The Extended N-Terminal Domain Confers Atypical Chemokine Receptor Properties to CXCR3-B.

peer reviewedThe chemokine receptor CXCR3 plays a critical role in immune cell recruitment and activation. CXCR3 exists as two main isoforms, CXCR3-A and CXCR3-B, resulting from alternative splicing. Although the two isoforms differ only by the presence of an N-terminal extension in CXCR3-B, they have been attributed divergent functional effects on cell migration and proliferation. CXCR3-B is the more enigmatic isoform and the mechanisms underlying its function and signaling remain elusive. We therefore undertook an in-depth cellular and molecular comparative study of CXCR3-A and CXCR3-B, investigating their activation at different levels of the signaling cascades, including G protein coupling, β-arrestin recruitment and modulation of secondary messengers as well as their downstream gene response elements. We also compared the subcellular localization of the two isoforms and their trafficking under resting and stimulated conditions along with their ability to internalize CXCR3-related chemokines. Here, we show that the N-terminal extension of CXCR3-B drastically affects receptor features, modifying its cellular localization and preventing G protein coupling, while preserving β-arrestin recruitment and chemokine uptake capacities. Moreover, we demonstrate that gradual truncation of the N terminus leads to progressive recovery of surface expression and G protein coupling. Our study clarifies the molecular basis underlying the divergent effects of CXCR3 isoforms, and emphasizes the β-arrestin-bias and the atypical nature of CXCR3-B

Production optimization of antibodies scFv fragments potentially recognizing mouse metalloproteinase ADAM17.

Proteolityczne uwalnianie ektodomen białkowych z powierzchni komórki jest kluczowym procesem w regulacji aktywności wielu białek błonowych w tym czynników wzrostu i cytokin. Metaloproteinaza błonowa ADAM17 została okryta jako główny czynnik odpowiedzialny za uwalnianie rozpuszczalnej formy TNF. Dodatkowo zaobserwowano, że jest zaangażowana w złuszczanie wielu innych białek, co uczyniło ją atrakcyjnym celem w terapii chorób nowotworowych czy chronicznych zapaleń. Otrzymane i scharakteryzowane syntetyczne inhibitory aktywności proteazy ADAM17 nie zostały jednak dopuszczone do klinicznego stosowania, co było związane z niską specyficznością ich działania i efektami ubocznymi.W Zakładzie Biochemii Komórki UJ od kilku lat prowadzone są próby uzyskania przeciwciał formatu scFv, specyficznie rozpoznających i hamujących aktywność mysiej sekretazy ADAM17. Do tego celu zastosowano metodę ekspresji fagowej (ang. phage display) i wykorzystano bibliotekę fagemidową Tomlinson I+J. Opierając się na wstępnych testach i analizach, wyselekcjonowano dziesięć fagemidów kodujących scFv, które z największym prawdopodobieństwem mogły hamować aktywność ADAM17. Głównym celem niniejszej pracy było uzyskanie wydajnej ekspresji rozpuszczalnych form wybranych przeciwciał w systemie bakteryjnym. Pierwszym etapem było poznanie sekwencji kodujących scFv oraz wyeliminowanie niepożądanych kodonów amber, które uniemożliwiały otrzymanie rozpuszczalnych form przeciwciał. W tym celu przeprowadzono ukierunkowaną mutagenezę metodą QuikChange®. Zaprojektowano i otrzymano następnie nowy wektor ekspresyjny oparty na systemie pET, który w połączeniu ze szczepem Rosetta(DE3)pLysS umożliwił wydajną ekspresję rozpuszczalnych przeciwciał scFv. Może on w przyszłości ułatwić otrzymanie rozpuszczalnych przeciwciał formatu scFv wyselekcjonowanych z biblioteki fagemidowej Tomlinson I+J. Etap oczyszczania wszystkich scFv przeprowadzono na złożu Białko L-sefaroza lub Białko A-sefaroza oraz wykonano analizę otrzymanych preparatów metodą SDS-PAGE. Przeprowadzono test ELISA komórkowa, który miał wykazać specyficzne wiązanie otrzymanych przeciwciał do mysiej skeretazy ADAM17, obecnej na powierzchni komórek.The proteolytic release of protein ectodomains from the cell surface is the key process in the regulation of activity of many membrane proteins, including growth factors and cytokines. The membrane metalloproteinase ADAM17 was discovered as the main enzyme responsible for the release of the soluble form of TNF. Additionally, it was found out, that it participates in the shedding of many other proteins such as growth factors from EGFR family, some cell adhesion molecules, and cytokine receptors which made this metalloproteinase a very attractive target in cancer or chronic inflammation therapies. However, the synthetic inhibitors of ADAM17 activity, although created and characterized, have not been approved for clinical use, due to their low specificity and numerous side effects.The scientists from the Department of Cell Biochemistry at the Jagiellonian University have been working on the creation of scFv antibodies, that would recognize and inhibit the activity of mouse ADAM17. In order to do that, the phage display method with the Tomlinson I+J library was used. Ten phagemids coding the scFvs, that most probably would interact with membrane ADAM17, were selected based on the preliminary results of tests and analyses. The goal of this master’s thesis project was to obtain efficient expression of soluble forms of given antibodies in a bacterial system.The first step included analyses of the sequences coding sfor different cFvs and the elimination of the unwanted amber codons, that prevented the obtaining of soluble antibodies. In order to replace these amber codons for codons for glutamic acid, the QuikChange® directed mutagenesis was performed. Next, the new expression vector based on the pET system was designed and generated. Together with the Rosetta(DE3)pLysS strain, it enabled the efficient expression of soluble scFv antibodies. This vector facilitate obtaining the variety of scFvs selected from the Tomlinson I+J phagemid library. ScFvs were purified using Protein L-Sepharose or Protein A-Sepharose and the obtained scFvs were analyzed on SDS-PAGE. The cellular ELISA test, that was expected to show the specific binding of scFvs to mouse ADAM17 present on the surface of the cells, was performed

Systematic reassessment of chemokine-receptor pairings confirms CCL20 but not CXCL13 and extends the spectrum of ACKR4 agonists to CCL22

Atypical chemokine receptors (ACKRs) have emerged as important regulators or scavengers of homeostatic and inflammatory chemokines. Among these atypical receptors, ACKR4 is reported to bind the homeostatic chemokines CCL19, CCL21, CCL25 and CXCL13. In a recent study by Matti et al., the authors show that ACKR4 is also a receptor for CCL20, previously established to bind to CCR6 only. They provide convincing evidence that, just as for its other chemokine ligands, ACKR4 rapidly internalizes CCL20 both in vitro and in vivo. Independently of this discovery, we undertook a screening program aiming at reassessing the activity of the 43 human chemokines toward ACKR4 using a highly sensitive β-arrestin recruitment assay. This systematic analysis confirmed CCL20 as a new agonist ligand for ACKR4 in addition to CCL19, CCL21, and CCL25. Furthermore, CCL22, which plays an important role in both homeostasis and inflammatory responses, and is known as a ligand for CCR4 and ACKR2 was found to also act as a potent partial agonist of ACKR4. In contrast, agonist activity of CXCL13 toward ACKR4 was disproved. This independent wide-range systematic study confirms the pairing of CCL20 with ACKR4 newly discovered by Matti and co-authors, and further refines the spectrum of chemokines activating ACKR4

Characterization of the multiples structural states of the multidrug transporter LmrP by conformational restriction

info:eu-repo/semantics/nonPublishe

STRUCTURAL STUDIES OF SECONDAIRY MULTIDRUGS TRANSPORTERS

info:eu-repo/semantics/nonPublishe