Aufklärung der dreidimensionalen Struktur der Sushi-Domäne des Interleukin-15-Rezeptor a mittels mehrdimensionaler NMR-Spektroskopie

IL-15 ist ein Mitglied der kleinen „Vier-alpha-Helix-Bündel“ -Zytokinfamilie. Zur Signaltransduktion verwenden IL-15 und IL-2, neben einen jeweils spezifischen alpha-Rezeptor, den beta- und den gamma-Rezeptor des IL-2. Zytokinrezeptoren sind gewöhnlich Multi-Domänen Proteine, welche ihre Liganden über eine Zytokin-Homologie-Region binden. Demgegenüber sind bei den alpha-Rezeptoren des IL-15 und IL-2 die Sushi-Domänen für die Zytokinbindung verantwortlich. Im Rahmen dieser Arbeit wurde die Sushi-Domäne des IL-15Ra rekombinant hergestellt und die Struktur des Proteins mittels multidimensionaler heteronuklearer NMR-Spektroskopie aufgeklärt. Die Generation eines Modells der Sushi-Domäne des IL-15Ra im Komplex mit IL-15 erlaubte einen Einblick in den Interaktionsbereich. Dabei zeigte sich, dass die Bindung zwischen IL-15 und dem IL-15Ra von einem Netzwerk aus ionischen Interaktionen dominiert wird. Diese Art der Interaktion erklärt die außergewöhnlich hohe Affinität des IL-15/IL-15Ra-Komplexes, welche für die biologischen Effekte des Zytokins von großer Bedeutung ist und bisher in anderen Zytokin/ Zytokinrezeptor-Komplexen nicht beobachtet wurde

A novel fluorescent pH probe for expression in plants

BACKGROUND: The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants. RESULTS: pHluorins have a higher pH-sensitivity. However, Pt-GFP has a broader pH-responsiveness, an excellent dynamic ratio range and a better acid stability. We demonstrate how Pt-GFP expressing Arabidopsis thaliana report cytosolic pH-clamp and changes of cytosolic pH in the response to anoxia and salt-stress. CONCLUSION: Pt-GFP appears to be the better choice when used for in vivo-recording of cellular pH in plants

Redox regulation of inflammatory processes is enzymatically controlled

Redox regulation depends on the enzymatically controlled production and decay of redox active molecules. NADPH oxidases, superoxide dismutases, nitric oxide synthases, and others produce the redox active molecules superoxide, hydrogen peroxide, nitric oxide, and hydrogen sulfide. These react with target proteins inducing spatiotemporal modifications of cysteine residues within different signaling cascades. Thioredoxin family proteins are key regulators of the redox state of proteins. They regulate the formation and removal of oxidative modifications by specific thiol reduction and oxidation. All of these redox enzymes affect inflammatory processes and the innate and adaptive immune response. Interestingly, this regulation involves different mechanisms in different biological compartments and specialized cell types. The localization and activity of distinct proteins including, for instance, the transcription factor NFκB and the immune mediator HMGB1 are redox-regulated. The transmembrane protein ADAM17 releases proinflammatory mediators, such as TNFα, and is itself regulated by a thiol switch. Moreover, extracellular redox enzymes were shown to modulate the activity and migration behavior of various types of immune cells by acting as cytokines and/or chemokines. Within this review article, we will address the concept of redox signaling and the functions of both redox enzymes and redox active molecules in innate and adaptive immune responses

CD9 controls integrin α5β1-mediated cell adhesion by modulating its association with the metalloproteinase ADAM17

Integrin α5β1 is a crucial adhesion molecule that mediates the adherence of many cell types to the extracellular matrix through recognition of its classic ligand fibronectin as well as to other cells through binding to an alternative counter-receptor, the metalloproteinase ADAM17/TACE. Interactions between integrin α5β1 and ADAM17 may take place both in trans (between molecules expressed on different cells) or in cis (between molecules expressed on the same cell) configurations. It has been recently reported that the cis association between α5β1 and ADAM17 keeps both molecules inactive, whereas their dissociation results in activation of their adhesive and metalloproteinase activities. Here we show that the tetraspanin CD9 negatively regulates integrin α5β1-mediated cell adhesion by enhancing the cis interaction of this integrin with ADAM17 on the cell surface. Additionally we show that, similarly to CD9, the monoclonal antibody 2A10 directed to the disintegrin domain of ADAM17 specifically inhibits integrin α5β1-mediated cell adhesion to its ligands fibronectin and ADAM17.This work has been supported by the grant SAF2016-77096-R from Ministerio Español de Economía y Competitividad (MINECO) awarded to CC, by a grant from Fundación Ramón Areces Ayudas a la Investigación en Ciencias de la Vida y de la Materia, 2014 awarded to MY-M, and by the Deutsche Forschungsgemeinschaft (SFB 877, A6, Z3 and SPP1710) to JG and I

Interleukin-6 receptor specific RNA aptamers for cargo delivery into target cells

Aptamers represent an emerging strategy to deliver cargo molecules, including dyes, drugs, proteins or even genes, into specific target cells. Upon binding to specific cell surface receptors aptamers can be internalized, for example by macropinocytosis or receptor mediated endocytosis. Here we report the in vitro selection and characterization of RNA aptamers with high affinity (Kd = 20 nM) and specificity for the human IL-6 receptor (IL-6R). Importantly, these aptamers trigger uptake without compromising the interaction of IL-6R with its natural ligands the cytokine IL-6 and glycoprotein 130 (gp130). We further optimized the aptamers to obtain a shortened, only 19-nt RNA oligonucleotide retaining all necessary characteristics for high affinity and selective recognition of IL-6R on cell surfaces. Upon incubation with IL-6R presenting cells this aptamer was rapidly internalized. Importantly, we could use our aptamer, to deliver bulky cargos, exemplified by fluorescently labeled streptavidin, into IL-6R presenting cells, thereby setting the stage for an aptamer-mediated escort of drug molecules to diseased cell populations or tissues

CD9 Controls Integrin α5β1-Mediated Cell Adhesion by Modulating Its Association With the Metalloproteinase ADAM17

Integrin α5β1 is a crucial adhesion molecule that mediates the adherence of many cell types to the extracellular matrix through recognition of its classic ligand fibronectin as well as to other cells through binding to an alternative counter-receptor, the metalloproteinase ADAM17/TACE. Interactions between integrin α5β1 and ADAM17 may take place both in trans (between molecules expressed on different cells) or in cis (between molecules expressed on the same cell) configurations. It has been recently reported that the cis association between α5β1 and ADAM17 keeps both molecules inactive, whereas their dissociation results in activation of their adhesive and metalloproteinase activities. Here we show that the tetraspanin CD9 negatively regulates integrin α5β1-mediated cell adhesion by enhancing the cis interaction of this integrin with ADAM17 on the cell surface. Additionally we show that, similarly to CD9, the monoclonal antibody 2A10 directed to the disintegrin domain of ADAM17 specifically inhibits integrin α5β1-mediated cell adhesion to its ligands fibronectin and ADAM17

Redundancy of protein disulfide isomerases in the catalysis of the inactivating disulfide switch in A Disintegrin and Metalloprotease 17

Abstract A Disintegrin and Metalloprotease 17 (ADAM17) can cause the fast release of growth factors and inflammatory mediators from the cell surface. Its activity has to be turned on which occurs by various stimuli. The active form can be inactivated by a structural change in its ectodomain, related to the pattern of the formed disulphide bridges. The switch-off is executed by protein disulfide isomerases (PDIs) that catalyze an isomerization of two disulfide bridges and thereby cause a disulfide switch. We demonstrate that the integrity of the CGHC-motif within the active site of PDIs is indispensable. In particular, no major variation is apparent in the activities of the two catalytic domains of PDIA6. The affinities between PDIA1, PDIA3, PDIA6 and the targeted domain of ADAM17 are all in the nanomolar range and display no significant differences. The redundancy between PDIs and their disulfide switch activity in ectodomains of transmembrane proteins found in vitro appears to be a basic characteristic. However, different PDIs might be required in vivo for disulfide switches in different tissues and under different cellular and physiological situations

KDEL Receptor 1 Contributes to Cell Surface Association of Protein Disulfide Isomerases

Background/Aims: Endoplasmic reticulum (ER)-resident proteins with a C-terminal KDEL ER-retention sequence are captured in the Golgi apparatus by KDEL receptors (KDELRs). The binding of such proteins to these receptors induces their retrograde transport. Nevertheless, some KDEL proteins, such as Protein Disulfide Isomerases (PDIs), are found at the cell surface. PDIs target disulfide bridges in the extracellular domains of proteins, such as integrins or A Disintegrin And Metalloprotease 17 (ADAM17) leading to changes in the structure and function of these molecules. Integrins become activated and ADAM17 inactivated upon disulfide isomerization. The way that PDIs escape from retrograde transport and reach the plasma membrane remains far from clear. Various mechanisms might exist, depending on whether a local cell surface association or a more global secretion is required. Methods: To get a more detailed insight in the transport of PDIs to the cell surface, methods such as cell surface biotinylation, flow cytometric analysis, immunoprecipitation, fluorescence microscopy as well as labeling of cells with fluorescence labled recombinant PDIA6 was performed. Results: Here, we show that the C-terminal KDEL ER retention sequence is sufficient to prevent secretion of PDIA6 into the extracellular space but is mandatory for its association with the cell surface. The cell surface trafficking of PDIA1, PDIA3, and PDIA6 is dependent on KDELR1, which travels in a dynamic manner to the cell surface. This transport is assumed to result in PDI cell surface association, which differs from PDI inducible secretion into the extracellular space. Distinct PDIs differ in their trafficking properties. Endogenous KDELR1, detectable at the cell surface, might be involved not only in the transport of cell-surface-associated PDIs, but also in their retrieval and internalization from the extracellular space. Conclusion: Beside their ER retention motive PDIs travel to the cell surface. Here they target different proteins to render their function. To escape the ER PDIs travel via various pathways. One of them depends on the KDELR1, which can transport its target to the cell surface, where it is to be expected to release its cargo in close vicinity to its target molecules. Hence, the KDEL sequence is needed for cell surface association of PDIs, such as PDIA6

Secreted Frizzled-related protein 3 (sFRP3)-mediated suppression of Interleukin-6 receptor release by A disintegrin and Metalloprotease 17 (ADAM17) is abrogated in the osteoarthritis-associated rare double variant of sFRP3

To avoid malformation and disease, tissue development and homeostasis are coordinated precisely in time and space. Secreted Frizzled-related protein 3 (sFRP3), encoded by the Frizzled-related protein gene (FRZB), acts as an antagonist of Wnt signalling in bone development by delaying the maturation of proliferative chondrocytes into hypertrophic chondrocytes. A disintegrin and metalloprotease 17 (ADAM17) is a transmembrane protease that is essential for developmental processes and promotes cartilage maturation into bone. sFRP3 is chondroprotective and is expressed in chondrocytes of healthy articular cartilage. Upon damage to cartilage, sFRP3 is down-regulated. Rare variants of sFRP3 are associated with osteoarthritis. This study demonstrates a novel function of sFRP3 in suppression of the enzymatic activity of ADAM17 which results in the inhibition of ADAM17-meditated interleukin-6 receptor (IL-6R) shedding. By contrast, the rare double variant of sFRP3 failed to suppress ADAM17. The shed soluble IL-6R is linked to inflammation, cartilage degeneration, and osteolysis. Accordingly, enhanced activity of ADAM17 in cartilage, caused by the expression of the rare double sFRP3 variant, provides an explanation for the genetic effect of sFRP3 variants in joint disease. The finding that sFRP3 interacts with the ADAM17 substrate IL-6R also suggests a new regulatory mechanism by which the substrate is protected against shedding.status: publishe