CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G0/G1 to S-phase
Abstract:ALDH1L1 is a folate-metabolizing enzyme abundant in liver and several other tissues. In human cancers and cell lines derived from malignant tumors, the ALDH1L1 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 fl… Show more
“…However, the effect of ALDH1L1 on purine biosynthesis would be crucial for rapidly proliferating cells but not quiescent cells. This is in agreement with the phenomenon that the ALDH1L1 expression is down-regulated in S-phase of the cell cycle but not in quiescent cells 14 . In the liver, overwhelming majority of cells are in non-proliferating state 51 , which would explain the lack of the effect of the Aldh1l1 KO on purine pools, and this is also in agreement with the 10-formyl-THF accumulation (otherwise 10-formyl-THF would be taken up by the purine biosynthesis).…”
Section: Discussionsupporting
confidence: 92%
“…Furthermore, the enzyme clears one-carbon groups, in the form of CO 2 , from the folate pool which might limit the overall biosynthetic capacity of folate-dependent reactions thus playing a regulatory role 11,13 . In support of such proliferation regulatory role, we have recently shown that ALDH1L1 is down-regulated in S-phase of the cell cycle in NIH 3T3 cells 14 . Several studies also implicated ALDH1L1 as a folate depot, the function likely important for preventing folate degradation 15–17 .…”
ALDH1L1 (10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism highly expressed in liver, metabolizes 10-formyltetrahydrofolate to produce tetrahydrofolate (THF). This reaction might have a regulatory function towards reduced folate pools, de novo purine biosynthesis, and the flux of folate-bound methyl groups. To understand the role of the enzyme in cellular metabolism, Aldh1l1−/− mice were generated using an ES cell clone (C57BL/6N background) from KOMP repository. Though Aldh1l1−/− mice were viable and did not have an apparent phenotype, metabolomic analysis indicated that they had metabolic signs of folate deficiency. Specifically, the intermediate of the histidine degradation pathway and a marker of folate deficiency, formiminoglutamate, was increased more than 15-fold in livers of Aldh1l1−/− mice. At the same time, blood folate levels were not changed and the total folate pool in the liver was decreased by only 20%. A two-fold decrease in glycine and a strong drop in glycine conjugates, a likely result of glycine shortage, were also observed in Aldh1l1−/− mice. Our study indicates that in the absence of ALDH1L1 enzyme, 10-formyl-THF cannot be efficiently metabolized in the liver. This leads to the decrease in THF causing reduced generation of glycine from serine and impaired histidine degradation, two pathways strictly dependent on THF.
“…However, the effect of ALDH1L1 on purine biosynthesis would be crucial for rapidly proliferating cells but not quiescent cells. This is in agreement with the phenomenon that the ALDH1L1 expression is down-regulated in S-phase of the cell cycle but not in quiescent cells 14 . In the liver, overwhelming majority of cells are in non-proliferating state 51 , which would explain the lack of the effect of the Aldh1l1 KO on purine pools, and this is also in agreement with the 10-formyl-THF accumulation (otherwise 10-formyl-THF would be taken up by the purine biosynthesis).…”
Section: Discussionsupporting
confidence: 92%
“…Furthermore, the enzyme clears one-carbon groups, in the form of CO 2 , from the folate pool which might limit the overall biosynthetic capacity of folate-dependent reactions thus playing a regulatory role 11,13 . In support of such proliferation regulatory role, we have recently shown that ALDH1L1 is down-regulated in S-phase of the cell cycle in NIH 3T3 cells 14 . Several studies also implicated ALDH1L1 as a folate depot, the function likely important for preventing folate degradation 15–17 .…”
ALDH1L1 (10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism highly expressed in liver, metabolizes 10-formyltetrahydrofolate to produce tetrahydrofolate (THF). This reaction might have a regulatory function towards reduced folate pools, de novo purine biosynthesis, and the flux of folate-bound methyl groups. To understand the role of the enzyme in cellular metabolism, Aldh1l1−/− mice were generated using an ES cell clone (C57BL/6N background) from KOMP repository. Though Aldh1l1−/− mice were viable and did not have an apparent phenotype, metabolomic analysis indicated that they had metabolic signs of folate deficiency. Specifically, the intermediate of the histidine degradation pathway and a marker of folate deficiency, formiminoglutamate, was increased more than 15-fold in livers of Aldh1l1−/− mice. At the same time, blood folate levels were not changed and the total folate pool in the liver was decreased by only 20%. A two-fold decrease in glycine and a strong drop in glycine conjugates, a likely result of glycine shortage, were also observed in Aldh1l1−/− mice. Our study indicates that in the absence of ALDH1L1 enzyme, 10-formyl-THF cannot be efficiently metabolized in the liver. This leads to the decrease in THF causing reduced generation of glycine from serine and impaired histidine degradation, two pathways strictly dependent on THF.
“…Of note, our previous work implicated CerS6 and C 16 -ceramide elevation in the cellular response to folate stress 11 . Similar to serum starvation experiments, p53 pulled down from cells experiencing folate stress (due to elevation of the folate-stress enzyme ALDH1L1 11 , 27 ) had high levels of bound C 16 -ceramide as well (Supplementary Figure 12 ). Fig.…”
Ceramides are important participants of signal transduction, regulating fundamental cellular processes. Here we report the mechanism for activation of p53 tumor suppressor by C16-ceramide. C16-ceramide tightly binds within the p53 DNA-binding domain (Kd ~ 60 nM), in close vicinity to the Box V motif. This interaction is highly selective toward the ceramide acyl chain length with its C10 atom being proximal to Ser240 and Ser241. Ceramide binding stabilizes p53 and disrupts its complex with E3 ligase MDM2 leading to the p53 accumulation, nuclear translocation and activation of the downstream targets. This mechanism of p53 activation is fundamentally different from the canonical p53 regulation through protein–protein interactions or posttranslational modifications. The discovered mechanism is triggered by serum or folate deprivation implicating it in the cellular response to nutrient/metabolic stress. Our study establishes C16-ceramide as a natural small molecule activating p53 through the direct binding.
“…It is not clear whether these enzymes have overlapping metabolic functions or whether each reaction serves different purposes. ALDH1L1 is suggested as a regulator of the folate metabolism, the function closely associated with the regulation of cellular proliferation [ 10 , 11 , 13 ]. In agreement with such function, ALDH1L1 is strongly and ubiquitously downregulated in many human cancers through the promoter methylation [ 35 ].…”
Section: Discussionmentioning
confidence: 99%
“…Merging a folate-binding domain with the aldehyde dehydrogenase catalytic machinery enabled a novel enzymatic reaction, the oxidation of folate-bound formyl group to CO 2 . Experimental data suggest that this reaction is important for the regulation of the overall cellular folate pool as well as the proliferative capacity of the cell [ 10 , 11 ]. We have recently generated mice with targeted Aldh1l1 knockout and demonstrated that the animals lacking the enzyme display metabolic signs of folate deficiency and have impaired glycine metabolism in the liver [ 12 ].…”
Background
Mitochondrial folate enzyme ALDH1L2 (aldehyde dehydrogenase 1 family member L2) converts 10-formyltetrahydrofolate to tetrahydrofolate and CO2 simultaneously producing NADPH. We have recently reported that the lack of the enzyme due to compound heterozygous mutations was associated with neuro-ichthyotic syndrome in a male patient. Here, we address the role of ALDH1L2 in cellular metabolism and highlight the mechanism by which the enzyme regulates lipid oxidation.
Methods
We generated Aldh1l2 knockout (KO) mouse model, characterized its phenotype, tissue histology, and levels of reduced folate pools and applied untargeted metabolomics to determine metabolic changes in the liver, pancreas, and plasma caused by the enzyme loss. We have also used NanoString Mouse Inflammation V2 Code Set to analyze inflammatory gene expression and evaluate the role of ALDH1L2 in the regulation of inflammatory pathways.
Results
Both male and female Aldh1l2 KO mice were viable and did not show an apparent phenotype. However, H&E and Oil Red O staining revealed the accumulation of lipid vesicles localized between the central veins and portal triads in the liver of Aldh1l2-/- male mice indicating abnormal lipid metabolism. The metabolomic analysis showed vastly changed metabotypes in the liver and plasma in these mice suggesting channeling of fatty acids away from β-oxidation. Specifically, drastically increased plasma acylcarnitine and acylglycine conjugates were indicative of impaired β-oxidation in the liver. Our metabolomics data further showed that mechanistically, the regulation of lipid metabolism by ALDH1L2 is linked to coenzyme A biosynthesis through the following steps. ALDH1L2 enables sufficient NADPH production in mitochondria to maintain high levels of glutathione, which in turn is required to support high levels of cysteine, the coenzyme A precursor. As the final outcome, the deregulation of lipid metabolism due to ALDH1L2 loss led to decreased ATP levels in mitochondria.
Conclusions
The ALDH1L2 function is important for CoA-dependent pathways including β-oxidation, TCA cycle, and bile acid biosynthesis. The role of ALDH1L2 in the lipid metabolism explains why the loss of this enzyme is associated with neuro-cutaneous diseases. On a broader scale, our study links folate metabolism to the regulation of lipid homeostasis and the energy balance in the cell.
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