Structural Study of the HD-PTP Bro1 Domain in a Complex with the Core Region of STAM2, a Subunit of ESCRT-0

<div><p>EGFR is a key player in cell proliferation and survival signaling, and its sorting into MVBs for eventual lysosomal degradation is controlled by the coordination of multiple ESCRT complexes on the endosomal membrane. HD-PTP is a cytosolic protein tyrosine phosphatase, and is associated with EGFR trafficking by interacting with the ESCRT-0 protein STAM2 and the ESCRT-III protein CHMP4B via its N-terminal Bro1 domain. Intriguingly, the homologous domain of two other human Bro1 domain-containing proteins, Alix and Brox, binds CHMP4B but not STAM2, despite their high structural similarity. To elucidate this binding specificity, we determined the complex structure of the HD-PTP Bro1 domain bound to the STAM2 core region. STAM2 binds to the hydrophobic concave pocket of the HD-PTP Bro1 domain, as CHMP4B does to the pocket of Alix, Brox, or HD-PTP but in the opposite direction. Critically, Thr145 of HD-PTP, corresponding to Lys151 of Alix and Arg145 of Brox, is revealed to be a determinant residue enabling this protein to bind STAM2, as the Alix- or Brox-mimicking mutations of this residue blocks the intermolecular interaction. This work therefore provides the structural basis for how HD-PTP recognizes the ESCRT-0 component to control EGFR sorting.</p></div

Data collection and structure refinement statistics.

<p>Data collection and structure refinement statistics.</p

Coimmunoprecipitation assay between HD-PTP and STAM2 or CHMP4B.

<p>HEK293 cells were transiently transfected with the indicated constructs, and the expression level of STAM2 proteins (<i>A</i>), or intermolecular interaction of HD-PTP with STAM2 (<i>B</i>) or with CHMP4B (<i>C</i>) was assessed by immunoprecipitation and immunoblotting.</p

Interaction of HD-PTP(1–361) with the STAM2 core region.

<p>(A) STAM2 constructs tested for binding to HD-PTP(1–361). Denoted beside the residue numbers is whether each construct interacted with HD-PTP(1–361) (B) SEC analysis results using a Superose 6 10/300 GL gel filtration column. The elution positions of standard protein size markers Blue dextran (void volume, V₀) and Conalbumin (75 kDa) are indicated by arrowheads. The proteins tested in each analysis are denoted (<i>Right</i>). The peak fractions from the HD-PTP and STAM2 mixture elution were analyzed and visualized by SDS-PAGE and Coomassie staining (<i>Left</i>). S, size marker; I, input. (C) ITC analysis. Each 0.5 mM STAM2 peptide was titrated into 50 μM HD-PTP(1–361). The <i>K</i>_D value was deduced from curve fittings of the integrated heat per mole of added ligand.</p

Structural analysis of the interaction between HD-PTP and STAM2.

<p>(A) Crystal structure of the HD-PTP(1–361;NAYA)−STAM2(350–370) complex. (<i>Left</i>) The two proteins are presented as ribbon drawings with the labels of secondary structures according to the order of their appearance in the primary sequence. (<i>Right</i>) α-helical wheel representation of the STAM2 fragment shown in the complex structure. STAM2 residues in contact with those of HD-PTP within 4 Å are covered by a gray semicircle. (B-C) Intermolecular hydrophobic interaction (<i>B</i>) and water-mediated hydrogen bonds (<i>C</i>). Shown in sticks are all STAM2 residues together with the HD-PTP residues involved in the complex formation step. Hydrogen bonds mediated by three water molecules (shown in red sphere) are represented as dotted lines. Labeled are the two protein residues participating in the intermolecular interaction.</p

Structural analysis of the binding selectivity of three Bro1 domains.

<p>(A) Structural comparison between HD-PTP(1–361;NAYA)−STAM2(350–370) and Alix(1–359)−CHMP4B(207–224) (PDB code 3C3Q) complexes. The key residues in the intermolecular hydrophobic interactions are shown in sticks and are labeled. The STAM2 and CHMP4B residues are reverse-aligned below, with the vertical lines matching the labeled residues. (B) Neither the Bro1 domain of Alix nor Brox binds STAM2(350–370). ITC measurements were carried out by titrating the 0.5 mM STAM2(350–370) peptide into the 50 μM Alix(1–359) and Brox(1–374) proteins. (C) Thr145 is the key residue in HD-PTP binding to STAM2. The HD-PTP(1–361;NAYA)−STAM2(350–370) structure is superposed on the Alix(1–359)−CHMP4B(207–224) (<i>Left</i>) and the Brox(1–377)−CHMP4B(207–224) (PDB code 3UM3) (<i>Middle</i>) complexes. Lys151 of Alix and Arg145 of Brox cause steric hindrance with STAM2, but Thr145 of HD-PTP does not. (<i>Right</i>) None of the three Bro1 domain residues brings about steric hindrance with CHMP4B. (D) Mutation of Thr145 prevents HD-PTP(1–361) from binding STAM2(350–370). ITC measurements were performed by titrating the 0.5 mM STAM2(350–370) peptide into 50 μM HD-PTP proteins. The left graph showing the interaction between STAM2(350–370) and HD-PTP(1–361) is identical to that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149113#pone.0149113.g001" target="_blank">Fig 1C</a>, which is included in this figure for comparison. (E) CHMP4B(207–224) interacts with HD-PTP(1–361) regardless of the Thr145 residue mutations. The 0.5 mM CHMP4B(207–224) peptide was titrated into 50 μM HD-PTP proteins, and the <i>K</i>_D values were deduced.</p

Nurr1 Represses Tyrosine Hydroxylase Expression via SIRT1 in Human Neural Stem Cells

<div><p>Nurr1 is an orphan nuclear receptor best known for its essential role in the development and maintenance of midbrain dopaminergic (DA) neurons. During DA neurogenesis, Nurr1 directly targets human tyrosine hydroxylase (hTH). Here we investigated this targeting to identify the molecular mechanisms by which Nurr1 regulates DA neurogenesis. We previously cloned the hTH promoter and found three consensus elements for Nurr1 binding: NBRE-A, -B, and -C. In the present study, gel retardation and luciferase assays using hTH constructs showed that Nurr1 preferentially bound to NBRE-A, through which it mediated transcriptional activity. Furthermore, Nurr1 displayed dual-function transcriptional activities depending on the cell type. In DA-like SH-SY5Y cells, Nurr1 dose-dependently stimulated hTH-3174 promoter activity by 7- to 11-fold. However, in the human neural stem cell (hNSC) line HB1.F3, Nurr1 strongly repressed transcription from the same promoter. This repression was relieved by mutation of only the NBRE-A element and by nicotinamide [an inhibitor of class III histone deacetylases (HDACs), such as SIRT1], but not by trichostatin A (an inhibitor of class I and II HDACs). SIRT1 was strongly expressed in the nucleus of HB1.F3 cells, while it was localized in the cytoplasm in SH-SY5Y cells. ChIP assays of HB1.F3 cells showed that Nurr1 overexpression significantly increased the SIRT1 occupancy of the NBRE-A hTH promoter region, while low SIRT1 levels were observed in control cells. In contrast, no significant SIRT1 recruitment was observed in SH-SY5Y cells. These results indicate that differential SIRT1 localization may be involved in hTH gene regulation. Overall, our findings suggest that Nurr1 exists in dual transcriptional complexes, including co-repressor complexes that can be remodeled to become co-activators and can fine-tune hTH gene transcription during human DA neurogenesis.</p></div

AvKTI is <i>O</i>-glycosylated and inhibits trypsin and chymotrypsin.

<p>(A) SDS-PAGE (left) and western blot analysis (right) of purified recombinant AvKTI expressed in baculovirus-infected Sf9 insect cells. Recombinant AvKTI was identified using a His-tag antibody. (B) Glycoprotein staining of AvKTI. Purified AvKTI and control protein samples were subjected to 12% SDS-PAGE (left) and then analyzed by glycoprotein staining (right). Horseradish peroxidase (5 µg), a glycosylated protein, was used as a positive control. Soybean trypsin inhibitor (5 µg), a non-glycosylated protein, was used as a negative control. (C) Enzyme inhibition by AvKTI. Trypsin or chymotrypsin was incubated with increasing amounts of AvKTI, and the residual enzyme activity was then determined (<i>n</i> = 3).</p

AvKTI is a Kunitz-type serine protease inhibitor.

<p>(A) The nucleotide and deduced amino acid sequences of <i>AvKTI</i> cDNA (GenBank accession no. JX844659). The start codon (ATG) is boxed, and the termination codon is indicated with an asterisk. The putative polyadenylation signal is underlined. The predicted signal sequence, a pro-peptide, and the mature peptide are indicated. The characteristic cysteine residues are indicated by squares. The P1 position is marked with a circle. (B) The alignment of the amino acid sequences for mature AvKTI with other known Kunitz-type serine protease inhibitors. The characteristic cysteine residues are shown in bold. The P1 position is marked with an asterisk. The sources of the aligned sequences were <i>A. ventricosus</i> (this study, GenBank accession no. JX844659), <i>Sarcophaga bullata</i> SBPI (P26228), <i>Bombyx mori</i> BmSPI1 (NP_001037044), <i>Anemonia sulcata</i> AsKC1 (Q9TWG0), <i>Haematobia irritans irritans</i> HiTI (AAL87009), <i>Anthopleura aff. xanthogrammica</i> AXPI-I (P81547), <i>Pseudonaja textilis textilis</i> Txln-1 (Q90WA1), <i>Hadrurus gertschi</i> Hg1 (P0C8W3), <i>Bos taurus</i> BPTI (P00974), <i>Pseudonaja textilis textilis</i> Txln-4 (Q90W98), <i>Haplopelma schmidti</i> HWTX-XI (P68425), and <i>Bombus ignitus</i> Bi-KTI (AEM68408). The AvKTI sequence was used as a reference for the identity/similarity (Id/Si) values. (C) Expression of <i>AvKTI</i> in <i>A. ventricosus</i>. Total RNA was isolated from the epidermis, fat body, silk gland, and venom gland of <i>A. ventricosus</i>. RNA was separated by 1.2% formaldehyde agarose gel electrophoresis, transferred onto a nylon membrane, and hybridized with radiolabeled <i>AvKTI</i> cDNA (lower panel). <i>AvKTI</i> transcripts are indicated with an arrow. The ethidium bromide-stained RNA gel shows uniform loading (upper panel).</p

AvKTI inhibits plasmin and elastase, but not factor Xa, thrombin, or tPA.

<p>(A) Inhibitory activity of AvKTI against several enzymes associated with the hemostatic system. Factor Xa, thrombin, or tPA was incubated with increasing amounts of AvKTI, and the residual enzyme activity was determined (<i>n</i> = 3). (B, C) The inhibitory activities of AvKTI against plasmin (B) and neutrophil elastase (C). Plasmin or neutrophil elastase was incubated with increasing amounts of AvKTI, and the residual enzyme activity was determined (<i>n</i> = 3).</p