Energetics of ¹⁴C-L-proline uptake into proteoliposomes containing purified HpPutP.

<p>Proteoliposomes in 100 mM KP_i, pH 7.5 (about 1 mg HpPutP ml^-1) were diluted 200fold into 100 mM Tris/Mes, pH 7.5 containing 2 mM β-mercaptoethanol, 5 mM MgSO₄, 0.2 μM valinomycin, 10 μM ¹⁴C-L-proline (26 Ci mol^-1) and 50 mM NaCl (smf), or LiCl (lmf), or no further additions (<i>Δ</i>ψ). In addition, proteoliposomes were diluted into 100 mM Tris/Mes, pH 6.0 containing 0.2 μM valinomycin (pmf), or 100 mM KP_i, pH 7.5 (diffusion). Proteoliposomes preloaded with 10 mM L-proline were diluted 200-fold into 100 mM KP_i, pH 7.5 containing ¹⁴C-L-proline (256 Ci mol^-1) (counterflow). Transport was assessed with a rapid filtration method as described in <i>Experimental </i><i>Procedures</i>, and data are shown as mean ± SEM of triplicate determinations.</p

Screen for inhibitors of HpPutP activity in <i>E. coli</i> WG170 (PutP^-A^-).

<p>Initial rates of uptake of 10 µM ¹⁴C-L-proline (10 Ci mol^-1) in <i>E</i>. <i>coli</i> WG170 harboring HpPutP with given substitutions were determined by transport measurements as described in the legend of <i>Figure 2A</i>. Putative inhibitors were added in 100fold molar access. NEM was added to the cells suspension at a concentration of 2 mM and incubated for 10 min prior to the start of ¹⁴C-L-proline uptake. Initial rates of triplicate determinations (shown as mean ± SEM) are represented as percentage of the rate in the absence of inhibitor. (L-Pro) L-proline; (D-Pro) D-proline; (DHP) 3,4-dehydro-D,L-proline; (AZC) L-azetidine-2-carboxylic acid; (GB) glycine betaine; (Pyr) pyrollidine; (His) histidine; (HOPro) hydroxy proline; (NEM) N-ethyl maleimide.</p

Homology model of HpPutP.

<p>(<i>A</i>) Overview of the homology model of HpPutP. The substrate binding site is enclosed by TMs 2, 3, 7, and 11, while the bound Na⁺ ion is coordinated by residues from TMs 2, 6, and 9. (<i>B</i>) The zoom-in view of the predicted Na⁺ and L-proline binding sites. The substrate and Na⁺ binding residues that have been mutated (see text) are shown in stick representation.</p

Effect of the placement of cysteine at given amino acid positions on HpPutP in <i>E. coli</i> WG170 (PutP^-A^-).

<p>(<i>A</i>) Initial rates of uptake (black columns) and maximum levels of accumulation (grey columns) of 10 µM ¹⁴C-L-proline (26 Ci mol^-1) in <i>E</i>. <i>coli</i> WG170 harboring HpPutP with given substitutions were determined by transport measurements as described in the legend of <i>Figure 2A</i>. (<i>B</i>) Relative amounts of HpPutP with given amino acid replacements in membranes of <i>E</i>. <i>coli</i> WG170 were estimated by Western blot analysis with HRP-linked anti-FLAG IgG directed against the FLAG epitope at the C terminus of HpPutP similar as described before [34].</p

Regulated Intramembrane Proteolysis and Degradation of Murine Epithelial Cell Adhesion Molecule mEpCAM

<div><p>Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at α-, β-, γ-, and ε-sites to generate soluble ectodomains, soluble Aβ-like-, and intracellular fragments termed mEpEX, mEp-β, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the α- and β-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble Aβ-like fragments mEp-β and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the γ-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified <i>in silico</i> in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent γ-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, γ-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation.</p></div

Cleavage of murine EpCAM in membrane assays.

<p>HEK293 cells were stably transfected with full-length mEpCAM in fusion with YFP (EpCAM-YFP). (<b>A</b>) Schematic representation of cleavage processes resulting in the generation of soluble EpEX, CTF-YFP, and intracellular EpICD-YFP fragments. (<b>B–C</b>) Membranes of stable transfectants were isolated and either kept at 0°C (0 h) or incubated at 37°C in reaction buffer for the indicated time points. Thereafter, pellets and supernatant were collected upon differential centrifugation. Pellets (<b>B</b>) and supernatants (<b>C</b>) of membrane assays were separated in a 10% SDS-PAGE and probed with mEpICD- and YFP-specific antibodies. (<b>D</b>) Embryonic stem cell line E14TG2a and teratocarcinoma cells mF9 were treated with DMSO (control) or the γ-secretase inhibitor DAPT before being subjected to a membrane assay. The total fraction of the membrane assay was separated in a 10% SDS-PAGE, and probed with a YFP-specific antibody. Treatment with DAPT resulted in the accumulation of CTF-YFP and in the inhibition of mEpICD-YFP formation. Protein bands corresponding to mEpCAM-YFP, CFT-YFP, and mEpICD-YFP are indicated in each immunoblot. Shown are the representative results of three independent experiments.</p

Determination of the sheddase cleavage amino acid sequence in mEpEX.

<p>(<b>A</b>) Schematic representation of mEpCAM-TF containing a TEV protease recognition site and a Flag-Tag in the mEpEX domain 42 amino acids before the predicted transmembrane domain. After cleavage by sheddases, the largest part of mEpEX can be removed through digestion with TEV protease and the resulting small fragment immunoprecipitated using Flag-specific antibodies. (<b>B</b>) Representative mass spectrometry spectrum of HEK293, NIH3T3, and mF9 cells stably expressing mEpCAM-TF and of vector control HEK293 cells as a control. Four major peak species are indicated. (<b>C</b>) Tabular overview of sheddase cleavage sites within mEpEX as determined upon mass spectrometric analysis and alignment to potential molecular weights. Calculated and determined masses are given in Dalton including error and charge of each peptide. (<b>D</b>) Representative mass spectrometry spectrum of HEK293 cells stably expressing mEpCAM-TF after treatment with DMSO, the metalloprotease protease inhibitor TAPI-1, and the phorbol ester PMA. (<b>E</b>) Representative mass spectrometry spectrum of HEK293 cells stably expressing mEpCAM-TF after treatment with DMSO, the BACE1 protease inhibitor C3, and after transient transfection of BACE1 expression plasmid. (<b>F</b>) Sequence alignment of murine and human EpCAM (top), and murine EpCAM and murine Trop-2 (bottom). metalloprotease protease cleavage (a-secretase) and BACE1 cleavage sites (b-secretase) are indicated.</p

Schematic representation of EpCAM presenilin-dependent regulated intramembrane proteolysis (PS-RIP) and endocytosis.

<p>Murine EpCAM (mEpCAM) is cleaved at the plasma membrane to release soluble EpEX (smEpEX). The resulting C-terminal fragment (mCTF) is a substrate for γ-secretase, which cleaves mCTF to generate soluble, extracellular mEp-β fragments (γ-cleavage) and intracellular mEpICD fragments, which are prone to proteasomal degradation. Additionally, mEpICD can be endocytosed and processed either by BACE1 in acidic intracellular compartments (endosome) and/or by acidic hydrolases in lysosomes.</p

Sequence conservation of cleavage sites in orthologs of EpCAM found in fishes, amphibians, birds, to placental mammals.

<p>Amino acid sequences of 52 orthologs of human EpCAM were aligned using ClustallW and sequence conservation of each amino acid was calculated (maximum score 11. Shown are the mean conservation score throughout all orthologs (mean) and conservation scores of single amino acids ranging positions p⁻³ to p⁺³ around determined cleavage sites of metalloproteases (<b>A</b>), BACE1 (<b>B</b>), γ-cleavage of γ-secretase (<b>C</b>), and ε-cleavage of γ-secretase (<b>D</b>).</p

Cleavage of endogeneous mEpCAM.

<p>Proteolytic cleavage of mEpCAM was addressed in mF9 (<b>A</b>) and E14TG2a (<b>B</b>) cells using membrane assays at pH 7 and pH 4. Membranes of mF9 and E14TG2a cells were incubated for 0 h and 24 h at 37°C and EpCAM fragments were detected in immunoblots using a mEpICD-specific antibody in combination with an HRP-conjugated secondary antibody. Inhibition of the γ-secretase complex was achieved upon treatment with DAPT where indicated. Shown are the representative results of three independent experiments.</p