CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G₀/G₁ to S-phase

<div><p>ALDH1L1 is a folate-metabolizing enzyme abundant in liver and several other tissues. In human cancers and cell lines derived from malignant tumors, the <i>ALDH1L1</i> gene is commonly silenced through the promoter methylation. It was suggested that ALDH1L1 limits proliferation capacity of the cell and thus functions as putative tumor suppressor. In contrast to cancer cells, mouse cell lines NIH3T3 and AML12 do express the ALDH1L1 protein. In the present study, we show that the levels of ALDH1L1 in these cell lines fluctuate throughout the cell cycle. During S-phase, ALDH1L1 is markedly down regulated at the protein level. As the cell cultures become confluent and cells experience increased contact inhibition, ALDH1L1 accumulates in the cells. In agreement with this finding, NIH3T3 cells arrested in G₁/S-phase by a thymidine block completely lose the ALDH1L1 protein. Treatment with the proteasome inhibitor MG-132 prevents such loss in proliferating NIH3T3 cells, suggesting the proteasomal degradation of the ALDH1L1 protein. The co-localization of ALDH1L1 with proteasomes, demonstrated by confocal microscopy, supports this mechanism. We further show that ALDH1L1 interacts with the chaperone-dependent E3 ligase CHIP, which plays a key role in the ALDH1L1 ubiquitination and degradation. In NIH3T3 cells, silencing of CHIP by siRNA halts, while transient expression of CHIP promotes, the ALDH1L1 loss. The downregulation of ALDH1L1 is associated with the accumulation of the ALDH1L1 substrate 10-formyltetrahydrofolate, which is required for <i>de novo</i> purine biosynthesis, a key pathway activated in S-phase. Overall, our data indicate that CHIP-mediated proteasomal degradation of ALDH1L1 facilitates cellular proliferation.</p></div

ALDH1L1 is ubiquitinated in NIH3T3 cells.

<p><b>A</b>, ALDH1L1 pulled-down from NIH3T3 cell lysates using ALDH1L1-specific antibody and protein A beads; elution with glycine buffer (<i>lane 1</i>), followed by elution with SDS-PAGE loading buffer (<i>lane 2</i>). Proteins were resolved on a 7.5% SDS-PAGE gel followed by Western blot assay with ubiquitin-specific antibody (<i>left panel</i>) or ALDH1L1-specific antibody (<i>right panel</i>). Lane <i>St</i> is purified recombinant ALDH1L1. <b>B</b>, ALDH1L1 was immunoprecipitated from NIH3T3 cell lysates using an ALDH1L1-specific antibody and Protein A Magnetic beads; samples were resolved on a 7.5% SDS-PAGE followed by Western blot assay with anti-ubiquitin monoclonal antibody. Cells were harvested at different time points after splitting (as indicated); lysates were treated with deubiquitinase inhibitor (4.0 μM recombinant human ubiquitin aldehyde C-terminal derivative) prior to immunoprecipitation. After immunoprecipitation, eluates were treated with deubiquitinase (200 nM of recombinant human USP2 catalytic domain); <i>control</i>, untreated lysates. <b>C</b>, ALDH1L1 was immunoprecipitated from NIH3T3 cells as in <b>B</b> and treated with USP7. Cells were treated with 10 μM MG-132 for 4 h before the pull-down. After treatment with USP7, we have repeated the pull-down with ALDH1L1-specific antibody and detected ubiquitinated species as in <b>B</b>.</p

Omitting components of ubiquitination machinery prevents <i>in vitro</i> ALDH1L1 ubiquitination.

<p>Purified ALDH1L1 (1.1 μg) was incubated for 1 h with the <i>in vitro</i> ubiquitination kit that included all components or with omission of ATP, E1, E2 or E3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199699#sec002" target="_blank">Materials and methods</a>). Reaction products were resolved by 7.5% SDS-PAGE followed by Western blot assay with ALDH1L1-specific antibody and Ubiquitin (Ub)-specific antibody. <i>St</i>, pre-stained molecular mass protein standards (numbers on the right indicates standard molecular masses, kDa). Experiment was performed three times with the same outcome.</p

Levels of ALDH1L1 protein in NIH3T3 cells arrested at difference phases.

<p><b>A</b>, NIH3T3 cells arrested in G₀/G₁ (serum starvation), S-phase (double thymidine block) or G₂/M (double thymidine block and nocodazole treatment) phase. Asynchronous cells shown as a control. Numbers on the panels indicate distribution of cells between cell cycle phases. Fitted peaks are: <i>Blue</i>, calculated G₀/G₁ phase; <i>yellow</i>, S phase; <i>green</i>, G₂/M phase. Cell cycle data were analyzed using FlowJo software. <b>B</b>, Western blot assay of ALDH1L1 in NIH3T3 cells arrested in indicated phase (20 μg of total cell lysate was loaded in each lane). Actin is shown as loading control. Arrows indicate molecular weight standards (St). Numbers show ALDH1L1 band intensity (arbitrary densitometry units) normalized to actin. Experiments were performed three times.</p

ALDH1L1 interacts with E3 ligase CHIP and CHIP-assisting chaperone HSP90 <i>in vivo</i>.

<p><b>A</b>, schematic depicting the pull-down assay on a folate affinity column. <b>B</b>, endogenous ALDH1L1 interacts strongly with immobilized folinic acid (after loading NIH3T3 cell lysate on the affinity column, the following eluted fractions were collected and analyzed by SDS-PAGE/Western blot assay with ALDH1L1-specific antibody: <i>lane 1</i>, washing buffer; <i>lane 2</i>, 0.5 M KCl; <i>lane 3</i>, 1.0 M KCl; <i>lane 4</i>, 2.0 KCl; <i>lane 5</i>, 5 mM folic acid in 2.0 KCl; <i>lane 6</i>, 20 mM folic acid in 2.0 M KCl; <i>lane 7</i>, purified ALDH1L1 standard; <i>St</i>, molecular masses standards (indicated by numbers in kDa on the <i>left</i>). <b>C</b>, samples as in <b>B</b> probed with CHIP-specific antibody. <b>D</b>, samples as in <i>B</i> probed with HSP90-specific antibody. <b>E</b>, Immunoprecipitation of NIH3T3 cell lysate with ALDH1L1-specific (<i>left panels</i>) or HSP90-specific (<i>right panels</i>) antibody followed by SDS-PAGE/Western blot assay with ALDH1L1-specific, HSP90-specific and CHIP-specific antibodies. In each experiment (ALDH1L1 or HSP90 pull-down) the same blot was stripped twice and reprobed. Numbers indicate molecular masses (kDa) for standards.</p

<i>In vitro</i> ubiquitination of purified ALDH1L1.

<p><b>A</b>, purified ALDH1L1 (1.1 μg) was ubiquitinated using the <i>in vitro</i> ubiquitination kit that included CHIP E3 ligase (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199699#sec002" target="_blank">Materials and methods</a>). Reaction products were resolved by 7.5% SDS-PAGE followed by Western blot assay with ALDH1L1-specific antibody (<i>left panel</i>) or Ub-specific antibody (<i>right panel</i>). <i>Arrows</i> indicate positions of 95 kDa and 130 kDa pre-stained molecular mass protein standards. <b>B</b>, lanes 1–4, increased amount of ALDH1L1 (0.6, 1.2, 2.4 and 4.8 μg) were subjected to <i>in vitro</i> ubiquitination with NIH3T3 cell lysate; ubiquitination with CHIP was the positive control. <b>C</b>, negative control for panel <b>B</b>, lanes 1–4, untreated purified ALDH1L1 (0.6, 1.2, 2.4 and 4.8 μg) prior to ubiquitination. Each experiment was repeated at least three times. Molecular mass standards (<i>St</i>) are the same for all panels.</p

Levels of ALDH1L1 protein fluctuate in proliferating NIH3T3 cells.

<p><b>A</b>, schematic depicting the ALDH1L1 metabolic pathway (the enzyme converts 10-formyl-THF to THF and CO₂ simultaneously producing NADPH; this pathway competes with <i>de novo</i> purine biosynthesis for the same substrate, 10-formyl-THF). <b>B-C</b>, levels of ALDH1L1 in proliferating NIH3T3 cells during the cell cycle progression. Time points on graphs correspond to those on the blot and indicate hours after splitting confluent cell culture. Quantification of ALDH1L1 bands (arbitrary densitometry units) normalized to actin is shown. Cell cycle data were analyzed using ModFit software.</p

Proteins relevant to the ubiquitin-proteasome pathway pulled down on ALDH1L1.

<p>Proteins relevant to the ubiquitin-proteasome pathway pulled down on ALDH1L1.</p