Protein Phosphatase 1 Dephosphorylates Profilin-1 at Ser-137

Profilin-1 (PFN1) plays an important role in the control of actin dynamics, and could represent an important therapeutic target in several diseases. We previously identified PFN1 as a huntingtin aggregation inhibitor, and others have implicated it as a tumor-suppressor. Rho-associated kinase (ROCK) directly phosphorylates PFN1 at Ser-137 to prevent its binding to polyproline sequences. This negatively regulates its anti-aggregation activity. However, the phosphatase that dephosphorylates PFN1 at Ser-137, and thus activates it, is unknown. Using a phospho-specific antibody against Ser-137 of PFN1, we characterized PFN1 dephosphorylation in cultured cells based on immunocytochemistry and a quantitative plate reader-based assay. Both okadaic acid and endothall increased pS137-PFN1 levels at concentrations more consistent with their known IC50s for protein phosphatase 1 (PP1) than protein phosphatase 2A (PP2A). Knockdown of the catalytic subunit of PP1 (PP1Cα), but not PP2A (PP2ACα), increased pS137-PFN1 levels. PP1Cα binds PFN1 in cultured cells, and this interaction was increased by a phosphomimetic mutation of PFN1 at Ser-137 (S137D). Together, these data define PP1 as the principal phosphatase for Ser-137 of PFN1, and provide mechanistic insights into PFN1 regulation by phosphorylation

Topical Application of the PI3Kβ-Selective Small Molecule Inhibitor TGX-221 Is an Effective Treatment Option for Experimental Epidermolysis Bullosa Acquisita.

Class I phosphoinositide 3-kinases (PI3K) have been implemented in pathogenesis of experimental epidermolysis bullosa acquisita (EBA), an autoimmune skin disease caused by type VII collagen (COL7) autoantibodies. Mechanistically, inhibition of specific PI3K isoforms, namely PI3Kβ or PI3Kδ, impaired immune complex (IC)-induced neutrophil activation, a key prerequisite for EBA pathogenesis. Data unrelated to EBA showed that neutrophil activation is also modulated by PI3Kα and γ, but their impact on the EBA has, so far, remained elusive. To address this and to identify potential therapeutic targets, we evaluated the impact of a panel of PI3K isoform-selective inhibitors (PI3Ki) on neutrophil function in vitro, and in pre-clinical EBA mouse models. We document that distinctive, and EBA pathogenesis-related activation-induced neutrophil in vitro functions depend on distinctive PI3K isoforms. When mice were treated with the different PI3Ki, selective blockade of PI3Kα (alpelisib), PI3Kγ (AS-604850), or PI3Kβ (TGX-221) impaired clinical disease manifestation. When applied topically, only TGX-221 impaired induction of experimental EBA. Ultimately, multiplex kinase activity profiling in the presence of disease-modifying PI3Ki identified unique signatures of different PI3K isoform-selective inhibitors on the kinome of IC-activated human neutrophils. Collectively, we here identify topical PI3Kβ inhibition as a potential therapeutic target for the treatment of EBA

Differential Calcium Signaling by Cone Specific Guanylate Cyclase-Activating Proteins from the Zebrafish Retina

Zebrafish express in their retina a higher number of guanylate cyclase-activating proteins (zGCAPs) than mammalians pointing to more complex guanylate cyclase signaling systems. All six zGCAP isoforms show distinct and partial overlapping expression profiles in rods and cones. We determined critical Ca2+-dependent parameters of their functional properties using purified zGCAPs after heterologous expression in E.coli. Isoforms 1–4 were strong, 5 and 7 were weak activators of membrane bound guanylate cyclase. They further displayed different Ca2+-sensitivities of guanylate cyclase activation, which is half maximal either at a free Ca2+ around 30 nM (zGCAP1, 2 and 3) or around 400 nM (zGCAP4, 5 and 7). Zebrafish GCAP isoforms showed also differences in their Ca2+/Mg2+-dependent conformational changes and in the Ca2+-dependent monomer-dimer equilibrium. Direct Ca2+-binding revealed that all zGCAPs bound at least three Ca2+. The corresponding apparent affinity constants reflect binding of Ca2+ with high (≤100 nM), medium (0.1–5 µM) and/or low (≥5 µM) affinity, but were unique for each zGCAP isoform. Our data indicate a Ca2+-sensor system in zebrafish rod and cone cells supporting a Ca2+-relay model of differential zGCAP operation in these cells

Engineered hexavalent Fc proteins with enhanced Fc-gamma receptor avidity provide insights into immune-complex interactions

Tania Rowley et al. present multivalent Fc molecules with enhanced avidity for Fc gamma receptors in order to improve the treatment of autoantibody-mediated human diseases. They found several key amino acids involved in Fc receptor binding interactions

Role of epididymal receptor HE6 in the regulation of sperm microenvironment.

HE6 (GPR64) is a highly conserved, tissue-specific heptahelical receptor of the human epididymis. The seven transmembrane (TM7) domains are a hallmark of G-protein-coupled receptors (GPCRs) which have a proven history of being excellent therapeutic targets. Of all currently marketed drugs, >30% are modulators of specific heptahelical receptors, emphasizing the potential of HE6 as a target for pharmaceutical intervention. Targeted mutation of the mouse HE6 counterpart resulted in male infertility, further emphasizing its role as a candidate target for male contraception. However, the precise function of HE6, together with its potential ligand(s), and signal transduction pathways have remained largely unknown. On the basis of shared sequence motifs within the TM7 region, HE6 has been grouped into the B class of GPCRs. Within this class, HE6 belongs to the 'large N-termini' family-B seven-transmembrane (LNB-TM7) receptors, also termed the adhesion-GPCRs. Members of this subgroup are 'orphan' receptors, and they all seem to be cleaved within a conserved GPCR proteolytic site (GPS) domain. The biological significance of the two-subunit architecture is still unknown. Clues to the function of HE6 within the epithelium of male excurrent ducts may come from its co-localisation with the apical actin cytoskeleton and from the down-regulation in "knockout" male mice of various proteins specific to the initial segment

Novel epididymis-specific mRNAs downregulated by HE6/Gpr64 receptor gene disruption.

Targeted disruption of the epididymis-specific HE6/Gpr64 receptor gene in mice led to male infertility. In order to characterize the phenotype at a molecular level, we compared the gene expression patterns of wild type (wt) versus knockout (KO) caput epididymides. The caput region of KO males, although morphologically normal, nevertheless showed an aberrant expression pattern. Combining micro array analysis, differential library screening, Northern blot analysis and quantitative RT-PCR, we found that the knockout of the HE6/Gpr64 receptor was mainly associated with the downregulation of genes specific to the initial segment. The list of KO downregulated transcripts comprised Enpp2/autotaxin, the lipocalins 8 and 9, the beta-defensin Defb42, cystatins 8 and 12, as well as the membrane proteins Adam (A Disintegrin And Metalloprotease) 28, claudin-10, EAAC1, and the novel Me9. Clusterin/ApoJ and osteopontin/Spp1 mRNAs, on the other hand, were upregulated in the KO tissues. The Me9 transcript was studied in further detail, and we report here a cluster of related epididymis-specific genes. Me9 is specifically expressed in the initial segment and is representative of a novel and highly conserved mammalian gene family. The family consists of single-exon genes only; intron-containing paralogs have not yet been ascertained. The cloned cDNA sequences predicted hydrophobic polytopic membrane proteins containing the DUF716 motif. Protein expression was shown in the rodent caput epididymidis but remained uncertain in primates

Gemeinsamkeiten und Unterschiede der Prozess- und Fertigungstechnik: Interdisziplinäre Aspekte der Produktionsmodellierung

Within the field of industrial production, discrete production and process technology have been established as mostly separated domains. Today´s development systematics and production requirements very often ask for an integration of the separated considerations in particular models. As a necessary presumption to enable a better, interdisciplinary integration up to an integrated automation control for hybrid plants, this paper compares and analyses the physical background, the technical task breakdown as well as established models and systems of both domains

Infection with Anaplasma phagocytophilum Activates the Phosphatidylinositol 3-Kinase/Akt and NF-kappa B Survival Pathways in Neutrophil Granulocytes

Anaplasma phagocytophilum, a Gram-negative, obligate intracellular bacterium infects primarily neutrophil granulocytes. Infection with A. phagocytophilum leads to inhibition of neutrophil apoptosis and consequently contributes to the longevity of the host cells. Previous studies demonstrated that the infection inhibits the executionary apoptotic machinery in neutrophils. However, little attempt has been made to explore which survival signals are modulated by the pathogen. The aim of the present study was to clarify whether the phosphatidylinositol 3-kinase (PI3K)/Akt and NF-κB signaling pathways, which are considered as important survival pathways in neutrophils, are involved in A. phagocytophilum-induced apoptosis delay. Our data show that infection of neutrophils with A. phagocytophilum activates the PI3K/Akt pathway and suggest that this pathway, which in turn maintains the expression of the antiapoptotic protein Mcl-1, contributes to the infection-induced apoptosis delay. In addition, the PI3K/Akt pathway is involved in the activation of NF-κB in A. phagocytophilum-infected neutrophils. Activation of NF-κB leads to the release of interleukin-8 (IL-8) from infected neutrophils, which, in an autocrine manner, delays neutrophil apoptosis. In addition, enhanced expression of the antiapoptotic protein cIAP2 was observed in A. phagocytophilum-infected neutrophils. Taken together, the data indicate that upstream of the apoptotic cascade, signaling via the PI3K/Akt pathway plays a major role for apoptosis delay in A. phagocytophilum-infected neutrophils