The NEDD4-2s residual C2 domain is sufficient to target some isolated HECT domains to Gag.

<p>(<b>A</b>) Residual C2 domain/HECT domain fusion proteins differ in their ability to induce the ubiquitination of Z_WT Gag, and to associate with Z_WT VLP. (<b>B</b>) The ubiquitination of Z_WT Gag by the isolated HECT domain of HECW2 and its association with Z_WT VLP were dependent on the presence of the NEDD4-2s residual C2 domain. 293T cells were transfected with the Z_WT Gag construct (2 µg) and empty vector, or vectors expressing FLAG-tagged versions of NEDD4-2s (2 µg) or of the indicated HECT domain constructs (6 µg each).</p

The residual C2 domain of NEDD4-2s functions as a Gag-targeting module.

<p>(<b>A</b>) NEDD4-2s sequences upstream of the first WW domain are sufficient to induce the ubiquitination of an L domain-independent HIV-1 Gag construct (Z_WT) by WWP1, and the association of WWP1 with Z_WT VLP. (<b>B</b>) Human ITCH and yeast Rsp5 ubiquitinate Z_WT Gag and associate with Z_WT VLP when their C2 domain is replaced by the NEDD4-2s residual C2 domain. 293T cells were transfected with the Z_WT Gag construct (2 µg) and vectors (2 µg each) expressing the indicated FLAG-tagged parental or chimeric ubiquitin ligase constructs, or the empty vector. VLP were analyzed by Western blotting with anti-CA to detect unmodified and ubiquitinated versions of Z_WT Gag, and with anti-FLAG to detect the incorporation of ubiquitin ligase constructs into VLP. The cell lysates were also examined with anti-FLAG.</p

Rescue of HIV-1_ΔPTAPP by isolated HECT domains fused to the NEDD4-2s residual C2 domain.

<p>293T cells were transfected with HXBH10_ΔPTAPP (0.5 µg) and empty vector, or vectors expressing FLAG-tagged versions of NEDD4-2s (2 µg) or of the indicated fusion proteins (6 µg each). Virion pellets and the cell lysates were analyzed by Western blotting to detect Gag products and FLAG-tagged proteins as indicated.</p

The residual C2 domain of NEDD4-2s is sufficient to transfer the ability to rescue HIV-1_ΔPTAPP.

<p>(<b>A</b>) The transfer of NEDD4-2s sequences upstream of the first WW domain to WWP1 is sufficient to confer activity in the ΔPTAPP rescue assay. (<b>B</b>) Human ITCH and yeast Rsp5 potently rescue HXBH10_ΔPTAPP when their C2 domain is replaced by the residual C2 domain of NEDD4-2s. 293T cells were transfected with HXBH10_ΔPTAPP (1 µg) and vectors (2 µg each) expressing the indicated FLAG-tagged parental or chimeric ubiquitin ligase constructs, or the empty vector. Virion pellets and the cell lysates were analyzed by Western blotting to detect Gag, Gag cleavage products, and FLAG-tagged proteins as indicated.</p

The isolated HECT domain of NEDD4-2s rescues HIV-1_ΔPTAPP when targeted to Gag.

<p>293T cells were transfected with HXBH10_ΔPTAPP (1 µg) and empty vector, or vectors (2 µg each) expressing FLAG-tagged WT NEDD4-2s, a version lacking the residual C2 domain (N_Δ1-31), or versions that have portions of NEDD4-2s replaced by the HIV-1 Gag-binding protein CypA as illustrated. Virion pellets and the cell lysates were analyzed by Western blotting to detect Gag, Gag cleavage products, and FLAG-tagged proteins as indicated.</p

Induction of Gag ubiquitination is not sufficient to rescue of HIV-1 release.

<p>(<b>A</b>) The isolated HECT domain of NEDD4-2s rescues HXBH10_ΔPTAPP when targeted to Gag via CypA (lane 2), whereas the isolated HECT domains of SMURF1, E6AP, or HERC6 do not (lanes 3–5). (<b>B</b>) The isolated HECT domains of NEDD4-2s (lane 2) and SMURF1 (lane 3) induced comparable levels of Gag ubiquitination when fused to CypA. 293T cells were transfected with HXBH10_ΔPTAPP (0.5 µg) or Z_WT (2 µg) and empty vector, or vectors expressing the indicated FLAG/HA-tagged CypA-HECT domain fusion proteins or FLAG-tagged NEDD4-2s (2 µg each). Gag proteins were detected with anti-CA, and the CypA-HECT fusion proteins were detected with anti-FLAG or anti-HA, as indicated.</p

Generation of transgenic rats.

<p>The transgene construct, pTet-shIR (A), contains two expression cassettes: One expresses shRNA against the insulin receptor (shIR) under the control of the human H1 promoter carrying a tetracycline operator (tetO) sequence. The second cassette consists of a tetracycline repressor (tetR) cDNA followed by a polyadenylation site (pA) and is driven by the CAGGS promoter. An RNase protection assay (RPA) probe was designed to bind to the loop and antisense strand of the hairpin. Primers TetRfor and TetRrev (arrowheads) were used for genotyping of rats. (B) Expression of the shRNA was detected by RPA in 20 µg of total RNA isolated from white adipose tissue (WAT) of wild-type (WT) and transgenic (Tet14 and Tet29) rats treated with doxycycline (DOX, 2 mg/mL) for 4 days. M: RNA Decade marker; Y: yeast RNA; Y-: yeast RNA without RNase digestion; nt: nucleotides. (C) Expression of insulin receptor (IR), tetracycline repressor (tetR), and ß-actin were detected by Western blot in 20 µg of WAT, brain and heart protein from the same rats.</p

Tissue-specificity of IR knockdown.

<p>Different tissues of both transgenic lines, Tet14 and Tet29, and WT were analysed for expression of IR by Western blot after treatment with doxycycline (2 mg/mL for 4 days). Quantification of the protein band intensities was carried out by the program TINA 2.08e and percentages of reduction of expression were calculated (WT, 100%). WAT, white adipose tissue; BAT, brown adipose tissue.</p

Chronic <i>diabetes mellitus</i> model in rats.

<p>Tet29 rats were treated with 5 µg/mL of doxycycline (DOX) for 8 days and with 1 µg/mL thereafter for in total 40 days. Blood glucose (A), body weight (B, BW), and drinking volume (C) were measured every second day; plasma insulin (D) was quantified by ELISA before and in the second week of DOX treatment, and urinary volume (E) and albumin (F) were determined weekly in the last three weeks. * p<0.05; ** p<0.01; *** p<0.001 <i>vs.</i> untreated Tet29 rats (Student's <i>t</i>-test).</p

Lack of toxicity of transgenic shRNA expression.

<p>(A) Tet29 rats were treated with doxycycline (DOX) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005124#pone-0005124-g003" target="_blank">Figure 3</a>. At the end of the experiment, 20 µg of total RNA from liver was used in an RPA for detection of mir122. M: RNA Decade marker; Y: yeast RNA; Y-: yeast RNA without RNase digestion; nt: nucleotides. Protein kinase R (PKR) expression was used as marker for interferon response in white adipose tissue of acutely (B, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005124#pone-0005124-g001" target="_blank">Figure 1</a>) or in heart of chronically (C, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005124#pone-0005124-g003" target="_blank">Figure 3</a>) DOX treated wild-type (WT), Tet14, and Tet29 rats. PKR was detected by Western blot in 20 µg protein; an unspecific band (indicated by *) was used as loading control. HEKi: positive control, 20 µg of protein of HEK cells treated with 1 µM interferon-α2a for 24 hours.</p