The Demethylase Activity of FTO (Fat Mass and Obesity Associated Protein) Is Required for Preadipocyte Differentiation

<div><p>FTO (fat mass and obesity associated gene) was genetically identified to be associated with body mass index (BMI), presumably through functional regulation of energy homeostasis. However, the cellular and molecular mechanisms by which FTO functions remain largely unknown. Using 3T3-L1 preadipocyte as a model to study the role of FTO in adipogenesis, we demonstrated that FTO is functionally required for 3T3-L1 differentiation. FTO knock-down with siRNA inhibited preadipocyte differentiation, whereas ectopic over-expression of FTO enhanced the process. The demethylase activity of FTO is required for differentiation. Level of N⁶-methyladenosine (m⁶A) is decreased in cells over-expressing FTO. In contrast, overexpression of R96Q, a FTO missense mutant lack of demethylase activity, had no effect on cellular m⁶A level and impeded differentiation. Treatment with Rosiglitazone, a PPARγ agonist, could overcome the differentiation inhibition imposed by R96Q mutant, suggesting the effect of FTO is mediated through PPARγ.</p></div

Confirmation of microarray findings with RT-qPCR.

<p>(A) Three genes from the focal adhesion, cytoskeleton and ECM gene set (Lamc1, Dock1 and Thbs2) were selected and confirmed to be decreased 6hr following adipogenic induction in FTO knock-down cells. (B) Three PPARγ target genes (CD36, Fabp5 and Adiponectin) were selected and verified to be down-regulated 3 days following adipogenic induction in siFTO transfected cells. All gene expression data are represented as mean ± SD of triplicates. * stands for p<0.05, ** stands for p<0.01 and *** stands for p<0.001in Student’s t-test (siFTO versus siCtrl).</p

Measurement of binding affinity between LidA and 9 kinds of Rabs by ITC.

<p>(A–G) Raw ITC data. Top panel: twenty injections of Rab2 (A), Rab4 (B), Rab6 (C), Rab7 (D), Rab9 (E), Rab11 (F), Rab20 (G) solutions were titrated into LidA(188-580) solution in ITC cell. The area of each injection peak corresponds to the total heat released for that injection. Bottom panel: the binding isotherm for these Rabs and LidA(188-580) interaction, the integrated heat is plotted against the stoichiometry of 1∶1, data fitting revealed a binding affinity as shown. (H) Raw ITC data. Top panel: twenty injections of Rab22 (H) solutions were titrated into LidA(FL) solution ITC cell, the experiment conditions was exactly the same as the top one unless the LidA is full-length. The <i>K_D</i> values of these Rabs and LidA are shown.</p

Structural Insights into a Unique <em>Legionella pneumophila</em> Effector LidA Recognizing Both GDP and GTP Bound Rab1 in Their Active State

<div><p>The intracellular pathogen <em>Legionella pneumophila</em> hijacks the endoplasmic reticulum (ER)-derived vesicles to create an organelle designated <em>Legionella</em>-containing vacuole (LCV) required for bacterial replication. Maturation of the LCV involved acquisition of Rab1, which is mediated by the bacterial effector protein SidM/DrrA. SidM/DrrA is a bifunctional enzyme having the activity of both Rab1-specific GDP dissociation inhibitor (GDI) displacement factor (GDF) and guanine nucleotide exchange factor (GEF). LidA, another Rab1-interacting bacterial effector protein, was reported to promote SidM/DrrA-mediated recruitment of Rab1 to the LCV as well. Here we report the crystal structures of LidA complexes with GDP- and GTP-bound Rab1 respectively. Structural comparison revealed that GDP-Rab1 bound by LidA exhibits an active and nearly identical conformation with that of GTP-Rab1, suggesting that LidA can disrupt the switch function of Rab1 and render it persistently active. As with GTP, LidA maintains GDP-Rab1 in the active conformation through interaction with its two conserved switch regions. Consistent with the structural observations, biochemical assays showed that LidA binds to GDP- and GTP-Rab1 equally well with an affinity approximately 7.5 nM. We propose that the tight interaction with Rab1 allows LidA to facilitate SidM/DrrA-catalyzed release of Rab1 from GDIs. Taken together, our results support a unique mechanism by which a bacterial effector protein regulates Rab1 recycling.</p> </div

Measurement of binding affinity between LidA and GTP-bound or GDP-bound Rab1 by ITC.

<p>(A) Raw ITC data. Top panel: twenty injections of GTP-bound Rab1(Q70L) solutions were titrated into LidA(188-580) solution in ITC cell. The area of each injection peak corresponds to the total heat released for that injection. Bottom panel: the binding isotherm for GTP-bound Rab1(Q70L) and LidA(188-580) interaction, the integrated heat is plotted against the stoichiometry of 1∶1, data fitting revealed a binding affinity of 7.5 nM. (B) Top panel: twenty injections of GDP-bound Rab1(S25N) solutions were titrated into LidA(188-580) solution in ITC cell. The area of each injection peak corresponds to the total heat released for that injection. Bottom panel: the binding isotherm for GDP-bound Rab1(S25N) and LidA(188-580) interaction, the integrated heat is plotted against the stoichiometry of 1∶1, data fitting revealed a binding affinity of 7.6 nM. Both titrations were performed in the absence of added Mg²⁺ and GDP/GTP.</p

Interaction of Rab1-LidA shares similar features with that of Rab4-Rabenosyn-5.

<p>(A) Superimposition of Rab1(S25N)-LidA(224-559) complex with Rab4-Rabenosyn-5 complex (PDB ID code 1Z0K). The superimposition of the similar anti-parallel coiled-coil regions is shown. Rab1(S25N) is in gray and LidA is in yellow orange; Rab4 is in light blue and Rabenosyn-5 is in green. (B and C) Similar interfaces of the two complexes. (B) represents the detailed interactions between Rab1(S25N) and LidA. (C) represents the detailed interactions between Rab4 and Rabenosyn-5. The residues involved in interaction are shown as stick representations and denoted by black labels.</p

The demethylase activity of FTO is required during 3T3-L1 differentiation.

<p>Two lines of 3T3-L1 stably expressing wt FTO (hFTO-3 and hFTO-4) or R96Q (R96Q-2 and R96Q-4) were generated. (A) The protein expression level was confirmed by Western-blot using FTO and Myc antibodies, with β-actin as a loading control. (B) The sub-cellular localization of hFTO-4 or R96Q-4 was determined with confocal fluorescence imaging. Scale bars of 10μm are shown in the bottom row of images. (C) 3T3-L1differentiation was enhanced by over-expression of wt FTO and inhibited by R96Q. Level of differentiation was measured by extent of Oil Red-O staining at day 6 post differentiation induction (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133788#sec002" target="_blank">materials and methods</a>). Demethylase activities of wt hFTO-4 and R96Q-4 in 3T3-L1 cells were determined by dot blotting (D) and quantified by Grayscale analysis with ImageJ software (E). Data are represented as means± SD of four replicates. * stands for p<0.05 and** stands for p<0.01in Student’s t-test.</p

13 kinds of Rab GTPase family members could be recognized by LidA <i>in vitro</i>.

<p>(A) Sequence alignment of fifteen different Rabs. The LidA-interacting residues in Rab1 are depicted by triangles. The two sequences underneath the black dashed lines correspond to the members unbound to LidA. (B) The GST-pull down of LidA(FL) by beads-immobilized 15 kinds of GST-Rab family members, Rab20 was His-tagged for exceptional.</p

Interaction interfaces between LidA and Rab1.

<p>(A) Rab1(S25N) is shown with electrostatic surface potentials. Blue and red represent the positive and negative charge potential, respectively. The extensive interactions between Rab1(S25N) switch regions and LidA index finger (smude), middle finger (wheat) and ring finger (light teal) are shown. This Figure is in the same orientation as the right panel of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002528#ppat-1002528-g002" target="_blank">Figure 2B</a>. (B) The detailed interactions between Rab1 switch I (magentas), switch II (marine), P-loop (limon) and LidA middle finger and ring finger. (C) The detailed interactions between Rab1 switch II and LidA index finger. The interacting residues of Rab1 and LidA are shown in sticks, residues labels are color-coded as corresponding cartoon chains, respectively. Hydrogen bonds are indicated by black dashed lines. Salt bridge is indicated by red dashed lines.</p

Rosiglitazone rescued adipogenesis inhibition in 3T3-L1 cells expressing R96Q.

<p>(A) Oil Red-O staining of 3T3-L1 cell lines treated with MDI (top and middle row), or MDI plus Rosiglitazone (bottom row). Scale bars are 500μm in 40x images and 50μm in 400x images respectively (left column). (B) Level of differentiation in 3T3-L1 lines expressing wt hFTO-4 or R96Q-4, measured by the extent of Oil Red-O staining. Expression levels of five PPARγ target genes (aP-2, Fabp5, Adiponectin, Perilipin and CD36) were evaluated in 3T3-L1 over expressing wt hFTO-4 (C) or R96Q-4(D) at day 3 post differentiation induction. All gene expression data are represented as mean ± SD of triplicates.* stands for p<0.05, ** stands for p<0.01 and *** stands for p<0.001 in Student’s t-test (Rosig versus DMSO)</p