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Protein ligation

Andrew R Swartz, Wilfred Chen
E2 nanocages functionalized with Z-domain-elastin-like polypeptide affinity ligands (Z-ELP40) using Sortase A (SrtA) ligation have been shown to be a promising scaffold for purifying monoclonal antibodies (mAbs) based on affinity precipitation. However, the reversible nature of SrtA reaction has been attributed to the low ligation efficiency (< 25%) and has significantly limited the practical utility of the technology. Here, we reported an improved conjugation platform using the SpyTag/SpyCatcher pair to enable the spontaneous isopeptide bond formation between SpyTag-E2 and Z-ELP-SpyCatcher fusion proteins of two different ELP chain-lengths...
August 10, 2018: Bioconjugate Chemistry
Fariborz Soroush, Yuan Tang, Kimberly Guglielmo, Alex Engelmann, Elisabetta Liverani, Jordan Langston, Shuang Sun, Satya Kunapuli, Mohammad F Kiani, Laurie E Kilpatrick
BACKGROUND: Neutrophil dysfunction plays an important role in inflammation-induced tissue injury. Previously, we identified Protein Kinase C-delta (PKCδ) as a critical controller of neutrophil activation and trafficking but how PKCδ is regulated in inflammation hasn't been delineated. PKCδ activity is regulated by tyrosine phosphorylation on multiple sites. Tyrosine155 is a key regulator of apoptosis and gene expression, but its role in proinflammatory signaling is not known. METHODS: In vitro studies: superoxide anion (O2) and neutrophil extracellular traps (NETs) were measured in bone marrow neutrophils (BMN) isolated from wild type (WT) and PKCδY155F knock-in mice (PKCδ tyrosine 155 →phenylalanine)...
August 7, 2018: Shock
Feng Hao, Lei Liu, Zi-Jian Wu, Ling Hu, Jie Wang, Qing Yu, Rong-Lin Cai, Lu He
OBJECTIVE: To compare the therapeutic effect of electroacupuncture (EA) of "Shenmen" (HT 7, Yuan point of the Heart Meridian), "Zhizheng" (SI 7, Luo point of the Small Intestine Meridian) and "Xinshu" (BL15, Back- shu point) on the expression of nerve growth factor (NGF) and its receptor, tyrosine kinase A (TrkA) in the cerebral cortex of myocardial ischemia (MI) rats. METHODS: A total of 70 male SD rats were randomized into sham operation (sham, n =10), model, HT 7, SI 7, and BL15 groups ( n =15 in each of the latter 4 groups)...
July 25, 2018: Zhen Ci Yan Jiu, Acupuncture Research
Brigitte Grouix, Francois Sarra-Bournet, Martin Leduc, Jean-Christophe Simard, Kathy Hince, Lilianne Geerts, Alexandra Blais, Liette Gervais, Alexandre Laverdure, Alexandra Felton, Jonathan Richard, Jugurtha Ouboudinar, William Gagnon, Francois Leblond, Pierre Laurin, Lyne Gagnon
Hepatic fibrosis is a major cause of morbidity and mortality for which there is currently no effective therapy. We have previously shown that PBI-4050 is a dual GPR40 agonist/GPR84 antagonist exerting anti-fibrotic, anti-inflammatory and anti-proliferative actions. We evaluated PBI-4050 for the treatment of liver fibrosis in vivo and elucidated its mechanism of action on human hepatic stellate cells (HSCs). The anti-fibrotic effect of PBI-4050 was evaluated in carbon tetrachloride and in bile duct ligation-induced liver fibrosis rodent models...
August 9, 2018: Journal of Pharmacology and Experimental Therapeutics
Franziska Thomas, Wolf Mathis Rink
Coiled Coils (CCs) are well-understood protein folding motifs. They appear in a variety of oligomer states and as homo- and heteromeric assemblies. This versatility and the general accessibility by de novo design makes them ideal building blocks for synthetic biology. This minireview highlights the efforts being made in designing small peptide catalysts or reaction templates based on the CC scaffold. First reports describe autocatalysis or mediation of peptide ligation based on CC recognition. Over the years, the designs became more advanced, catalyzing ester hydrolysis, acyl transfer and redox reactions with in part enzyme-like reactivity...
August 9, 2018: Chemistry: a European Journal
Mengqiu Dang, Xiangjun Zeng, Buxing Chen, Hongxia Wang, Huihua Li, Fenghe Du, Caixia Guo
The ubiquitin-proteasome system (UPS) is essential for protein degradation and plays critical roles in myocardial ischemia/reperfusion (MI/R) injuries. Previous studies have demonstrated that the soluble receptor for advanced glycation end-product (sRAGE) inhibited MI/R-induced apoptosis by upregulating proteasome subunits. However, the mechanism remains unknown. An MI/R model was established by left anterior descending (LAD) coronary artery ligation in mice. Recombinant sRAGE protein or saline was injected intramyocardially with or without neutralizing interferon-γ (IFN-γ) antibody injected intraperitoneally before ligation...
August 8, 2018: Laboratory Investigation; a Journal of Technical Methods and Pathology
Adisak Romsang, Jintana Duang-Nkern, Khwannarin Khemsom, Lampet Wongsaroj, Kritsakorn Saninjuk, Mayuree Fuangthong, Paiboon Vattanaviboon, Skorn Mongkolsuk
During the translation process, transfer RNA (tRNA) carries amino acids to ribosomes for protein synthesis. Each codon of mRNA is recognized by a specific tRNA, and enzyme-catalysed modifications to tRNA regulate translation. TtcA is a unique tRNA-thiolating enzyme that requires an iron-sulfur ([Fe-S]) cluster to catalyse thiolation of tRNA. In this study, the physiological functions of a putative ttcA in Pseudomonas aeruginosa, an opportunistic human pathogen that causes serious problems in hospitals, were characterized...
August 8, 2018: Scientific Reports
Yohei Hirano, Yasumasa Ode, Mahendar Ochani, Ping Wang, Monowar Aziz
Sepsis is a severe inflammatory condition associated with high mortality. Transmigration of neutrophils into tissues increases their lifespan to promote deleterious function. Junctional adhesion molecule-C (JAM-C) plays a pivotal role in neutrophil transmigration into tissues. We aim to study the role of JAM-C on the aging of neutrophils to cause sepsis-induced acute lung injury (ALI). Sepsis was induced in C57BL/6J mice by cecal ligation and puncture (CLP) and JAM-C expression in serum was assessed. Bone marrow-derived neutrophils (BMDN) were treated with recombinant mouse JAM-C (rmJAM-C) ex vivo and their viability was assessed...
August 8, 2018: Journal of Leukocyte Biology
Sarah Riman, Chin Hong Shek, Michelle A Peck, Jaclyn Benjamin, Daniele Podini
The positive identification of seminal fluids in sexual assault crimes is considered crucial evidence to determine whether a sexual act occurred or not. However, current presumptive methods lack specificity and sensitivity. Confirmation of semen by microscopic examination of spermatozoa is laborious, time consuming, and can sometimes lead to negative or inconclusive results. Here we report the use of the Proximity Ligation Real-Time PCR (PLiRT-PCR) assay as an attractive and promising confirmatory method for the identification of semen and sperm proteins using two polyclonal antibodies, Prostate Specific Antigen (PSA) and Sperm-Specific Protein (SP10), respectively...
July 25, 2018: Forensic Science International. Genetics
Peter van Ulsen, Katinka M Zinner, Wouter S P Jong, Joen Luirink
The classical monomeric autotransporters are ubiquitously used by Gram-negative bacteria to export virulence and colonization factors to their cell surface or into their surroundings. They are expressed as monomeric proteins that pass the inner and outer membrane in two consecutive steps facilitated by the Sec translocon and the Bam complex, respectively. In this mini-review we discuss how autotransporters translocate their secreted functional domains across the outer membrane. We highlight the interactions with the Bam complex and discuss how specific features of the recently solved structure of Bam lead to a mechanistic model for autotransporter secretion...
July 30, 2018: FEMS Microbiology Letters
Jose A Rodriguez, Josune Orbe, Goren Saenz-Pipaon, Gloria Abizanda, Natalia Gebara, Flavia Radulescu, Pedro M Azcarate, Luis Alonso-Perez, David Merino, Felipe Prosper, Jose A Paramo, Carmen Roncal
INTRODUCTION: Wound healing after myocardial infarction (MI) is mediated by different cell types, secreted proteins, components of the extracellular matrix (ECM) and, as increasing evidences suggest, extracellular vesicles (EVs). We aim to determine the dynamics of release and origin of EVs after MI, as well as their biological activity on endothelial cells (ECs). METHODS: MI was induced in WT mice and blood and tissues collected at baseline, 3, 15 and 30 days post-ligation for cardiac function (echocardiography) and histological evaluation...
August 1, 2018: Thrombosis Research
Christopher H Bowen, Bin Dai, Cameron J Sargent, Wenqin Bai, Pranay Ladiwala, Huibao Feng, Wenwen Huang, David L Kaplan, Jonathan M Galazka, Fuzhong Zhang
Despite significant efforts to engineer their heterologous production, recombinant spider silk proteins (spidroins) have yet to replicate the unparalleled combination of high strength and toughness exhibited by natural spider silks, preventing their use in numerous mechanically-demanding applications. To overcome this long-standing challenge, we have developed a synthetic biology approach combining standardized DNA part assembly and split intein-mediated ligation to produce recombinant spidroins of previously unobtainable size (556 kDa), containing 192 repeat motifs of the Nephila clavipes dragline spidroin...
August 6, 2018: Biomacromolecules
Wolfgang Ott, Ellis Durner, Hermann E Gaub
Covalent surface immobilization of proteins for binding assays is typically performed non-specifically via lysine residues. However, receptors that either have lysines near their binding pockets, or whose presence at the sensor surface is electrostatically disfavoured, can be hard to probe. To overcome these limitations and to improve the homogeneity of surface functionalization, we adapted and optimized three different enzymatic coupling strategies (4´-phosphopantetheinyl transferase, Sortase A and asparaginyl endopeptidase) for biolayer interferometry surface modification...
August 6, 2018: Angewandte Chemie
Xue Yang, Thomas J M Michiels, Coen de Jong, Marjolein Soethoudt, Niek Dekker, Euan Gordon, Mario van der Stelt, Laura H Heitman, Daan van der Es, Adriaan P IJzerman
Using activity-based protein profiling (ABPP) functional proteins can be interrogated in their native environment. Despite their pharmaceutical relevance G protein-coupled receptors (GPCRs), have been difficult to address through ABPP. In the current study, we took the prototypical human adenosine A2A receptor (hA2A R) as the starting point for the construction of a chemical toolbox allowing two-step affinity-based labeling of GPCRs. Firstly, we equipped an irreversibly binding hA2A R ligand with a terminal alkyne to serve as probe...
August 6, 2018: Journal of Medicinal Chemistry
Nam Chu, Antonieta L Salguero, Albert Z Liu, Zan Chen, Daniel R Dempsey, Scott B Ficarro, William M Alexander, Jarrod A Marto, Yana Li, L Mario Amzel, Sandra B Gabelli, Philip A Cole
Akt is a critical protein kinase that drives cancer proliferation, modulates metabolism, and is activated by C-terminal phosphorylation. The current structural model for Akt activation by C-terminal phosphorylation has centered on intramolecular interactions between the C-terminal tail and the N lobe of the kinase domain. Here, we employ expressed protein ligation to produce site-specifically phosphorylated forms of purified Akt1 that are well suited for mechanistic analysis. Using biochemical, crystallographic, and cellular approaches, we determine that pSer473-Akt activation is driven by an intramolecular interaction between the C-tail and the pleckstrin homology (PH)-kinase domain linker that relieves PH domain-mediated Akt1 autoinhibition...
July 27, 2018: Cell
Andrea E Vasconez, Patrick Janetzko, James A Oo, Beatrice Pflüger-Müller, Corina Ratiu, Lunda Gu, Kristian Helin, Gerd Geisslinger, Ingrid Fleming, Katrin Schröder, Christian Fork, Ralf P Brandes, Matthias S Leisegang
AIM: The histone demethylase Jarid1b limits gene expression by removing the active methyl-mark from histone3-lysine4 at gene promoter regions. A vascular function of Jarid1b is unknown, but a vasoprotective function to inflammatory and hypertrophic stimuli, like Angiotensin II (AngII) could be inferred. This hypothesis was tested using Jarid1b knockout mice and the inhibitor PBIT. METHODS: Mice or aortic segments were treated with AngII to induce endothelial dysfunction...
August 4, 2018: Acta Physiologica
Wenwen She, Jing Ni, Ke Shui, Fei Wang, Ruyi He, Jinhui Xue, Manfred T Reetz, Aitao Li, Lixin Ma
The quality and efficiency of any PCR-based mutagenesis technique may not be optimal due to, inter alia, amino acid bias, which means that the development of efficient PCR-free methods is desirable. Here, we present a highly efficient in vitro CRISPR/Cas9-mediated mutagenic (ICM) system that allows rapid construction of designed mutants in a PCR-free manner. First, it involves plasmid digestion by utilizing a complex of Cas9 with specific single guide RNA (sgRNA), followed by degradation with T5 exonuclease to generate 15 nt homologous region...
August 3, 2018: ACS Synthetic Biology
Lu Yang, Sai-Hua Wang, Yan Hu, Yan-Fang Sui, Tao Peng, Tie-Cheng Guo
This study investigated the effects of different frequencies of repetitive transcranial magnetic stimulation (rTMS) on chronic neuropathic pain in rats. The behavior of rats with experimental chronic neuropathic pain was observed, and the expression of neuronal nitric oxide synthase (nNOS) in the ipsilateral dorsal root ganglions (DRGs) and the activation and proliferation of astrocytes in the ipsilateral spinal dorsal horn were detected. Thirty-two male Sprague-Dawley rats were randomly divided into four groups: sham-operated group, sham-rTMS group, 1 Hz group and 20 Hz group (8 rats in each group)...
June 2018: Current medical science
Kristian Strømgaard
All proteins contain characteristic backbones formed of consecutive amide bonds, which can engage in hydrogen bonds. However, the importance of these is not easily addressed by conventional technologies that only allow for side chain substitutions. By contrast, technologies such as nonsense suppression mutagenesis and protein ligation allow for manipulation of the protein backbone. In particular, replacing the backbone amide with an ester, i.e. amide-to-ester mutations, are powerful tools to examine backbone-mediated hydrogen bonds...
August 3, 2018: Chembiochem: a European Journal of Chemical Biology
Apon Zaenal Mustopa, Siti Mariyah, Fatimah, Sri Budiarti, Hidayah Murtiyaningsih, Wida Nurul Alfisyahrin
Lactobacillus plantarum produces bacteriocin called plantaricin that can kill or inhibit other bacteria. Plantaricin E (Pln E), a recombinant bacteriocin, has been successfully constructed and produced by a GRAS host, Lactococcus lactis. A polymerase chain reaction (PCR) overlapping technique has been used to construct a ligation of signal peptide gene, Pln A and bacteriocin encoding gene, Pln E. Furthermore, the fusion fragment were cloned into pNZ8148 vector and transformed into L. lactis NZ3900. Molecular expression study shows that recombinant L...
July 31, 2018: Molecular Biology Reports
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