RESUMO
Human programmed cell death protein 1 (hPD-1) is an essential receptor in the immune checkpoint pathway. It has played an important role in cancer therapy. However, not all patients respond positively to the PD-1 antibody treatment, and the underlying mechanism remains unknown. PD-1 is a transmembrane glycoprotein, and its extracellular domain (ECD) is reported to be responsible for interactions and signal transduction. This domain contains 4 N-glycosylation sites and 25 potential O-glycosylation sites, which implicates the importance of glycosylation. The structure of hPD-1 has been intensively studied, but the glycosylation of this protein, especially the glycan on each glycosylation site, has not been comprehensively illustrated. In this study, hPD-1 ECD expressed by human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells was analyzed; not only N- and O-glycosylation sites but also the glycans on these sites were comprehensively analyzed using mass spectrometry. In addition, hPD-1 ECD binding to different anti-hPD-1 antibodies was tested, and N-glycans were found functioned differently. All of this glycan information will be beneficial for future PD-1 studies.
Assuntos
Cricetulus , Glicômica , Polissacarídeos , Receptor de Morte Celular Programada 1 , Humanos , Glicosilação , Células CHO , Animais , Receptor de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/química , Células HEK293 , Polissacarídeos/metabolismo , Polissacarídeos/química , Polissacarídeos/análise , Glicômica/métodos , Proteômica/métodos , Domínios Proteicos , Glicoproteínas/metabolismo , Glicoproteínas/química , Ligação ProteicaRESUMO
Chemical synthesis can generate homogeneous glycoproteins with well-defined and modifiable glycan structures at designated sites. The precision and flexibility of the chemical synthetic approach provide a solution to the heterogeneity problem of glycopeptides/glycoproteins obtained through biological approaches. In this study, we reported that the conserved N-glycosylation sequon (Asn-Xaa-Ser/Thr) of glycoproteins can serve as a general site for performing Ser/Thr ligation to achieve N-linked glycoprotein synthesis. We developed an N + 2 strategy to prepare the corresponding glycopeptide salicylaldehyde esters for Ser/Thr ligation and demonstrated that Ser/Thr ligation at the sequon was not affected by the steric hindrance brought about by the large-sized glycan structures. The effectiveness of this strategy was showcased by the total synthesis of the glycosylated receptor-binding domain (RBD) of the SARS-CoV-2 spike protein.
Assuntos
Glicopeptídeos , Glicoproteínas , Glicosilação , Glicopeptídeos/química , Glicopeptídeos/síntese química , Glicoproteínas/química , Glicoproteínas/síntese química , Glicoproteína da Espícula de Coronavírus/química , Humanos , Aldeídos/químicaRESUMO
The rapid spread of drug-resistant pathogens and the declining discovery of new antibiotics have created a global health crisis and heightened interest in the search for novel antibiotics. Beyond their discovery, elucidating mechanisms of action has necessitated new approaches, especially for antibiotics that interact with lipidic substrates and membrane proteins. Here, we develop a methodology for real-time reaction monitoring of the activities of two bacterial membrane phosphatases, UppP and PgpB. We then show how we can inhibit their activities using existing and newly discovered antibiotics such as bacitracin and teixobactin. Additionally, we found that the UppP dimer is stabilized by phosphatidylethanolamine, which, unexpectedly, enhanced the speed of substrate processing. Overall, our results demonstrate the potential of native mass spectrometry for real-time biosynthetic reaction monitoring of membrane enzymes, as well as their in situ inhibition and cofactor binding, to inform the mode of action of emerging antibiotics.
Assuntos
Antibacterianos , Bacitracina , Antibacterianos/química , Testes de Sensibilidade Microbiana , BactériasRESUMO
Pseudaminic acid (Pse) is found in the polysaccharide structures of the cell surface of various Gram-negative pathogenic bacteria including Acinetobacter baumannii and considered as an important component of cell surface glycans including oligosaccharides and glycoproteins. However, the glycosyltransferase that is responsible for the Pse glycosylation in A. baumannii remains unknown yet. In this study, through comparative genomics analysis of Pse-positive and negative A. baumannii clinical isolates, we identified a potential glycosyltransferase, KpsS1, located right downstream of the Pse biosynthesis genetic locus. Deletion of this gene in an Pse-positive A. baumannii strain, Ab8, impaired the glycosylation of Pse to the surface CPS and proteins, while the gene knockout strain, Ab8ΔkpsS1, could still produce Pse with 2.86 folds higher amount than that of Ab8. Furthermore, impairment of Pse glycosylation affected the morphology and virulence potential of A. baumannii, suggesting the important role of this protein. This study will provide insights into the further understanding of Pse in bacterial physiology and pathogenesis.
Assuntos
Acinetobacter baumannii , Glicosiltransferases , Acinetobacter baumannii/metabolismo , Glicosilação , Glicosiltransferases/metabolismo , Glicosiltransferases/genética , Açúcares Ácidos/metabolismo , Açúcares Ácidos/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , VirulênciaRESUMO
The scavenger receptors (SRs) gene family is considered as the membrane-associated pattern recognition receptors that plays important roles in the immune responses of organisms. However, there is currently limited research on the systematic identification of the SRs gene family in teleost and their role in the innate immunity of S. schegelii. In this study, we identified and annotated 15 SRs genes in S. schegelii. Through phylogenetic analysis, analysis of conserved domains, gene structure, and motif composition, we found that SRs gene family within different classes were relatively conserved. Additionally, we used qRT-PCR to analyze the expression patterns of SRs genes in immune-related tissues from healthy and Acinetobacter johnsonii-infected S. schegelii. The results showed that SRs genes exhibited different tissue expression patterns and the expression of SRs genes significantly changed after A. johnsonii infection. These results provided a valuable basis for further understanding of the functions of SRs in the innate immune response of S. schegelii.
Assuntos
Evolução Molecular , Doenças dos Peixes , Proteínas de Peixes , Perfilação da Expressão Gênica , Imunidade Inata , Filogenia , Receptores Depuradores , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/química , Imunidade Inata/genética , Doenças dos Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Receptores Depuradores/genética , Receptores Depuradores/imunologia , Receptores Depuradores/química , Perciformes/genética , Perciformes/imunologia , Regulação da Expressão Gênica/imunologia , Peixes/genética , Peixes/imunologia , Alinhamento de Sequência/veterináriaRESUMO
Although solid-phase peptide synthesis combining with chemical ligation provides a way to build up customized polypeptides in general, many targets are still presenting challenges for the conventional synthetic process, such as hydrophobic proteins. New methods and strategies are still required to overcome these obstacles. In this study, kinetic studies of Cys/Pen ligation and its acidolysis were performed, from which the fast acidolysis of substituted N,S-benzylidene thioacetals (NBTs) was discovered. The study demonstrates the potential of NBTs as a promising Cys switchable protection, facilitating the chemical synthesis of peptides and proteins by efficiently disrupting peptide aggregation. The compatibility of NBTs with other commonly adopted Cys protecting groups and their applications in sequential disulfide bond formation were also investigated. The first chemical synthesis of the native human programmed death ligand 1 immunoglobulin V-like (PD-L1 IgV) domain was achieved using the NBT strategy, showcasing its potential in difficult protein synthesis.
Assuntos
Cisteína , Peptídeos , Cisteína/química , Peptídeos/química , Peptídeos/síntese química , Humanos , Acetais/química , Compostos de Benzilideno/química , Compostos de Benzilideno/síntese química , Proteínas/química , Proteínas/síntese químicaRESUMO
Diketopiperazine (DKP) derived cyclic amidine structures widely exist in peptide natural products according to the genome mining result. The largely unknown bioactivity and mode of action are partially caused by the poor availability of the compounds via microbiological and chemical approaches. To tackle this challenge, in this work, we have developed the on-resin ring-closing amidine formation strategy to synthesize peptides containing N-terminal DKP derived cyclic amidine structure, in which the 6-exo-trig cyclization mediated by HgCl2 activation of thioamides was the key step. Leveraging from this new strategy, we finished the total syntheses of streptamidine and klebsazolicin. Meanwhile, eleven klebsazolicin analogues were synthesized for its structure-activity relationship study.
Assuntos
Amidinas , Amidinas/química , Amidinas/síntese química , Ciclização , Biomimética , Relação Estrutura-Atividade , Estrutura Molecular , Dicetopiperazinas/química , Dicetopiperazinas/síntese química , Materiais Biomiméticos/química , Materiais Biomiméticos/síntese químicaRESUMO
Cooperation between organelles is essential to maintain the normal functions of cells. Lipid droplets (LDs) and nucleoli, as important organelles, play an important role in the normal activities of cells. However, due to the lack of appropriate tools, in situ observation of the interaction between them has been rarely reported. In this work, taking into full consideration the pH and charge differences between LDs and nucleoli, a pH-triggered charge reversible fluorescent probe (LD-Nu) was constructed based on a cyclization-ring-opening mechanism. The in vitro pH titration experiment and 1H NMR showed that LD-Nu gradually transferred from the charged form to the electroneutral form with the increase of pH, and thus, the conjugate plane was reduced and its fluorescence blue-shifted. Most importantly, the physical contact between LDs and nucleoli was visualized for the first time. Meanwhile, the relationship between LDs and nucleoli was also further investigated, and the results showed that their interaction was more liable to be affected by the abnormality of LDs than those of nucleoli. Moreover, the cell imaging results displayed that the LDs both in the cytoplasm and nucleus were observed using the probe LD-Nu, and interestingly, the LDs in the cytoplasm were more susceptible to external stimuli than those in the nucleus. In a word, the probe LD-Nu can serve as a powerful tool for further exploration of the interaction mechanism between LDs and nucleoli in living cells.
Assuntos
Corantes Fluorescentes , Gotículas Lipídicas , Gotículas Lipídicas/química , Corantes Fluorescentes/química , Fluorescência , Diagnóstico por Imagem , Concentração de Íons de HidrogênioRESUMO
The S-palmitoylation on Cys residue and O-acetylation on Ser/Thr residues are two types of base-labile post-translational modifications (PTMs) in cells. The lability of these PTMs to bases and nucleophiles makes the peptides/proteins bearing S-palmitoyl or O-acetyl groups challenging synthetic targets, which cannot be prepared via the standard Fmoc-SPPS and native chemical ligation. In this review, we summarized the efforts towards their preparation in the past 40â years, with the focus on the evolution of synthetic methods.
Assuntos
Peptídeos , Proteínas , Proteínas/química , Peptídeos/química , Processamento de Proteína Pós-TraducionalRESUMO
With the advance of deep learning technology, convolutional neural network (CNN) has been wildly used and achieved the state-of-the-art performances in the area of medical image classification. However, most existing medical image classification methods conduct their experiments on only one public dataset. When applying a well-trained model to a different dataset selected from different sources, the model usually shows large performance degradation and needs to be fine-tuned before it can be applied to the new dataset. The goal of this work is trying to solve the cross-domain image classification problem without using data from target domain. In this work, we designed a self-supervised plug-and-play feature-standardization-block (FSB) which consisting of image normalization (INB), contrast enhancement (CEB) and boundary detection blocks (BDB), to extract cross-domain robust feature maps for deep learning framework, and applied the network for chest x-ray-based lung diseases classification. Three classic deep networks, i.e. VGG, Xception and DenseNet and four chest x-ray lung diseases datasets were employed for evaluating the performance. The experimental result showed that when employing feature-standardization-block, all three networks showed better domain adaption performance. The image normalization, contrast enhancement and boundary detection blocks achieved in average 2%, 2% and 5% accuracy improvement, respectively. By combining all three blocks, feature-standardization-block achieved in average 6% accuracy improvement.
Assuntos
Aprendizado Profundo , Pneumopatias , Humanos , Pulmão , Pneumopatias/diagnóstico por imagem , Redes Neurais de Computação , Padrões de ReferênciaRESUMO
Proteins with highly hydrophobic regions or aggregation-prone sequences are typically difficult targets for chemical synthesis at the current stage, as obtaining such type of peptides via solid-phase peptide synthesis requires sophisticated operations. Herein, we report N,O-benzylidene acetal dipeptides (NBDs) as robust and effective building blocks to allow the direct synthesis of difficult peptides and proteins via a kinked backbone strategy. The effectiveness and easy accessibility of NBDs have been well demonstrated in our chemical syntheses of various challenging peptides and proteins, including chemokine, therapeutic hormones, histone, and glycosylated erythropoietin.
Assuntos
Acetais , Dipeptídeos , Dipeptídeos/química , Peptídeos/química , Proteínas , Técnicas de Síntese em Fase SólidaRESUMO
Recently, ortho-phthalaldehyde (OPA) is experiencing a renascence for the modification of proteins and peptides through OPA-amine two-component reactions for bioconjugation and intramolecular OPA-amine-thiol three-component reactions for cyclization. Historically, small thiol molecules were used in large excess to allow for the intermolecular OPA-amine-thiol reaction forming 1-thio-isoindole derivatives. In this study, we discovered that guanidine could serve as an effective additive to switch the intermolecular OPA-amine-thiol three-component reaction to a stoichiometric process and enable the modular construction of peptide-peptide, and peptide-drug conjugate structures. Thus, 12 model peptide-peptide conjugates have been synthesized from unprotected peptides featuring all proteinogenic residues. Besides, 6 peptide-drug conjugates have been prepared in one step, with excellent conversions and isolated yields. In addition, a conjugate product has been further functionalized by utilizing a premodified OPA derivative, demonstrating the versatility and flexibility of this reaction.
RESUMO
Chemical synthesis of proteins bearing base-labile post-translational modifications (PTMs) is a challenging task. For instance, O-acetylation and S-palmitoylation PTMs cannot survive Fmoc removal conditions during Fmoc-solid phase peptide synthesis (SPPS). In this work, we developed a new Boc-SPPS-based strategy for the synthesis of peptide C-terminal salicylaldehyde (SAL) esters, which are the key reaction partner in Ser/Thr ligation and Cys/Pen ligation. The strategy utilized the semicarbazone-modified aminomethyl (AM) resin, which could support the Boc-SPPS and release the peptide SAL ester upon treatment with TFA/H2 O and pyruvic acid. The non-oxidative aldehyde regeneration was fully compatible with all the canonical amino acids. Armed with this strategy, we finished the syntheses of the O-acetylated protein histone H3(S10ac, T22ac) and the hydrophobic S-palmitoylated peptide derived from caveolin-1.
Assuntos
Peptídeos , Proteínas , Peptídeos/química , Aldeídos , Ésteres/químicaRESUMO
Chemical synthesis of proteins with aggregable or colloidal peptide segments presents a formidable task, as such peptides prove to be difficult for both solid-phase peptide synthesis and peptide ligation. To address this issue, we have developed ligation embedding aggregation disruptor (LEAD) as an effective strategy for the chemical synthesis of difficult-to-obtain proteins. The N,O/S-benzylidene acetals generated from Ser/Thr ligation and Cys/Pen ligation are found to effectively disrupt peptide aggregation, and they can be carried for sequential ligations toward protein synthesis. The effectiveness and generality of this strategy have been demonstrated with total syntheses of programmed cell death protein 1 immunoglobulin like V-type domain and extracellular domain.
Assuntos
Peptídeos , Receptor de Morte Celular Programada 1 , Imunoglobulinas , Peptídeos/química , Proteínas/química , Técnicas de Síntese em Fase SólidaRESUMO
Herein, we report the synthesis and antibacterial evaluation of a series of daptomycin lactam-based analogues. As compared with daptomycin, the daptomycin analogue with singly modified lactam has an eightfold increase in its minimum inhibitory concentration (MIC) against methicillin-resistant Staphylococcus aureus. Incorporating effective modifications found in previous daptomycin structure-activity relationship studies to produce lactam-based analogues with multiple modifications did not improve the antibacterial activity of the analogues. Instead, the antibacterial activity was greatly reduced when a rather rigid 4-(phenylethynyl)benzoyl group replaced the flexible n-decanoyl group. The fact that the lactam analogue with the 4-(phenylethynyl)benzoyl group did not exhibit the antibacterial activity comparable to the two respective singly modified analogues showed that the inactivity was probably due to the deviation from the active conformation. This series of lactam analogues may generate insights on the importance of studying the active conformation of daptomycin and how the structural modifications affect the active conformation.
Assuntos
Daptomicina , Staphylococcus aureus Resistente à Meticilina , Daptomicina/farmacologia , Lactamas/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , Testes de Sensibilidade MicrobianaRESUMO
Xanthine oxidoreductase (XOR) is a clinically validated target for the treatment of hyperuricemia and gout. A series of novel 1,2,4-triazoles were identified as potent XO inhibitors via a fused-pharmacophore strategy based on the interaction modes of febuxostat and topiroxostat. Among them, compound 7i showed an IC50 value of 0.20 nM against XOR, which was superior to febuxostat and topiroxostat. Furthermore, 7i exhibited significant hypouricemic and serum XOR inhibitory effects in potassium oxonate induced hyperuricemia mouse models. A single-dose toxicity assessment of 7i showed no noticeable toxicity at the dose of 50 mg/kg. These results demonstrated that 7i could be a promising lead compound for the treatment of hyperuricemia and gout.
Assuntos
Gota , Hiperuricemia , Camundongos , Animais , Febuxostat/farmacologia , Xantina Desidrogenase/uso terapêutico , Hiperuricemia/induzido quimicamente , Hiperuricemia/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Triazóis/farmacologia , Triazóis/uso terapêutico , Gota/tratamento farmacológico , Xantina OxidaseRESUMO
As a ubiquitous degradation process in cells, autophagy plays important roles in various biological activities. However, the abnormality of autophagy is closely related to many diseases, such as aging, neurological disorder, and cancer. Thus, monitoring the process of autophagy in living cells has high significance in biological studies and diagnosis of related diseases. In order to real-time and in situ monitor the process of autophagy, various organic fluorescent probes have been explored in recent years owing to the advantages such as handy staining processes, flexible molecular design strategies, and near-nondestructive detection. However, this interesting and frontier topic has not been reviewed so far. In this tutorial review, we will focus on the latest breakthrough results of organic fluorescent probes in monitoring autophagy of living cells, especially the probe design strategies based on the several microenvironment changes of the autophagy process, and the responding mechanisms and bio-imaging applications in the autophagy process. In addition, we will discuss the shortcomings and limitations of the probes developed, such as susceptible to interference, unable to monitor the whole process, and lack of clinical applications. Finally, we will highlight some challenges and further opportunities in this field. This tutorial review may promote the development of more robust fluorescent probes to further reveal the mechanisms of autophagy, which is the basis of degradation and recycling of cell components.
Assuntos
Autofagia , Corantes Fluorescentes/química , Neoplasias/diagnóstico por imagem , Doenças do Sistema Nervoso/diagnóstico por imagem , Compostos Orgânicos/química , Linhagem Celular Tumoral , Senescência Celular , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Estrutura Molecular , Compostos Orgânicos/síntese química , Compostos Orgânicos/metabolismoRESUMO
The homogeneously glycosylated 76-amino acid adiponectin collagenous domains (ACDs) with all of the possible 15 glycoforms have been chemically and individually synthesized using stereoselective glycan synthesis and chemical peptide ligation. The following biological and pharmacological studies enabled correlating glycan pattern to function in the inhibition of cancer cell growth as well as the regulation of systemic energy metabolism. In particular, hAdn-WM6877 was tested in detail with different mouse models and it exhibited promising in vivo antitumor, insulin sensitizing, and hepatoprotective activities. Our studies demonstrated the possibility of using synthetic glycopeptides as the adiponectin downsized mimetic for the development of novel therapeutics to treat diseases associated with deficient adiponectin.
Assuntos
Adiponectina/síntese química , Adiponectina/metabolismo , Adiponectina/química , Glicosilação , Humanos , Modelos Moleculares , Estrutura MolecularRESUMO
Nonribosomal peptide synthesis in bacteria has endowed cyclic peptides with fascinating structural complexity via incorporating nonproteinogenic amino acids. These bioactive cyclic peptides provide interesting structural motifs for exploring total synthesis and medicinal chemistry studies. Cyclic glycopeptide mannopeptimycins exhibit antibacterial activity against antibiotic-resistant Gram-positive pathogens and act as the lipid II binder to stop bacterial cell wall biosynthesis. Here, we report a strategy streamlining solution phase-solid phase synthesis and chemical ligation-mediated peptide cyclization for the total synthesis of mannopeptimycin ß.
Assuntos
Aminoácidos/química , Glicopeptídeos/síntese química , Imidazolidinas/química , Glicopeptídeos/química , Estrutura MolecularRESUMO
Visualizing cholesterol (CL) fluctuation in plasma membranes is a crucially important yet challenging task in cell biology. Here, we proposed a new imaging strategy based on permeability changes of plasma membranes triggered by different CL contents to result in controllable spatial distribution of single fluorescent probes (SF-probes) in subcellular organelles. Three spatial distribution-controllable SF-probes (PMM-Me, PMM-Et, and PMM-Bu) for imaging CL fluctuation in plasma membranes were rationally developed. These SF-probes target plasma membranes and mitochondria at normal CL levels, while they display solely staining in plasma membranes and mitochondria at increased and decreased CL levels, respectively. These polarity-sensitive probes also show distinct emission colors with fluorescence peaks of 575 and 620 nm in plasma membranes and mitochondria, respectively. Thus, the CL fluctuation in plasma membranes can be clearly visualized by means of the spatially distributed and two-color emissive SF-probes.