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1.
Magnes Res ; 26(2): 56-66, 2013.
Article in English | MEDLINE | ID: mdl-23823179

ABSTRACT

The Na(+)/Mg(2+) exchanger SLC41A1 is involved in the pathophysiology of various disease conditions. It forms high-molecular-mass, possibly hetero-oligomeric protein complexes in transgenic HEK293 cells. Therefore, we attempted to identify binding partners of SLC41A1 by utilizing the split-ubiquitin modification of the yeast two-hybrid assay. As the most prominent binding partners in our experimental system, we identified 3-beta-hydroxysteroid-Δ(8),Δ(7)-isomerase and B-cell receptor associated-protein 31. Other polytons (interactors appearing in the screen more than once) included: IER3IP1, PPIB, UPF0480 protein C15orf24, SPINT2, C14orf1/PEBP28, NIFIE14, YIPF6, and KCP2. In total, 20 polytons and 38 singletons (interactors appearing in the screen only once) were identified. The polytons identified were mostly endoplasmic reticulum-located, integral proteins involved in protein maturation, N-glycosylation, protein folding, anterograde transport of proteins, protein secretion, and the regulation of apoptosis. Among the singletons, we identified SLC31A2, SLC35B1, SLC39A13, CRACM1, and MTCH2 as putative binding partners of SLC41A1. Interestingly, we did not identify interactions among SLC41A1 molecules. Most of the identified interactors are integral proteins localized in cellular compartments other than the cytoplasmic membrane, whereas SLC41A1 is targeted to the cytoplasmic membrane where it performs its core function. None of the interactors was confirmed by mass spectrometry. Instead, we identified among the proteins co-purified with strep-tagged SLC41A1: ACCA1, UBB, ATX2L, HSP7C and TBB. We therefore conclude that: (1) identified interactors form transient rather than stable complexes with SLC41A1, (2) the molecular interactors identified primarily among the polytons might contribute to the production, proper folding, and maturation of SLC41A1 in the endoplasmic reticulum and Golgi apparatus, (3) most of the interactors identified among singletons might undergo similar maturation steps (post-translational modification), anterograde transport, and protein sorting as SLC41A1.


Subject(s)
Cation Transport Proteins/metabolism , Multiprotein Complexes/metabolism , Two-Hybrid System Techniques , Ubiquitin/metabolism , Blotting, Western , Chromatography, Affinity , Electrophoresis, Polyacrylamide Gel , HEK293 Cells , Humans , Protein Binding , Steroid Isomerases/metabolism
2.
ACS Chem Biol ; 8(3): 549-58, 2013 Mar 15.
Article in English | MEDLINE | ID: mdl-23301619

ABSTRACT

Glaucoma and age-related macular degeneration are ocular diseases targeted clinically by anecortave acetate (AA). AA and its deacetylated metabolite, anecortave desacetate (AdesA), are intraocular pressure (IOP)-lowering and angiostatic cortisenes devoid of glucocorticoid activity but with an unknown mechanism of action. We used a methotrexate-anchored yeast three-hybrid (Y3H) technology to search for binding targets for AA in human trabecular meshwork (TM) cells, the target cell type that controls IOP, a major risk factor in glaucoma. Y3H hits were filtered by competitive Y3H screens and coimmunoprecipitation experiments and verified by surface plasmon resonance analysis to yield a single target, phosphodiesterase 6-delta (PDE6D). PDE6D is a prenyl-binding protein with additional function outside the PDE6 phototransduction system. Overexpression of PDE6D in mouse eyes caused elevated IOP, and this elevation was reversed by topical ocular application of either AA or AdesA. The identification of PDE6D as the molecular binding partner of AA provides insight into the role of this drug candidate in treating glaucoma.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 6/antagonists & inhibitors , Methotrexate/metabolism , Pregnadienediols/pharmacology , Two-Hybrid System Techniques , Animals , Cell Line , Cyclic Nucleotide Phosphodiesterases, Type 6/biosynthesis , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , High-Throughput Screening Assays , Humans , Methotrexate/chemistry , Mice , Mice, Inbred BALB C , Molecular Structure , Pregnadienediols/chemistry , Structure-Activity Relationship
3.
Magnes Res ; 26(4): 176-81, 2013.
Article in English | MEDLINE | ID: mdl-24491491

ABSTRACT

Membrane topology is an important parameter for understanding the function and regulation of any integral protein. This aspect of the NME SLC41A1 is currently under debate. The most probable model, which has been computer-predicted, exhibits ten TMh with both termini being oriented intracellularly. However, other freely accessible online prediction programs predict that SLC41A1 possesses eleven ("outside-in" configuration), nine ("outside-in" configuration), or eight ("inside-in" configuration) TMh. The consensus based on published experimental data acquired by independent research teams is that the N-terminal flanking region is located intracellularly. However, controversy remains about the orientation of the C-terminus, which has lately been proposed to be extracellular in peer-reviewed bibliography. Here, we performed split-ubiquitin functional assays with transgenic SLC41A1 fused N- or C-terminally to a Cub-LexA-VP16 reporter cassette. The bait constructs were co-expressed in S. cerevisiae st. NMY51 with positive recombinant membrane markers (Ost1, Fur4, Alg5, Tom20) tagged with NubI (or NubG). Ubiquitin could only be reconstituted if the reporter moiety was exposed to the cytosol. Functional reconstitution of ubiquitin was observed when SLC41A1 C-terminally tagged with Cub was co-expressed with NubI-tagged membrane markers, thereby, indicating a cytosolic orientation of the C-terminus of SLC41A1. Thus, our experimental data are in favor of the - the in silico analyses being strongly preferred - ten TMh model of SLC41A1 topology, with both termini being oriented intracellularly.


Subject(s)
Cation Transport Proteins/chemistry , Cation Transport Proteins/metabolism , Cell Membrane/metabolism , Cell Membrane/chemistry , Protein Conformation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Ubiquitin/metabolism
4.
Am J Pathol ; 181(5): 1621-33, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22959909

ABSTRACT

Pulmonary hypertension (PH) is a fatal disease characterized by remodeling processes such as increased migration and proliferation of pulmonary arterial smooth muscle cells (PASMC), enhanced matrix deposition, and dysregulation of cytoskeletal proteins. However, the contribution of cytoskeletal proteins in PH is still not fully understood. In this study, we have used a yeast two-hybrid screen to identify novel binding partners of the cytoskeletal adaptor protein four-and-a-half LIM domains 1 (Fhl-1). This identified paxillin as a new Fhl-1 interacting partner, and consequently we assessed its contribution to vascular remodeling processes. Native protein-protein binding was confirmed by co-immunoprecipitation studies in murine and human PASMC. Both proteins co-localized in PASMC in vitro and in vivo. In lung samples from idiopathic pulmonary arterial hypertension patients, paxillin expression was increased on mRNA and protein levels. Laser-microdissection of murine intrapulmonary arteries revealed elevated paxillin expression in hypoxia-induced PH. Furthermore, hypoxia-dependent upregulation of paxillin was HIF-1α dependent. Silencing of paxillin expression led to decreased PASMC adhesion, proliferation, and increased apoptosis. Regulation of these processes occurred via Akt and Erk1/2 kinases. In addition, adhesion of PASMC to the extracellular matrix protein fibronectin was critically dependent on paxillin expression. To summarize, we identified paxillin as a new regulator protein of PASMC growth.


Subject(s)
Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/physiopathology , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Paxillin/metabolism , Pulmonary Artery/pathology , Adult , Animals , Apoptosis , Cell Adhesion , Cell Proliferation , Disease Models, Animal , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , Familial Primary Pulmonary Hypertension , Female , Fibronectins/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Humans , Hypertension, Pulmonary/complications , Hypertension, Pulmonary/pathology , Hypoxia/complications , Hypoxia/metabolism , Hypoxia/pathology , Hypoxia/physiopathology , Intracellular Signaling Peptides and Proteins/metabolism , LIM Domain Proteins/metabolism , Lung/metabolism , Lung/pathology , Lung/physiopathology , Male , Mice , Middle Aged , Muscle Proteins/metabolism , Myocytes, Smooth Muscle/enzymology , Phosphorylation , Protein Binding , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction
5.
Am J Respir Crit Care Med ; 180(4): 353-64, 2009 Aug 15.
Article in English | MEDLINE | ID: mdl-19498059

ABSTRACT

RATIONALE: Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism by which pulmonary gas exchange is optimized by the adaptation of blood flow to alveolar ventilation. In chronic hypoxia, in addition to HPV a vascular remodeling process leads to pulmonary hypertension. A complex of heme oxygenase-2 (HO-2) and the BK channel has been suggested as a universal oxygen sensor system. OBJECTIVES: We investigated whether this complex serves as an oxygen sensor for the vascular effects of alveolar hypoxia in the lung. METHODS: The investigations were performed in chronically hypoxic mice, in isolated perfused and ventilated lungs, and on the cellular level, including HO-2- and BK-channel deficient mice. MEASUREMENTS AND MAIN RESULTS: Immunohistochemical analysis of mouse lungs identified HO-2 mainly in pulmonary arteries, the bronchial epithelium, and alveolar epithelial cells. BK channel alpha-subunit (BKalpha) immunoreactivity was found primarily in the bronchial and vascular smooth muscle layer. Immunofluorescence staining and coimmunoprecipitation suggested only a weak complexation of HO-2 and BKalpha in pulmonary arterial smooth muscle cells. The strength of acute and sustained HPV, determined in isolated perfused and ventilated lungs, was not different among wild-type, HO-2-deficient, and BKalpha-deficient mice. Exposure of mice to 3 weeks of chronic hypoxia resulted in a slight down-regulation of HO-2 and no alteration in BKalpha expression. The degree of pulmonary hypertension that developed, quantified on the basis of right ventricular pressure, right-heart hypertrophy, and the degree of muscularization of precapillary pulmonary arteries, was not different among wild-type, HO-2-deficient, and BKalpha-deficient mice. CONCLUSIONS: It is demonstrated that neither deletion of HO-2 nor BK channels affect acute, sustained, and chronic vascular responses to alveolar hypoxia in the lung.


Subject(s)
Heme Oxygenase (Decyclizing)/genetics , Heme Oxygenase (Decyclizing)/physiology , Hypoxia/physiopathology , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/genetics , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/physiology , Lung/blood supply , Pulmonary Alveoli/blood supply , Vasoconstriction/physiology , Animals , Hypertension, Pulmonary/pathology , Hypertension, Pulmonary/physiopathology , Hypoxia/pathology , In Vitro Techniques , Lung/pathology , Mice , Microscopy, Fluorescence , Muscle, Smooth, Vascular/pathology , Muscle, Smooth, Vascular/physiopathology , Polymerase Chain Reaction , Pulmonary Alveoli/pathology , Pulmonary Wedge Pressure/physiology , RNA, Messenger/genetics
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