RAB3GAP1 und RAB3GAP2 (RAB3 GTPase-activating Protein 1/2) beeinflussen die Autophagie in C. elegans und humanen Zellen

Die Proteinhomöostase wird in der Zelle von drei Stoffwechselwegen reguliert: den molekularen Chaperonen, dem Ubiquitin-Proteasom-System und dem autophagosomalen Abbauweg. Die (Makro)Autophagie verpackt und transportiert zytosolische Komponenten in Autophagosomen zu den Lysosomen, wo sie abgebaut werden. Eine Störung dieses Abbauwegs wirkt auf die Proteostase.rnIn dieser Dissertation wurde C. elegans als Modellorganismus zur Erforschung von Proteinstabilität genutzt. In einer RNAi-vermittelten Proteostase-Analyse von Chromosom I und ausgewählter zusätzlicher Gene wurde ein Wurmstamm, der ein Luc::GFP-Konstrukt im Muskel exprimiert, genutzt. Dieses Reporterprotein aggregiert unter Hitzestressbedingungen und diese Aggregation kann durch Modulatoren der Proteostase beeinflusst werden. Dabei wurden mögliche neue Faktoren der Proteinhomöostase entdeckt. Durch weitere Experimente bei denen die Aggregation von PolyQ35::YFP im AM140-System, der Paralyse-Phänotyp und die Akkumulation Thioflavin S-gefärbter Aggregate von Aβ42 im CL2006-Wurmstamm und die Effekte auf die Autophagie mittels eines GFP::LGG1-Konstrukt analysiert wurden, konnten rbg-1 und rbg-2 als neue Modulatoren der Proteinhomöostase, insbesondere der Autophagie, identifiziert werden.rnIm Säuger bilden beide Orthologe dieser Gene, RAB3GAP1 und RAB3GAP2 den heterodimeren RAB3GAP-Komplex, der bisher nur bekannt war für die Stimulation der Umwandlung der GTP-gebundenen aktiven Form zur GDP-gebundenen inaktiven Form der RAB GTPase RAB3. In Immunoblot-Analysen und mikroskopischen Darstellungen im Säugersystem konnte gezeigt werden, dass die Effekte auf die Proteostase über den autophagosomalen Abbauweg wirken. RAB3GAP1/2 wirken als positive Stimulatoren, wenn die Lipidierung von LC3-I und der autophagische Flux von LC3-II und p62/SQSTM1 betrachtet werden. Diese Effekte werden aber nicht über die RAB GTPase RAB3 vermittelt. Die Proteine FEZ1 und FEZ2 haben einen antagonistischen Effekt auf die Autophagie und wenn alle vier Komponenten RAB3GAP1, RAB3GAP2, FEZ1 und FEZ2 zusammen herunter- oder hochreguliert werden, heben sich diese Effekte auf. In Co-Immunopräzipitationen und proteomischen Analysen konnte keine direkte Interaktion zwischen dem RAB3GAP-Komplex und FEZ1/2 oder zu anderen Autophagie-Genen nachgewiesen werden.rnHier konnte der RAB3GAP-Komplex funktionell mit Proteostase und Autophagie in C. elegans und Säugerzellen assoziiert werden. Dieser Komplex zeigt Einflüsse auf die autophagosomale Biogenese indem sie die Proteostase und die Bildung von (prä)autophagosomalen Strukturen in C. elegans und die Lipidierung von LC3 und damit den autophagischen Flux der Autophagiesubstrate LC3-II und p62/SQSTM1 in Säugerzellen beeinflusst. Darüber hinaus wirkt RAB3GAP der komplexen Autophagie-Unterdrückung durch FEZ1 und FEZ2 entgegen. Somit konnte gezeigt werden, dass RAB3GAP als neuartiger Faktor auf die autophagosomale Biogenese und somit auf die Proteostase wirkt.rn

Biomimetic coating of organic polymers with a protein-functionalized layer of calcium phosphate: the surface properties of the carrier influence neither the coating characteristics nor the incorporation mechanism or release kinetics of the protein

Polymers that are used in clinical practice as bone-defect-filling materials possess many essential qualities, such as moldability, mechanical strength and biodegradability, but they are neither osteoconductive nor osteoinductive. Osteoconductivity can be conferred by coating the material with a layer of calcium phosphate, which can be rendered osteoinductive by functionalizing it with an osteogenic agent. We wished to ascertain whether the morphological and physicochemical characteristics of unfunctionalized and bovine-serum-albumin (BSA)-functionalized calcium-phosphate coatings were influenced by the surface properties of polymeric carriers. The release kinetics of the protein were also investigated. Two sponge-like materials (Helistat® and Polyactive®) and two fibrous ones (Ethisorb and poly[lactic-co-glycolic acid]) were tested. The coating characteristics were evaluated using state-of-the-art methodologies. The release kinetics of BSA were monitored spectrophotometrically. The characteristics of the amorphous and the crystalline phases of the coatings were not influenced by either the surface chemistry or the surface geometry of the underlying polymer. The mechanism whereby BSA was incorporated into the crystalline layer and the rate of release of the truly incorporated depot were likewise unaffected by the nature of the polymeric carrier. Our biomimetic coating technique could be applied to either spongy or fibrous bone-defect-filling organic polymers, with a view to rendering them osteoconductive and osteoinductive.

Epigallocatechin-3-gallate induces cell death in acute myeloid leukaemia cells and supports all-trans retinoic acid-induced neutrophil differentiation via death-associated protein kinase 2

Acute promyelocytic leukaemia (APL) patients are successfully treated with all-trans retinoic acid (ATRA). However, concurrent chemotherapy is still necessary and less toxic therapeutic approaches are needed. Earlier studies suggested that in haematopoietic neoplasms, the green tea polyphenol epigallocatechin-3-gallate (EGCG) induces cell death without adversely affecting healthy cells. We aimed at deciphering the molecular mechanism of EGCG-induced cell death in acute myeloid leukaemia (AML). A significant increase of death-associated protein kinase 2 (DAPK2) levels was found in AML cells upon EGCG treatment paralleled by increased cell death that was significantly reduced upon silencing of DAPK2. Moreover, combined ATRA and EGCG treatment resulted in cooperative DAPK2 induction and potentiated differentiation. EGCG toxicity of primary AML blasts correlated with 67 kDa laminin receptor (67LR) expression. Pretreatment of AML cells with ATRA, causing downregulation of 67LR, rendered these cells resistant to EGCG-mediated cell death. In summary, it was found that (i) DAPK2 is essential for EGCG-induced cell death in AML cells, (ii) ATRA and EGCG cotreatment significantly boosted neutrophil differentiation, and 67LR expression correlates with susceptibility of AML cells to EGCG. We thus suggest that EGCG, by selectively targeting leukaemic cells, may improve differentiation therapies for APL and chemotherapy for other AML subtypes.

The contribution of surface residues to membrane binding and ligand transfer by the -tocopherol transfer protein ( -TTP)

Previous work has shown that the -tocopherol transfer protein ( -TTP) can bind to vesicular or immobilized phospholipid membranes. Revealing the molecular mechanisms by which -TTP associates with membranes is thought to be critical to understanding its function and role in the secretion of tocopherol from hepatocytes into the circulation. Calculations presented in the Orientations of Proteins in Membranes database have provided a testable model for the spatial arrangement of -TTP and other CRAL-TRIO family proteins with respect to the lipid bilayer. These calculations predicted that a hydrophobic surface mediates the interaction of -TTP with lipid membranes. To test the validity of these predictions, we used site-directed mutagenesis and examined the substituted mutants with regard to intermembrane ligand transfer, association with lipid layers and biological activity in cultured hepatocytes. Substitution of residues in helices A8 (F165A and F169A) and A10 (I202A, V206A and M209A) decreased the rate of intermembrane ligand transfer as well as protein adsorption to phospholipid bilayers. The largest impairment was observed upon mutation of residues that are predicted to be fully immersed in the lipid bilayer in both apo (open) and holo (closed) conformations such as Phe165 and Phe169. Mutation F169A, and especially F169D, significantly impaired -TTP-assisted secretion of -tocopherol outside cultured hepatocytes. Mutation of selected basic residues (R192H, K211A, and K217A) had little effect on transfer rates, indicating no significant involvement of nonspecific electrostatic interactions with membranes.

Characterisation of gp34, a GPI-anchored protein expressed by schizonts of Theileria parva and T. annulata

Using bioinformatics tools, we searched the predicted Theileria annulata and T. parva proteomes for putative schizont surface proteins. This led to the identification of gp34, a GPI-anchored protein that is stage-specifically expressed by schizonts of both Theileria species and is downregulated upon induction of merogony. Transfection experiments in HeLa cells showed that the gp34 signal peptide and GPI anchor signal are also functional in higher eukaryotes. Epitope-tagged Tp-gp34, but not Ta-gp34, expressed in the cytosol of COS-7 cells was found to localise to the central spindle and midbody. Overexpression of Tp-gp34 and Ta-gp34 induced cytokinetic defects and resulted in accumulation of binucleated cells. These findings suggest that gp34 could contribute to important parasite-host interactions during host cell division.

Level of G protein-coupled receptor kinase-2 determines myocardial ischemia/reperfusion injury via pro- and anti-apoptotic mechanisms

Activation of prosurvival kinases and subsequent nitric oxide (NO) production by certain G protein-coupled receptors (GPCRs) protects myocardium in ischemia/reperfusion injury (I/R) models. GPCR signaling pathways are regulated by GPCR kinases (GRKs), and GRK2 has been shown to be a critical molecule in normal and pathological cardiac function.

Protein level expression of Toll-like receptors 2, 4 and 9 in renal disease

Toll-like receptors (TLR) recognize a variety of ligands, including pathogen-associated molecular patterns and link innate and adaptive immunity. Individual receptors can be up-regulated during infection and inflammation. We examined the expression of selected TLRs at the protein level in various types of renal disease.

Deubiquitylating enzyme USP2 counteracts Nedd4-2-mediated downregulation of KCNQ1 potassium channels

KCNQ1 (Kv7.1), together with its KCNE β subunits, plays a pivotal role both in the repolarization of cardiac tissue and in water and salt transport across epithelial membranes. Nedd4/Nedd4-like (neuronal precursor cell-expressed developmentally downregulated 4) ubiquitin-protein ligases interact with the KCNQ1 potassium channel through a PY motif located in the C terminus of KCNQ1. This interaction induces ubiquitylation of KCNQ1, resulting in a reduced surface density of the channel. It was reported recently that the epithelial sodium channel is regulated by the reverse process-deubiquitylation-mediated by USP2 (ubiquitin-specific protease 2).

Nedd4-2-dependent ubiquitylation and regulation of the cardiac potassium channel hERG1

The voltage-gated cardiac potassium channel hERG1 (human ether-à-gogo-related gene 1) plays a key role in the repolarization phase of the cardiac action potential (AP). Mutations in its gene, KCNH2, can lead to defects in the biosynthesis and maturation of the channel, resulting in congenital long QT syndrome (LQTS). To identify the molecular mechanisms regulating the density of hERG1 channels at the plasma membrane, we investigated channel ubiquitylation by ubiquitin ligase Nedd4-2, a post-translational regulatory mechanism previously linked to other ion channels. We found that whole-cell hERG1 currents recorded in HEK293 cells were decreased upon neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2) co-expression. The amount of hERG1 channels in total HEK293 lysates and at the cell surface, as assessed by Western blot and biotinylation assays, respectively, were concomitantly decreased. Nedd4-2 and hERG1 interact via a PY motif located in the C-terminus of hERG1. Finally, we determined that Nedd4-2 mediates ubiquitylation of hERG1 and that deletion of this motif affects Nedd4-2-dependent regulation. These results suggest that ubiquitylation of the hERG1 protein by Nedd4-2, and its subsequent down-regulation, could represent an important mechanism for modulation of the duration of the human cardiac action potential.

Neuronal precursor cell-expressed developmentally down-regulated 4-1 (NEDD4-1) controls the sorting of newly synthesized Ca(V)1.2 calcium channels

Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) proteins are ubiquitin ligases, which attach ubiquitin moieties to their target proteins, a post-translational modification that is most commonly associated with protein degradation. Nedd4 ubiquitin ligases have been shown to down-regulate both potassium and sodium channels. In this study, we investigated whether Nedd4 ubiquitin ligases also regulate Ca(v) calcium channels. We expressed three Nedd4 family members, Nedd4-1, Nedd4-2, and WWP2, together with Ca(v)1.2 channels in tsA-201 cells. We found that Nedd4-1 dramatically decreased Ca(v) whole-cell currents, whereas Nedd4-2 and WWP2 failed to regulate the current. Surface biotinylation assays revealed that Nedd4-1 decreased the number of channels inserted at the plasma membrane. Western blots also showed a concomitant decrease in the total expression of the channels. Surprisingly, however, neither the Ca(v) pore-forming α1 subunit nor the associated Ca(v)β and Ca(v)α(2)δ subunits were ubiquitylated by Nedd4-1. The proteasome inhibitor MG132 prevented the degradation of Ca(v) channels, whereas monodansylcadaverine and chloroquine partially antagonized the Nedd4-1-induced regulation of Ca(v) currents. Remarkably, the effect of Nedd4-1 was fully prevented by brefeldin A. These data suggest that Nedd4-1 promotes the sorting of newly synthesized Ca(v) channels for degradation by both the proteasome and the lysosome. Most importantly, Nedd4-1-induced regulation required the co-expression of Ca(v)β subunits, known to antagonize the retention of the channels in the endoplasmic reticulum. Altogether, our results suggest that Nedd4-1 interferes with the chaperon role of Ca(v)β at the endoplasmic reticulum/Golgi level to prevent the delivery of Ca(v) channels at the plasma membrane.

Bone morphogenetic protein receptor 2 in patients with idiopathic portal hypertension

In idiopathic portal hypertension (IPH) typical vascular lesions are present in the branches of the portal vein or in the perisinusoidal area of the liver. Similar histological alterations have been reported in the pulmonary vasculature of patients with idiopathic pulmonary artery hypertension (IPAH). As IPAH is associated with mutations of the bone morphogenetic protein receptor 2 (BMPR2) gene, the aim of this study was to investigate whether this association might also be found in patients with IPH. Twenty-three samples belonging to 21 unrelated caucasian patients with IPH followed in the hepatic haemodynamic laboratory of the Hospital Clinic in Barcelona were included in the study. All patients were studied for the entire open reading frame and splice site of the BMPR2 gene by direct sequencing and multiple ligation probe amplification (MLPA) in order to detect large deletions/duplications. None of the 23 patients had pulmonary artery hypertension. Four patients presented one single nucleotide polymorphism (SNP) in intron 5, four patients had a SNP in exon 12 and a SNP in exon 1 was found in two cases. Two patients had both intron 5 and exon 12 polymorphisms. All SNPs were previously described. Except for these three SNPs, neither mutations nor rearrangements have been identified in the BMPR2 gene in this population. We did not detect mutations or rearrangements in the coding region of the BMPR2 gene in our patients with IPH. These findings suggest that, in contrast to IPAH, mutations in BMPR2 are not involved in the pathogenesis of IPH.

Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody

The Nef protein of HIV-1 is important for AIDS pathogenesis, but it is not targeted by current antiviral strategies. Here, we describe a single-domain antibody (sdAb) that binds to HIV-1 Nef with a high affinity (K(d) = 2 × 10(-9)M) and inhibited critical biologic activities of Nef both in vitro and in vivo. First, it interfered with the CD4 down-regulation activity of a broad panel of nef alleles through inhibition of the Nef effects on CD4 internalization from the cell surface. Second, it was able to interfere with the association of Nef with the cellular p21-activated kinase 2 as well as with the resulting inhibitory effect of Nef on actin remodeling. Third, it counteracted the Nef-dependent enhancement of virion infectivity and inhibited the positive effect of Nef on virus replication in peripheral blood mononuclear cells. Fourth, anti-Nef sdAb rescued Nef-mediated thymic CD4(+) T-cell maturation defects and peripheral CD4(+) T-cell activation in the CD4C/HIV-1(Nef) transgenic mouse model. Because all these Nef functions have been implicated in Nef effects on pathogenesis, this anti-Nef sdAb may represent an efficient tool to elucidate the molecular functions of Nef in the virus life cycle and could now help to develop new strategies for the control of AIDS.

Type 2 diabetes is associated with reduced ATP-binding cassette transporter A1 gene expression, protein and function

Objective Increasing plasma glucose levels are associated with increasing risk of vascular disease. We tested the hypothesis that there is a glycaemia-mediated impairment of reverse cholesterol transport (RCT). We studied the influence of plasma glucose on expression and function of a key mediator in RCT, the ATP binding cassette transporter-A1 (ABCA1) and expression of its regulators, liver X receptor-α (LXRα) and peroxisome proliferator-activated receptor–γ (PPARγ). Methods and Results Leukocyte ABCA1, LXRα and PPARγ expression was measured by polymerase chain reaction in 63 men with varying degrees of glucose homeostasis. ABCA1 protein concentrations were measured in leukocytes. In a sub-group of 25 men, ABCA1 function was quantified as apolipoprotein-A1-mediated cholesterol efflux from 2–3 week cultured skin fibroblasts. Leukocyte ABCA1 expression correlated negatively with circulating HbA1c and glucose (rho = −0.41, p<0.001; rho = −0.34, p = 0.006 respectively) and was reduced in Type 2 diabetes (T2DM) (p = 0.03). Leukocyte ABCA1 protein was lower in T2DM (p = 0.03) and positively associated with plasma HDL cholesterol (HDL-C) (rho = 0.34, p = 0.02). Apolipoprotein-A1-mediated cholesterol efflux correlated negatively with fasting glucose (rho = −0.50, p = 0.01) and positively with HDL-C (rho = 0.41, p = 0.02). It was reduced in T2DM compared with controls (p = 0.04). These relationships were independent of LXRα and PPARγ expression. Conclusions ABCA1 expression and protein concentrations in leukocytes, as well as function in cultured skin fibroblasts, are reduced in T2DM. ABCA1 protein concentration and function are associated with HDL-C levels. These findings indicate a glycaemia- related, persistent disruption of a key component of RCT.

Caenorhabditis elegans Heterochromatin protein 1 (HPL-2) links developmental plasticity, longevity and lipid metabolism

Background Heterochromatin protein 1 (HP1) family proteins have a well-characterized role in heterochromatin packaging and gene regulation. Their function in organismal development, however, is less well understood. Here we used genome-wide expression profiling to assess novel functions of the Caenorhabditis elegans HP1 homolog HPL-2 at specific developmental stages. Results We show that HPL-2 regulates the expression of germline genes, extracellular matrix components and genes involved in lipid metabolism. Comparison of our expression data with HPL-2 ChIP-on-chip profiles reveals that a significant number of genes up- and down-regulated in the absence of HPL-2 are bound by HPL-2. Germline genes are specifically up-regulated in hpl-2 mutants, consistent with the function of HPL-2 as a repressor of ectopic germ cell fate. In addition, microarray results and phenotypic analysis suggest that HPL-2 regulates the dauer developmental decision, a striking example of phenotypic plasticity in which environmental conditions determine developmental fate. HPL-2 acts in dauer at least partly through modulation of daf-2/IIS and TGF-β signaling pathways, major determinants of the dauer program. hpl-2 mutants also show increased longevity and altered lipid metabolism, hallmarks of the long-lived, stress resistant dauers. Conclusions Our results suggest that the worm HP1 homologue HPL-2 may coordinately regulate dauer diapause, longevity and lipid metabolism, three processes dependent on developmental input and environmental conditions. Our findings are of general interest as a paradigm of how chromatin factors can both stabilize development by buffering environmental variation, and guide the organism through remodeling events that require plasticity of cell fate regulation.

Cardiac G-protein-coupled receptor kinase 2 ablation induces a novel Ca2+ handling phenotype resistant to adverse alterations and remodeling after myocardial infarction

G-protein-coupled receptor kinase 2 (GRK2) is a primary regulator of β-adrenergic signaling in the heart. G-protein-coupled receptor kinase 2 ablation impedes heart failure development, but elucidation of the cellular mechanisms has not been achieved, and such elucidation is the aim of this study.