Xiangyuan He

Memapsin 2 (β‐secretase) cytosolic domain binds to the VHS domains of GGA1 and GGA2: implications on the endocytosis mechanism of memapsin 2

Memapsin 2, or β‐secretase, is a membrane‐anchored aspartic protease that initiates the cleavage of β‐amyloid precursor protein (APP) leading to the production of β‐amyloid peptide in the brain and the onset of Alzheimers disease. Memapsin 2 and APP are both endocytosed into endosomes for cleavage. Here we show that the cytosolic domain of memapsin 2, but not that of memapsin 1, binds the VHS domains of GGA1 and GGA2. Gel‐immobilized VHS domains of GGA1 and GGA2 also bound to full‐length memapsin 2 from cell mammalian lysates. Mutagenesis studies established that Asp496, Leu499 and Leu500 were essential for the binding. The spacing of these three residues in memapsin 2 is identical to those in the cytosolic domains of mannose‐6‐phosphate receptors, sortilin and low density lipoprotein receptor‐related protein 3. These observations suggest that the endocytosis and intracellular transport of memapsin 2, mediated by its cytosolic domain, may involve the binding of GGA1 and GGA2.

Apolipoprotein Receptor 2 and X11α/β Mediate Apolipoprotein E-Induced Endocytosis of Amyloid-β Precursor Protein and β-Secretase, Leading to Amyloid-β Production

The homeostasis of amyloid-β (Aβ) in the brain is critical to the pathogenesis of Alzheimers disease (AD). Aβ is a fragment of amyloid-β precursor protein (APP) generated in neurons by two proteases, β- and γ-secretases. APP and β-secretase, both present on cell surface, are endocytosed into endosomes to produce Aβ. The molecular mechanism by which neurons trigger the production of Aβ is poorly understood. We describe here evidence that the binding of lipid-carrying apolipoprotein E (ApoE) to receptor apolipoprotein E receptor 2 (ApoER2) triggers the endocytosis of APP, β-secretase, and ApoER2 in neuroblastoma cells, leading to the production of Aβ. This mechanism, mediated by adaptor protein X11α or X11β (X11α/β), whose PTB (phosphotyrosine-binding) domain binds to APP and a newly recognized motif in the cytosolic domain of ApoER2. Isomorphic form ApoE4 triggers the production of more Aβ than by ApoE2 or ApoE3; thus, it may play a role in the genetic risk of ApoE4 for the sporadic AD. The mechanism, which functions independently from Reelin–ApoER2 interaction, also provides a link between lipid uptake and Aβ production, which may be important for the regulation of neuronal activity.

Stabilin-1 localizes to endosomes and the trans-Golgi network in human macrophages and interacts with GGA adaptors

Stabilin‐1 and stabilin‐2 constitute a novel family of fasciclin domain‐containing hyaluronan receptor homologues recently described by us. Whereas stabilin‐1 is expressed in sinusoidal endothelial cells and in macrophages in vivo, stabilin‐2 is absent from the latter. In the present study, we analyzed the subcellular distribution of stabilin‐1 in primary human macrophages. Using flow cytometry, expression of stabilin‐1 was demonstrated on the surface of interleukin‐4/dexamethasone‐stimulated macrophages (MΦ2). By immunofluorescense and confocal microscopy, we established that stabilin‐1 is preferentially localized in early endosome antigen‐1‐positive early/sorting endosomes and in recycling endosomes identified by transferrin endocytosis. Association of stabilin‐1 was infrequently seen with p62 lck ligand‐positive late endosomes and with CD63‐positive lysosomes but not in lysosome‐associated membrane protein‐1‐positive lysosomes. Stabilin‐1 was also found in the trans‐Golgi network (TGN) but not in Golgi stack structures. Glutathione S‐transferase pull‐down assay revealed that the cytoplasmic tail of stabilin‐1 but not stabilin‐2 binds to recently discovered Golgi‐localized, γ‐ear‐containing, adenosine 5′‐diphosphate‐ribosylation factor‐binding (GGA) adaptors GGA1, GGA2, and GGA3 long, mediating traffic between Golgi and endosomal/lysosomal compartments. Stabilin‐1 did not bind to GGA3 short, which lacks a part of the Vps27p/Hrs/STAM domain. Deletion of DDSLL and LL amino acid motifs resulted in decreased binding of stabilin‐1 with GGAs. A small portion of stabilin‐1 colocalized with GGA2 and GGA3 in the TGN in MΦ2. Treatment with brefeldin A resulted in accumulation of stabilin‐1 in the TGN. Our results suggest that stabilin‐1 is involved in the GGA‐mediated sorting processes at the interface of the biosynthetic and endosomal pathways; similarly to other GGA‐interacting proteins, stabilin‐1 may thus function in endocytic and secretory processes of human macrophages.

Crystal structure of human GGA1 GAT domain complexed with the GAT-binding domain of Rabaptin5

GGA proteins coordinate the intracellular trafficking of clathrin‐coated vesicles through their interaction with several other proteins. The GAT domain of GGA proteins interacts with ARF, ubiquitin, and Rabaptin5. The GGA–Rabaptin5 interaction is believed to function in the fusion of trans‐Golgi‐derived vesicles to endosomes. We determined the crystal structure of a human GGA1 GAT domain fragment in complex with the Rabaptin5 GAT‐binding domain. In this structure, the Rabaptin5 domain is a 90‐residue‐long helix. At the N‐terminal end, it forms a parallel coiled‐coil homodimer, which binds one GAT domain of GGA1. In the C‐terminal region, it further assembles into a four‐helix bundle tetramer. The Rabaptin5‐binding motif of the GGA1 GAT domain consists of a three‐helix bundle. Thus, the binding between Rabaptin5 and GGA1 GAT domain is based on a helix bundle–helix bundle interaction. The current structural observation is consistent with previously reported mutagenesis data, and its biological relevance is further confirmed by new mutagenesis studies and affinity analysis. The four‐helix bundle structure of Rabaptin5 suggests a functional role in tethering organelles.

Crystal structure of GGA2 VHS domain and its implication in plasticity in the ligand binding pocket

Golgi‐localized, γ‐ear‐containing, ARF binding (GGA) proteins regulate intracellular vesicle transport by recognizing sorting signals on the cargo surface in the initial step of the budding process. The VHS (VPS27, Hrs, and STAM) domain of GGA binds with the signal peptides. Here, a crystal structure of the VHS domain of GGA2 is reported at 2.2 Å resolution, which permits a direct comparison with that of homologous proteins, GGA1 and GGA3. Significant structural difference is present in the loop between helices 6 and 7, which forms part of the ligand binding pocket. Intrinsic fluorescence spectroscopic study indicates that this loop undergoes a conformational change upon ligand binding. Thus, the current structure suggests that a conformational change induced by ligand binding occurs in this part of the ligand pocket.

Analysis of the Interaction between GGA1 GAT Domain and Rabaptin‐5

GGAs are a family of adaptor proteins involved in vesicular transport. As an effector of the small GTPase Arf, GGA interacts using its GAT domain with the GTP-bound form of Arf. The GAT domain is also found to interact with ubiquitin and rabaptin-5. Rabaptin-5 is, in turn, an effector of another small GTPase, Rab5, which regulates early endosome fusion. The interaction between GGAs and rabaptin-5 is likely to take place in a pathway between the trans-Golgi network and early endosomes. This chapter describes in vitro biochemical characterization of the interaction between the GGA1 GAT domain and rabaptin-5. Combining with the complex crystal structure, we reveal that the binding mode is helix bundle-to-helix bundle in nature.

Expression and processing of fluorescent fusion proteins of amyloid precursor protein (APP)

Processing of β-amyloid precursor protein (APP) by β- and γ-secretases in neurons produces amyloid-β (Aβ), whose excess accumulation leads to Alzheimers disease (AD). Knowledge on subcellular trafficking pathways of APP and its fragments is important for the understanding of AD pathogenesis. We designed fusion proteins comprising a C-terminal fragment of APP (app) and fluorescent proteins GFP (G) and DsRed (D) to permit the tracking of the fusion proteins and fragments in cells. CAD cells expressing these proteins emitted colocalized green and red fluorescence and produce ectodomains, sGapp and sRapp, and Aβ, whose level was reduced by inhibitors of β- and γ-secretases. The presence of GappR in endosomes was observed via colocalization with Rab5. These observations indicated that the fusion proteins were membrane inserted, transported in vesicles and proteolytically processed by the same mechanism for APP. By attenuating fusion protein synthesis with cycloheximide, individual fluorescent colors from the C-terminus of the fusion proteins appeared in the cytosol which was strongly suppressed by β-secretase inhibitor, suggesting that the ectodomains exit the cell rapidly (t1/2 about 20min) while the C-terminal fragments were retained longer in cells. In live cells, we observed the fluorescence of the ectodomains located between parental fusion proteins and plasma membrane, suggesting that these ectodomain positions are part of their secretion pathway. Our results indicate that the native ectodomain does not play a decisive role for the key features of APP trafficking and processing and the new fusion proteins may lead to novel insights in intracellular activities of APP.

Erratum to: Memapsin 2 (β-secretase) cytosolic domain binds to the VHS domains of GGA1 and GGA2: implications on the endocytosis mechanism of memapsin 2 (FEBS 26327)

Erratum to: Memapsin 2 (L-secretase) cytosolic domain binds to the VHS domains of GGA1 and GGA2: implications on the endocytosis mechanism of memapsin 2 (FEBS 26327) [FEBS Letters 524 (2002) 183^187]C Xiangyuan Hea, Wan-Pin Changa, Gerard Koelscha;b, Jordan Tanga;c; aProtein Studies Program, Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104, USA bZapaq, Inc., Oklahoma City, OK 73104, USA cDepartment of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA