a simple salting out method followed by pH changing. The final yield of Oxysterlin 1 was about 1.2 mg/L, and the subsequent antimicrobial experiment showed the expected antimicrobial activity. This study holds promise for large-scale production of antimicrobial peptides and the in-depth study of its antimicrobial mechanism.


Assuntos
Elastina , Escherichia coli , Escherichia coli/genética , Inteínas , Peptídeos/genética , Peptídeos/farmacologia , Proteínas Citotóxicas Formadoras de Poros , Proteínas Recombinantes de Fusão/genética
2.
Int J Mol Sci ; 22(17)2021 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-34502016

RESUMO

Antisense peptide technology (APT) is based on a useful heuristic algorithm for rational peptide design. It was deduced from empirical observations that peptides consisting of complementary (sense and antisense) amino acids interact with higher probability and affinity than the randomly selected ones. This phenomenon is closely related to the structure of the standard genetic code table, and at the same time, is unrelated to the direction of its codon sequence translation. The concept of complementary peptide interaction is discussed, and its possible applications to diagnostic tests and bioengineering research are summarized. Problems and difficulties that may arise using APT are discussed, and possible solutions are proposed. The methodology was tested on the example of SARS-CoV-2. It is shown that the CABS-dock server accurately predicts the binding of antisense peptides to the SARS-CoV-2 receptor binding domain without requiring predefinition of the binding site. It is concluded that the benefits of APT outweigh the costs of random peptide screening and could lead to considerable savings in time and resources, especially if combined with other computational and immunochemical methods.


Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Algoritmos , Sequência de Aminoácidos/genética , Sítios de Ligação/genética , COVID-19/sangue , COVID-19/virologia , Humanos , Imunoquímica/métodos , Simulação de Acoplamento Molecular , Peptídeos/genética , Ligação Proteica/genética , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/metabolismo
3.
Sci Rep ; 11(1): 15650, 2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34341401

RESUMO

SARS-CoV-2 is coronavirus causing COVID-19 pandemic. To enter human cells, receptor binding domain of S1 subunit of SARS-CoV-2 (SARS-CoV-2-RBD) binds to peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2) receptor. Employing peptides to inhibit binding between SARS-CoV-2-RBD and ACE2-PD is a therapeutic solution for COVID-19. Previous experimental study found that 23-mer peptide (SBP1) bound to SARS-CoV-2-RBD with lower affinity than ACE2. To increase SBP1 affinity, our previous study used residues 21-45 of α1 helix of ACE2-PD (SPB25) to design peptides with predicted affinity better than SBP1 and SPB25 by increasing interactions of residues that do not form favorable interactions with SARS-CoV-2-RBD. To design SPB25 with better affinity than ACE2, we employed computational protein design to increase interactions of residues reported to form favorable interactions with SARS-CoV-2-RBD and combine newly designed mutations with the best single mutations from our previous study. Molecular dynamics show that predicted binding affinities of three peptides (SPB25Q22R, SPB25F8R/K11W/L25R and SPB25F8R/K11F/Q22R/L25R) are better than ACE2. Moreover, their predicted stabilities may be slightly higher than SBP1 as suggested by their helicities. This study developed an approach to design SARS-CoV-2 peptide binders with predicted binding affinities better than ACE2. These designed peptides are promising candidates as SARS-CoV-2 inhibitors.


Assuntos
Enzima de Conversão de Angiotensina 2/química , Simulação de Dinâmica Molecular , Peptídeos/química , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Enzima de Conversão de Angiotensina 2/genética , COVID-19 , Humanos , Peptídeos/genética , Ligação Proteica , Domínios Proteicos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética
4.
Nat Commun ; 12(1): 5090, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429421

RESUMO

CRISPR-Cas9 mediated genome editing offers unprecedented opportunities for treating human diseases. There are several reports that demonstrate pre-existing immune responses to Cas9 which may have implications for clinical development of CRISPR-Cas9 mediated gene therapy. Here we use 209 overlapping peptides that span the entire sequence of Staphylococcus aureus Cas9 (SaCas9) and human peripheral blood mononuclear cells (PBMCs) from a cohort of donors with a distribution of Major Histocompatibility Complex (MHC) alleles comparable to that in the North American (NA) population to identify the immunodominant regions of the SaCas9 protein. We also use an MHC Associated Peptide Proteomics (MAPPs) assay to identify SaCas9 peptides presented by MHC Class II (MHC-II) proteins on dendritic cells. Using these two data sets we identify 22 SaCas9 peptides that are both presented by MHC-II proteins and stimulate CD4+ T-cells.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Sistemas CRISPR-Cas , Proliferação de Células/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Sequência de Aminoácidos , Proteína 9 Associada à CRISPR/genética , Citocinas , Edição de Genes , Humanos , Staphylococcus aureus/genética , Linfócitos T/imunologia
5.
Yi Chuan ; 43(8): 737-746, 2021 Aug 20.
Artigo em Chinês | MEDLINE | ID: mdl-34413014

RESUMO

Existing research has shown that there are a large amount of non-coding RNAs (ncRNAs) in organisms. Short open reading frames (sORFs) abundantly exist in molecular sequences inaccurately annotated as ncRNAs. Several sORFs can be transcribed and translated into evolutionarily conserved micropeptides, which were ignored in previous studies due to short sequence lengths and the limitations of research techniques. To date, sORF-encoded micropeptides with various functions have been found to play important roles in regulating vital biological activities. This article reviews the functional micropeptides which have been found in recent years, introduces the new micropeptide designated as MIAC that we have discovered and describes the related technologies for mining potential micropeptides, thereby providing insights and references for new micropeptide discovery for researchers.


Assuntos
Peptídeos , RNA não Traduzido , Fases de Leitura Aberta/genética , Peptídeos/genética
6.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445285

RESUMO

N-glycosylation is essential for many biological processes in mammals. A variety of N-glycan structures exist, of which, the formation of bisecting N-acetylglucosamine (GlcNAc) is catalyzed by N-acetylglucosaminyltransferase-III (GnT-III, encoded by the Mgat3 gene). We previously identified various bisecting GlcNAc-modified proteins involved in Alzheimer's disease and cancer. However, the mechanisms by which GnT-III acts on the target proteins are unknown. Here, we performed comparative glycoproteomic analyses using brain membranes of wild type (WT) and Mgat3-deficient mice. Target glycoproteins of GnT-III were enriched with E4-phytohemagglutinin (PHA) lectin, which recognizes bisecting GlcNAc, and analyzed by liquid chromatograph-mass spectrometry. We identified 32 N-glycosylation sites (Asn-Xaa-Ser/Thr, Xaa ≠ Pro) that were modified with bisecting GlcNAc. Sequence alignment of identified N-glycosylation sites that displayed bisecting GlcNAc suggested that GnT-III does not recognize a specific primary amino acid sequence. The molecular modeling of GluA1 as one of the good cell surface substrates for GnT-III in the brain, indicated that GnT-III acts on N-glycosylation sites located in a highly flexible and mobile loop of GluA1. These results suggest that the action of GnT-III is partially affected by the tertiary structure of target proteins, which can accommodate bisecting GlcNAc that generates a bulky flipped-back conformation of the modified glycans.


Assuntos
Acetilglucosamina/metabolismo , Encéfalo/metabolismo , Membrana Celular/metabolismo , Peptídeos/metabolismo , Receptores de AMPA/metabolismo , Análise de Sequência de Proteína , Acetilglucosamina/genética , Animais , Membrana Celular/genética , Glicosilação , Camundongos , Camundongos Knockout , N-Acetilglucosaminiltransferases/deficiência , N-Acetilglucosaminiltransferases/metabolismo , Mapeamento de Peptídeos , Peptídeos/genética , Receptores de AMPA/genética
7.
Int J Mol Sci ; 22(16)2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34445382

RESUMO

Natural and de novo designed peptides are gaining an ever-growing interest as drugs against several diseases. Their use is however limited by the intrinsic low bioavailability and poor stability. To overcome these issues retro-inverso analogues have been investigated for decades as more stable surrogates of peptides composed of natural amino acids. Retro-inverso peptides possess reversed sequences and chirality compared to the parent molecules maintaining at the same time an identical array of side chains and in some cases similar structure. The inverted chirality renders them less prone to degradation by endogenous proteases conferring enhanced half-lives and an increased potential as new drugs. However, given their general incapability to adopt the 3D structure of the parent peptides their application should be careful evaluated and investigated case by case. Here, we review the application of retro-inverso peptides in anticancer therapies, in immunology, in neurodegenerative diseases, and as antimicrobials, analyzing pros and cons of this interesting subclass of molecules.


Assuntos
Peptídeos/genética , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Humanos , Peptídeos/síntese química , Conformação Proteica
8.
Anal Chem ; 93(30): 10627-10634, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34292722

RESUMO

In multiple myeloma diseases, monoclonal immunoglobulin light chains (LCs) are abundantly produced, with, as a consequence in some cases, the formation of deposits affecting various organs, such as the kidney, while in other cases remaining soluble up to concentrations of several g·L-1 in plasma. The exact factors crucial for the solubility of LCs are poorly understood, but it can be hypothesized that their amino acid sequence plays an important role. Determining the precise sequences of patient-derived LCs is therefore highly desirable. We establish here a novel de novo sequencing workflow for patient-derived LCs, based on the combination of bottom-up and top-down proteomics without database search. PEAKS is used for the de novo sequencing of peptides that are further assembled into full length LC sequences using ALPS. Top-down proteomics provides the molecular masses of proteoforms and allows the exact determination of the amino acid sequence including all posttranslational modifications. This pipeline is then used for the complete de novo sequencing of LCs extracted from the urine of 10 patients with multiple myeloma. We show that for the bottom-up part, digestions with trypsin and Nepenthes digestive fluid are sufficient to produce overlapping peptides able to generate the best sequence candidates. Top-down proteomics is absolutely required to achieve 100% final sequence coverage and characterize clinical samples containing several LCs. Our work highlights an unexpected range of modifications.


Assuntos
Mieloma Múltiplo , Sequência de Aminoácidos , Humanos , Cadeias Leves de Imunoglobulina/genética , Peptídeos/genética , Proteômica , Análise de Sequência de Proteína
9.
Methods Mol Biol ; 2312: 253-276, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228295

RESUMO

Recent studies revealed the biological significance of dynamic multicomponent assemblies of biomolecules inside living cells. Protein and nucleic acid assemblies are biomolecular condensates or non-membrane-bound organelles that have attracted increasing attention. Synthetic tools that manipulate the dynamic assembly/disassembly process of the structures are useful in elucidating both biophysical mechanisms of their assembly/disassembly and physiological roles of the condensates. In this report, general protocols to form and observe synthetic polymer-based condensates in living cells are described using the tool iPOLYMER. Taking advantage of the modular design of the tool, both chemical and light stimuli can induce formation of synthetic condensates inside living cells, which are observed by laser-scanning confocal microscopy. The experimental design described herein should help those who conduct experiments on synthetic manipulation of biomolecular condensates using iPOLYMER and other tools for synthetic manipulation of condensates. Technical notes for using iPOLYMER, including basic protocols of chemical- or light-inducible dimerization techniques (CID/LID), choice of proper control experiments, and advantages/disadvantages are also presented.


Assuntos
Engenharia Celular , Grânulos Citoplasmáticos/genética , Regulação da Expressão Gênica , Mimetismo Molecular , Optogenética , Peptídeos/genética , RNA/genética , Biologia Sintética , Antígeno-1 Intracelular de Células T/genética , Animais , Células COS , Técnicas de Cultura de Células , Chlorocebus aethiops , Grânulos Citoplasmáticos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Células HEK293 , Humanos , Hidrogéis , Luz , Microscopia Confocal , Microscopia de Fluorescência , Peptídeos/metabolismo , Domínios Proteicos , RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sirolimo/farmacologia , Antígeno-1 Intracelular de Células T/metabolismo , Transfecção
10.
Nat Commun ; 12(1): 4365, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34272369

RESUMO

Activating RAS missense mutations are among the most prevalent genomic alterations observed in human cancers and drive oncogenesis in the three most lethal tumor types. Emerging evidence suggests mutant KRAS (mKRAS) may be targeted immunologically, but mKRAS epitopes remain poorly defined. Here we employ a multi-omics approach to characterize HLA class I-restricted mKRAS epitopes. We provide proteomic evidence of mKRAS epitope processing and presentation by high prevalence HLA class I alleles. Select epitopes are immunogenic enabling mKRAS-specific TCRαß isolation. TCR transfer to primary CD8+ T cells confers cytotoxicity against mKRAS tumor cell lines independent of histologic origin, and the kinetics of lytic activity correlates with mKRAS peptide-HLA class I complex abundance. Adoptive transfer of mKRAS-TCR engineered CD8+ T cells leads to tumor eradication in a xenograft model of metastatic lung cancer. This study validates mKRAS peptides as bona fide epitopes facilitating the development of immune therapies targeting this oncoprotein.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Carcinogênese/imunologia , Epitopos de Linfócito T/imunologia , Neoplasias Pulmonares/imunologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Transferência Adotiva , Alelos , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Mutação , Peptídeos/genética , Peptídeos/imunologia , Proteômica , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Biomolecules ; 11(6)2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207317

RESUMO

One of the treatment strategies for Alzheimer's disease (AD) is based on the use of pharmacological agents capable of binding to beta-amyloid (Aß) and blocking its aggregation in the brain. Previously, we found that intravenous administration of the synthetic tetrapeptide Acetyl-His-Ala-Glu-Glu-Amide (HAEE), which is an analogue of the 35-38 region of the α4 subunit of α4ß2 nicotinic acetylcholine receptor and specifically binds to the 11-14 site of Aß, reduced the development of cerebral amyloidogenesis in a mouse model of AD. In the current study on three types of laboratory animals, we determined the biodistribution and tissue localization patterns of HAEE peptide after single intravenous bolus administration. The pharmacokinetic parameters of HAEE were established using uniformly tritium-labeled HAEE. Pharmacokinetic data provided evidence that HAEE goes through the blood-brain barrier. Based on molecular modeling, a role of LRP1 in receptor-mediated transcytosis of HAEE was proposed. Altogether, the results obtained indicate that the anti-amyloid effect of HAEE, previously found in a mouse model of AD, most likely occurs due to its interaction with Aß species directly in the brain.


Assuntos
Peptídeos/farmacologia , Peptídeos/farmacocinética , Receptores Nicotínicos/genética , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/efeitos dos fármacos , Peptídeos beta-Amiloides/metabolismo , Animais , Transporte Biológico , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptídeos/genética , Coelhos , Ratos , Ratos Wistar , Receptores Nicotínicos/fisiologia
12.
Interdiscip Sci ; 13(3): 521-534, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34324157

RESUMO

The prolific spread of COVID-19 caused by a novel coronavirus (SARS-CoV-2) from its epicenter in Wuhan, China, to every nook and cranny of the world after December 2019, jeopardize the prevailing health system in the world and has raised serious concerns about human safety. Multi-directional efforts are made to design small molecule inhibitors, and vaccines and many other therapeutic options are practiced, but their final therapeutic potential is still to be tested. Using the old drug or vaccine or peptides could aid this process to avoid such long experimental procedures. Hence, here, we have repurposed a small peptide (ATLQAIAS) from the previous study, which reported the inhibitory effects of this peptide. We used in silico mutagenesis approach to design more peptides from the native wild peptide, which revealed that substitutions (T2W, T2Y, L3R, and A5W) could increase the binding affinity of the peptide towards the 3CLpro. Furthermore, using MD simulation and free energy calculation confirmed its dynamics stability and stronger binding affinities. Per-residue energy decomposition analysis revealed that the specified substitution significantly increased the binding affinity at the residue level. Our wide-ranging analyses of binding affinities disclosed that our designed peptide owns the potential to hinder the SARS-CoV-2 and will reduce the progression of SARS-CoV-2-borne pneumonia. Our research strongly suggests the experimental and clinical validation of these peptides to curtail the recent corona outbreak.


Assuntos
Simulação por Computador , Proteases 3C de Coronavírus/antagonistas & inibidores , Simulação de Dinâmica Molecular , Mutagênese , Peptídeos/química , Peptídeos/farmacologia , Vírus da SARS , SARS-CoV-2/efeitos dos fármacos , Antivirais/química , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , COVID-19/virologia , Humanos , Simulação de Acoplamento Molecular , Peptídeos/genética , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Vírus da SARS/química , Vírus da SARS/genética , SARS-CoV-2/enzimologia , Termodinâmica
13.
Commun Biol ; 4(1): 849, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34239038

RESUMO

Huntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Yet, we find filaments in both types of aggregates under ~2 nm in width, thinner than previously reported, and regions forming large sheets. In addition, our data show a prevalent subpopulation of filaments exhibiting a lumpy slab morphology in both aggregates, supportive of the polyQ core model. This provides a basis for future cryoET studies of various aggregated mHTT and polyQ constructs to improve their structure-based modeling as well as their identification in cells without fusion tags.


Assuntos
Tomografia com Microscopia Eletrônica/métodos , Éxons/genética , Proteína Huntingtina/genética , Mutação , Peptídeos/genética , Humanos , Proteína Huntingtina/química , Proteína Huntingtina/ultraestrutura , Doença de Huntington/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/ultraestrutura , Agregados Proteicos , Agregação Patológica de Proteínas , Conformação Proteica
14.
J Agric Food Chem ; 69(31): 8758-8767, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34314160

RESUMO

Lasso peptides, a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) secreted by bacteria, have antimicrobial activity. Here, a novel lasso peptide, microcin Y (MccY), was discovered and characterized. The gene cluster for MccY synthesis was cloned for expression in Escherichia coli. This peptide was purified by HPLC and characterized by Q-TOF. MIC assays showed that some Bacillus, Staphylococcus, Pseudomonas, Shigella, and Salmonella strains were sensitive to MccY. Interestingly, Salmonellatyphimurium and Salmonella infantis were efficiently inhibited by MccY, while they were not affected by MccJ25, a lasso peptide that has antibacterial effects on many Salmonella strains. Furthermore, MccY-resistant strains of S. typhimurium were screened, and mutations were found in FhuA and SbmA, indicating the importance of these transporters for MccY absorption. This novel peptide can greatly broaden the antimicrobial spectrum of MccJ25 in Salmonella and is expected to be used in food preservation and animal feed additive areas.


Assuntos
Bacteriocinas , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa , Bacteriocinas/genética , Bacteriocinas/farmacologia , Escherichia coli/genética , Peptídeos/genética , Peptídeos/farmacologia
15.
Methods Enzymol ; 656: 429-458, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34325794

RESUMO

Backbone N-methylation as a posttranslational modification was recently discovered in a class of ribosomally encoded peptides referred to as borosins. The founding members of the borosins are the omphalotins (A-I), backbone N-methylated, macrocyclic dodecapeptides produced by the mushroom Omphalotus olearius. Omphalotins display a strong and selective toxicity toward the plant parasitic nematode Meloidogyne incognita. The primary product omphalotin A is synthesized via a concerted action of the omphalotin precursor protein (OphMA) and the dual function prolyloligopeptidase/macrocyclase (OphP). OphMA consists of α-N-methyltransferase domain that autocatalytically methylates the core peptide fused to its C-terminus via a clasp domain. Genome mining uncovered over 50 OphMA homologs from the fungal phyla Ascomycota and Basidiomycota. However, the derived peptide natural products have not been described yet, except for lentinulins, dendrothelins and gymnopeptides produced by the basidiomycetes Lentinula edodes, Dendrothele bispora and Gymnopus fusipes, respectively. In this chapter, we describe methods used to isolate and characterize these backbone N-methylated peptides and their precursor proteins both in their original hosts and in the heterologous hosts Escherichia coli and Pichia pastoris. These methods may pave the path for both the discovery of novel borosins with interesting bioactivities. In addition, understanding of borosin biosynthetic pathways may allow setting up a biotechnological platform for the production of pharmaceutical leads for orally available peptide drugs.


Assuntos
Peptídeos , Processamento de Proteína Pós-Traducional , Agaricales , Metilação , Peptídeos/genética , Peptídeos/metabolismo , Saccharomycetales
16.
Methods Enzymol ; 656: 521-544, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34325797

RESUMO

Here we comprehensively summarize the most recent efforts in our research team, aiming at installing N-methyl and azole backbones into peptides expressed in translation. The genetic code reprogramming using the Flexible In-vitro Translation system (FIT system) has proven to be the most reliable and versatile approach for ribosomally installing various exotic amino acids. However, it had been yet difficult in translating diverse kinds of multiple and consecutive sequences of N-methyl amino acids (MeAAs). We have recently reported that a semi-rational fine tuning of MeAA-tRNA affinities for EF-Tu by altering tRNA T-stem sequence achieves efficient delivery of MeAA-tRNAs to the ribosome. Indeed, this approach has made it possible to express N-methyl-peptides containing multiple MeAAs with a remarkably high fidelity. Another interesting backbone modification in peptides is azole moieties often found in natural products, but they are explicitly installed by post-translational modifying enzymes. We have recently devised a method to bypass such enzymatic processes where a bromovinyl group-containing amino acid is incorporated into the peptide by genetic code reprogramming and then chemically converted to an azole group via an intramolecular heterocyclization reaction. These methods will grant more drug-like properties to peptides than ordinary peptides in terms of protease resistance and cell membrane permeability. Particularly when they can be integrated with in vitro mRNA display, such as the RaPID system, the discovery of de novo bioactive peptides can be realized.


Assuntos
Código Genético , Ribossomos , Peptídeos/genética , RNA Mensageiro/genética , RNA de Transferência , Ribossomos/genética
17.
Nat Commun ; 12(1): 3613, 2021 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-34127656

RESUMO

The development of biomedical glues is an important, yet challenging task as seemingly mutually exclusive properties need to be combined in one material, i.e. strong adhesion and adaption to remodeling processes in healing tissue. Here, we report a biocompatible and biodegradable protein-based adhesive with high adhesion strengths. The maximum strength reaches 16.5 ± 2.2 MPa on hard substrates, which is comparable to that of commercial cyanoacrylate superglue and higher than other protein-based adhesives by at least one order of magnitude. Moreover, the strong adhesion on soft tissues qualifies the adhesive as biomedical glue outperforming some commercial products. Robust mechanical properties are realized without covalent bond formation during the adhesion process. A complex consisting of cationic supercharged polypeptides and anionic aromatic surfactants with lysine to surfactant molar ratio of 1:0.9 is driven by multiple supramolecular interactions enabling such strong adhesion. We demonstrate the glue's robust performance in vitro and in vivo for cosmetic and hemostasis applications and accelerated wound healing by comparison to surgical wound closures.


Assuntos
Peptídeos/química , Peptídeos/genética , Proteínas/química , Proteínas/genética , Adesivos/química , Adulto , Animais , Cianoacrilatos/química , Modelos Animais de Doenças , Feminino , Regeneração Tecidual Guiada/métodos , Hemostasia , Humanos , Fígado/patologia , Camundongos , Ratos , Ratos Wistar , Pele/patologia , Suínos , Adesivos Teciduais/química , Cicatrização
18.
Acta Trop ; 221: 106013, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34146538

RESUMO

AIM: This study is looking for a common pathogenicity between SARS-CoV-2 and Plasmodium species, in individuals with certain HLA serotypes. METHODS: 1. Tblastx searches of SARS-CoV-2 are performed by limiting searches to five Plasmodium species that infect humans. 2. Aligned sequences in the respective organisms' proteomes are searched with blastp. 3. Binding predictions of the identified SARS-CoV-2 peptide to HLA supertype representatives are performed. 4. Blastp searches of predicted epitopes that bind strongly to the identified HLA allele are performed by limiting searches to H. sapiens and Plasmodium species, separately. 5. Peptides with minimum 60% identity to the predicted epitopes are found in results. 6. Peptides among those, which bind strongly to the same HLA allele, are predicted. 7. Step-4 is repeated by limiting searches to H. sapiens, followed by the remaining steps until step-7, for peptides sourced by Plasmodium species after step-6. RESULTS: SARS-CoV-2 peptide with single letter amino acid code CFLGYFCTCYFGLFC has the highest identity to P. vivax. Its YFCTCYFGLF part is predicted to bind strongly to HLA-A*24:02. Peptides in the human proteome both homologous to YFCTCYFGLF and with a strong binding affinity to HLA-A*24:02 are YYCARRFGLF, YYCHCPFGVF, and YYCQQYFFLF. Such peptides in the Plasmodium species' proteomes are FFYTFYFELF, YFVACLFILF, and YFPTITFHLF. The first one belonging to P. falciparum has a homologous peptide (YFYLFSLELF) in the human proteome, which also has a strong binding affinity to the same HLA allele. CONCLUSION: Immune responses to the identified-peptides with similar sequences and strong binding affinities to HLA-A*24:02 can be related to autoimmune response risk in individuals with HLA-A*24:02 serotypes, upon getting infected with SARS-CoV-2 or P. falciparum.


Assuntos
COVID-19 , Antígeno HLA-A24 , Malária Vivax , Peptídeos , Epitopos de Linfócito T , Humanos , Peptídeos/genética , SARS-CoV-2
19.
Front Immunol ; 12: 613365, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34149681

RESUMO

Hyla annectans is a tree frog living in the southwestern plateau area of China where there is strong ultraviolet radiation and long duration of sunshine. So their naked skin may possess chemical defense components that protect it from acute photo-damage. However, no such peptide or components has been identified till to date. In the current work, two novel peptides (FW-1, FWPLI-NH2 and FW-2, FWPMI-NH2) were identified from the skin of the tree frog. Five copies of FW-1 and four copies of FW-2 are encoded by an identical gene and released from the same protein precursor, which possess 167 amino acid residues. FW-1 and -2 can exert significant anti-inflammatory functions by directly inhibiting Ultraviolet B irradiation (UVB)-induced secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). They may achieve this function by modulating the UV-induced stress signaling pathways such as Mitogen-activated protein kinases (MAPK) and Nuclear Factor Kappa B (NF-κB). Besides, FW-1 and -2 showed potential antioxidant effects on epidermis by attenuating the UVB-induced reactive oxygen species (ROS) production through an unknown mechanism. Considering small peptides' easy production, storage, and potential photo-protective activity, FW-1/2 might be exciting leading compounds or templates for the development of novel pharmacological agents for the suppression of UVB-induced skin inflammation. Moreover, this study might expand our knowledge on skin defensive mechanism of tree frog upon UVB irradiation.


Assuntos
Proteínas de Anfíbios/metabolismo , Anti-Inflamatórios/metabolismo , Queratinócitos/fisiologia , Peptídeos/metabolismo , Pele/metabolismo , Raios Ultravioleta/efeitos adversos , Proteínas de Anfíbios/genética , Animais , Antioxidantes , Anuros , China , Clonagem Molecular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Peptídeos/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Pele/patologia , Fator de Necrose Tumoral alfa/metabolismo
20.
Int J Biol Macromol ; 184: 522-529, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34119553

RESUMO

Sericin, as the main component of silkworm cocoon silk, surrounds and protects the silk fibroin. Sericin is a natural macromolecular protein complex encoded by the genes Ser1, Ser2, and Ser3. At present, there are no available antibodies against sericin that may be used to identify and locate it at the protein level, hindering the study of its secretion mechanism and materials application. Therefore, the development of effective antibodies against sericin is an urgent necessity. To address this problem, we prepared polyclonal antibodies against the Ser1, Ser2 and Ser3 proteins using synthesized peptides for the first time. The specificity of the antibodies was confirmed using dot blot, immunoblotting and mass spectrometry on the hybrid bands of the middle silk gland. The immunoblotting results of anti-sericin antibodies showed that sericin has different molecular weights in different regions of the middle silk gland and strains in the 5th instar. Through immunohistochemistry, anti-sericin antibodies revealed that sericin presented different distributions in the anterior part of the middle silk gland of 872 strain at the 7th day of 5th instar. In addition, the prepared antibodies not only detected intact sericin molecules, but also detected degraded sericin that was dissolved in five different solvents. In summary, this work prepared effective sericin antibodies for silk protein synthesis and secretion research and provides a possible molecular detection method for biological products containing silkworm sericin.


Assuntos
Anticorpos/análise , Bombyx/crescimento & desenvolvimento , Peptídeos/imunologia , Sericinas/química , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Bombyx/imunologia , Bombyx/metabolismo , Peso Molecular , Família Multigênica , Peptídeos/genética , Sericinas/genética , Sericinas/imunologia , Especificidade da Espécie
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