John Wagner

The Architecture of the Multisubunit TRAPP I Complex Suggests a Model for Vesicle Tethering

Transport protein particle (TRAPP) I is a multisubunit vesicle tethering factor composed of seven subunits involved in ER-to-Golgi trafficking. The functional mechanism of the complex and how the subunits interact to form a functional unit are unknown. Here, we have used a multidisciplinary approach that includes X-ray crystallography, electron microscopy, biochemistry, and yeast genetics to elucidate the architecture of TRAPP I. The complex is organized through lateral juxtaposition of the subunits into a flat and elongated particle. We have also localized the site of guanine nucleotide exchange activity to a highly conserved surface encompassing several subunits. We propose that TRAPP I attaches to Golgi membranes with its large flat surface containing many highly conserved residues and forms a platform for protein-protein interactions. This study provides the most comprehensive view of a multisubunit vesicle tethering complex to date, based on which a model for the function of this complex, involving Rab1-GTP and long, coiled-coil tethers, is presented.

Neuroendocrine dysplasia in mice lacking protein tyrosine phosphatase σ

Protein tyrosine phosphatase σ (PTP-σ, encoded by the Ptprs gene) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases that is highly expressed during mammalian embryonic development in the germinal cell layer lining the lateral ventricles of the developing brain, dorsal root ganglia, Rathkes pouch, olfactory epithelium, retina and developing lung and heart. On the basis of its expression and homology with the Drosophila melanogaster orthologues DPTP99 and DPTP100A (Refs 5,6), which have roles in the targeting of axonal growth cones, we hypothesized that PTP-σ may also have a modulating function in cell-cell interactions, as well as in axon guidance during mammalian embryogenesis. To investigate its function in vivo, we generated Ptprs-deficient mice. The resulting Ptprs-/- animals display retarded growth, increased neonatal mortality, hyposmia and hypofecundity. Anatomical and histological analyses showed a decrease in overall brain size with a severe depletion of luteinizing hormone-releasing hormone (LHRH)-immunoreactive cells in Ptprs-/- hypothalamus. Ptprs-/- mice have an enlarged intermediate pituitary lobe, but smaller anterior and posterior lobes. These results suggest that tyrosine phosphorylation-dependent signalling pathways regulated by PTP-σ influence the proliferation and/or adhesiveness of various cell types in the developing hypothalamo-pituitary axis.

Murine embryonic fibroblasts lacking TC-PTP display delayed G1 phase through defective NF-κB activation

Previous results suggested a potential role for T-cell protein tyrosine phosphatase (TC-PTP) in cell proliferation. However, no conclusive data has supported such a function in the modulation of this process. In order to clarify this issue, we isolated TC-PTP−/− murine embryonic fibroblasts (MEFs) as well as cell lines to characterize the role of TC-PTP in the control of cell proliferation and cell cycle. Both TC-PTP−/− primary MEFs and cell lines proliferate slower than TC-PTP+/+ cells. We also demonstrated that TC-PTP−/− cells have a slow progression through the G1 phase of the cell cycle. Further characterization of the G1 defect indicates that the kinetics of cyclin D1 induction was delayed and that p27KIP1 remains at higher levels for an extended period of time. Moreover, cells lacking TC-PTP showed a delayed activation of CDK2. This slow progression through the early G1-phase resulted in decreased phosphorylation of the RB protein and subsequent delay into the S phase transition. In contrast, no further defects were detected in other phases of the cell cycle. Survey of the potential signaling pathways leading to this delayed cyclin D1 expression indicated that NF-κB activation was compromised and that IKKβ activity was also reduced following PDGF stimulation. Reintroduction of wild-type TC-PTP into the TC-PTP−/− cells rescued the defective proliferation, cyclin D1 expression, NF-κB activation as well as IκB phosphorylation. Together, these results confirm that TC-PTP plays a positive role in the progression of early G1 phase of the cell cycle through the NF-κB pathway.

Coupling of the murine protein tyrosine phosphatase PEST to the epidermal growth factor (EGF) receptor through a Src homology 3 (SH3) domain-mediated association with Grb2.

The involvement of murine protein tyrosine phosphatase-PEST (MPTP – PEST) in signal transduction pathways is suggested by its ability to dephosphorylate phosphotyrosine residues, its interaction with the adaptor protein SHC and by the presence of five proline-rich stretches in its non-catalytic carboxyl terminus. Proline-rich sequences have been identified as binding sites for Src homology 3 (SH3) domains found in proteins associated with signal transduction events. The ability of these sequences to act as SH3 domain recognition motifs was investigated using bacterially expressed SH3 domains derived from several different signalling proteins. In vitro binding assays indicate that four of these proline-rich sequences constitute specific binding sites for both SH3 domains of the adaptor molecule Grb2. Wild type Grb2, but not Grb2 proteins corresponding to loss-of-function mutants in the Caenorhabditis elegans sem-5 protein, associate with MPTP – PEST in vivo. Experiments in EGF receptor expressing cells show that the interaction between MPTP – PEST and Grb2 results in the binding of this complex to activated EGF receptors. In addition, identification of putative substrate(s) of MPTP – PEST have revealed a candidate protein of ∼120 kDa which is tyrosine phosphorylated upon EGF stimulation. Together, these results describe a novel SH3 domain-dependent recruitment of a protein tyrosine phosphatase to an activated receptor tyrosine kinase and establish a potential role for MPTP – PEST in signalling pathways at the molecular level.

Conditional disruption of mouse HFE2 gene: Maintenance of systemic iron homeostasis requires hepatic but not skeletal muscle hemojuvelin†‡

Mutations of the HFE2 gene are linked to juvenile hemochromatosis, a severe hereditary iron overload disease caused by chronic hyperabsorption of dietary iron. HFE2 encodes hemojuvelin (Hjv), a membrane‐associated bone morphogenetic protein (BMP) coreceptor that enhances expression of the liver‐derived iron regulatory hormone hepcidin. Hjv is primarily expressed in skeletal muscles and at lower levels in the heart and the liver. Moreover, a soluble Hjv form circulates in plasma and is thought to act as a decoy receptor, attenuating BMP signaling to hepcidin. To better understand the regulatory function of Hjv, we generated mice with tissue‐specific disruption of this protein in hepatocytes or in muscle cells. The hepatic ablation of Hjv resulted in iron overload, quantitatively comparable to that observed in ubiquitous Hjv−/− mice. Serum iron and ferritin levels, transferrin saturation, and liver iron content were significantly (P < 0.001) elevated in liver‐specific Hjv−/− mice. Hepatic Hjv mRNA was undetectable, whereas hepcidin expression was markedly suppressed (12.6‐fold; P < 0.001) and hepatic BMP6 mRNA up‐regulated (2.4‐fold; P < 0.01), as in ubiquitous Hjv−/− counterparts. By contrast, the muscle‐specific disruption of Hjv was not associated with iron overload or altered hepcidin expression, suggesting that muscle Hjv mRNA is dispensable for iron metabolism. Our data do not support any significant iron‐regulatory function of putative muscle‐derived soluble Hjv in mice, at least under physiological conditions. Conclusion: The hemochromatotic phenotype of liver‐specific Hjv−/− mice suggests that hepatic Hjv is necessary and sufficient to regulate hepcidin expression and control systemic iron homeostasis. (HEPATOLOGY 2011;)

Structure-function analysis of Escherichia coli MnmG (GidA), a highly conserved tRNA-modifying enzyme.

The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-A resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.

Trapping open and closed forms of FitE-A group III periplasmic binding protein.

Periplasmic binding proteins (PBPs) are essential components of bacterial transport systems, necessary for bacterial growth and survival. The two‐domain structures of PBPs are topologically classified into three groups based on the number of crossovers or hinges between the globular domains: group I PBPs have three connections, group II have two, and group III have only one. Although a large number of structures for group I or II PBPs are known, fewer group III PBPs have been structurally characterized. Group I and II PBPs exhibit significant domain motions during transition from the unbound to ligand‐bound form, however, no large conformational changes have been observed to date in group III PBPs. We have solved the crystal structure of a periplasmic binding protein FitE, part of an iron transport system, fit, recently identified in a clinical E. coli isolate. The structure, determined at 1.8 Å resolution, shows that FitE is a group III PBP containing a single α‐helix bridging the two domains. Among the individual FitE molecules present in two crystal forms we observed three different conformations (open, closed, intermediate). Our crystallographic and molecular dynamics results strongly support the notion that group III PBPs also adopt the same Venus flytrap mechanism as do groups I and II PBPs. Unlike other group III PBPs, FitE forms dimers both in solution and in the crystals. The putative siderophore binding pocket is lined with arginine residues, suggesting an anionic nature of the iron‐containing siderophore. Proteins 2009.

Biochemical and Crystallographic Studies Reveal a Specific Interaction between Trapp Subunits Trs33P and Bet3P

Transport protein particle (TRAPP) comprises a family of two highly related multiprotein complexes, with seven common subunits, that serve to target different classes of transport vesicles to their appropriate compartments. Defining the architecture of the complexes will advance our understanding of the functional differences between these highly related molecular machines. Genetic analyses in yeast suggested a specific interaction between the TRAPP subunits Bet3p and Trs33p. A mammalian bet3–trs33 complex was crystallized, and the structure was solved to 2.2 Å resolution. Intriguingly, the overall fold of the bet3 and trs33 monomers was similar, although the proteins had little overall sequence identity. In vitro experiments using yeast TRAPP subunits indicated that Bet3p binding to Trs33p facilitates the interaction between Bet3p and another TRAPP subunit, Bet5p. Mutational analysis suggests that yeast Trs33p facilitates other Bet3p protein–protein interactions. Furthermore, we show that Trs33p can increase the Golgi‐localized pool of a mutated Bet3 protein normally found in the cytosol. We propose that one of the roles of Trs33p is to facilitate the incorporation of the Bet3p subunit into assembling TRAPP complexes.

Hfe and Hjv exhibit overlapping functions for iron signaling to hepcidin

Functional inactivation of HFE or hemojuvelin (HJV) is causatively linked to adult or juvenile hereditary hemochromatosis, respectively. Systemic iron overload results from inadequate expression of hepcidin, the iron regulatory hormone. While HJV regulates hepcidin by amplifying bone morphogenetic protein (BMP) signaling, the role of HFE in the hepcidin pathway remains incompletely understood. We investigated the pathophysiological implications of combined Hfe and Hjv ablation in mice. Isogenic Hfe−/− and Hjv−/− mice were crossed to generate double Hfe−/−Hjv−/− progeny. Wild-type control and mutant mice of all genotypes were analyzed for serum, hepatic, and splenic iron content, expression of iron metabolism proteins, and expression of hepcidin and Smad signaling in the liver, in response to a standard or an iron-enriched diet. As expected, Hfe−/− and Hjv−/− mice developed relatively mild or severe iron overload, respectively, which corresponded to the degree of hepcidin inhibition. The double Hfe−/−Hjv−/− mice exhibited an indistinguishable phenotype to single Hjv−/− counterparts with regard to suppression of hepcidin, serum and hepatic iron overload, splenic iron deficiency, tissue iron metabolism, and Smad signaling, under both dietary regimens. We conclude that the hemochromatotic phenotype caused by disruption of Hjv is not further aggravated by combined Hfe/Hjv deficiency. Our results provide genetic evidence that Hfe and Hjv operate in the same pathway for the regulation of hepcidin expression and iron metabolism.Key messagesCombined disruption of Hfe and Hjv phenocopies single Hjv deficiency.Single Hjv−/− and double Hfe−/−Hjv−/− mice exhibit comparable iron overload.Hfe and Hjv regulate hepcidin via the same pathway.

[A contribution to the pseudo-LE-syndrome. Investigations of the cardiovascular system, abdominal organs, lymphatic system and drug histories (author's transl)].

13 of 30 patients suffering from pseudo-LE-syndrome showed a usually reversible enlargement of the heart during the acute stage of the disease. In two patients carditis occurring in pseudo-LE-syndrome lead to congestive heart failure and an additional patient died in the acute stage of carditis. As opposed to Systemic Lupus Erythematosus hypertension did not occur in a higher frequency than normal. In five cases cardiac catheter examinations showed slight elevation of the end-diastolic pressure in the right ventricle, in two cases an abnormal high mean pressure in the right atrium and a systolic gradient at the pulmonic valve was found. -Scintigrams showed definite enlargement of the spleen and to a lesser degree enlargement of the liver was seen. Laparascopy showed multiple concretions after peritonitis. Lymphographic changes in the retroperitoneal lymph nodes and lymphatic ducts were not observed in contrast to rheumatic diseases. Drug histories in most cases discovered intake of Venopyronum dragees prior to onset of the disease. But recurrent attacks of the disease also occurred without further intake of the drug.Summary13 of 30 patients suffering from pseudo-LE-syndrome showed a usually reversible enlargement of the heart during the acute stage of the disease. In two patients carditis occurring in pseudo-LE-syndrome lead to congestive heart failure and an additional patient died in the acute stage of carditis. As opposed to Systemic Lupus Erythematosus hypertension did not occur in a higher frequency than normal. In five cases cardiac catheter examinations showed slight elevation of the end-diastolic pressure in the right ventricle, in two cases an abnormal high mean pressure in the right atrium and a systolic gradient at the pulmonic valve was found. — Scintigrams showed definite enlargement of the spleen and to a lesser degree enlargement of the liver was seen. Laparascopy showed multiple concretions after peritonitis. Lymphographic changes in the retroperitoneal lymph nodes and lymphatic ducts were not observed in contrast to rheumatic diseases. Drug histories in most cases discovered intake of Venopyronum® dragees prior to onset of the disease. But recurrent attacks of the disease also occurred without further intake of the drug.ZusammenfassungVon 30 Patienten mit Pseudo-LE-Syndrome wiesen im akuten Schub der Erkrankung 13 eine meist reversible Herzvergrößerung auf. Bei zwei Patienten hatte die im Rahmen der Erkrankung Über Jahre dauernde Carditis zur klinisch manifesten Herzinsuffizienz geführt und ein weiterer Patient war im akuten Stadium an der Carditis verstorben. Im Gegensatz zum Lupus erythematodes disseminatus wurde eine Hypertonie nicht gehäuft beobachtet. Rechtsherzkatheteruntersuchungen an 5 Patienten zeigten durchgehend geringgradige Erhöhungen des enddiastolischen Druckes im rechten Ventrikel und bei je zwei Patienten einen erhöhten Mitteldruck im rechten Vorhof und einen systolischen Druckgradienten über der Pulmonalis. — Szintigraphisch wurden teilweise deutliche Milzvergrößerungen und geringgradige Lebervergrößerungen nachgewiesen. Laparoskopisch fanden sich nach abgelaufener Peritonitis ausgedehnte Verwachsungen. — Lymphographisch konnten Veränderungen an den retroperitonealen Lymphknoten und Lymphbahnen im Gegensatz zu anderen rheumatischen Erkrankungen nicht festgestellt werden. — Arzneimittelanamnesen deckten in den meisten Fällen eine Einnahme von Venopyronum® Dragees vor Erkrankungsbeginn auf, doch kam es auch zum Auftreten neuer Krankheitsschübe, ohne daß jeweils das Medikament zuvor erneut eingenommen worden war.