Proteomic analysis of ubiquitination substrates reveals a CTLH E3 ligase complex‐dependent regulation of glycolysis

Maitland, Matthew E. R.; Kuljanin, Miljan; Wang, Xu; Lajoie, Gilles; Schild-Poulter, Caroline

doi:10.1096/fj.202100664r

Cited by 26 publications

(39 citation statements)

References 59 publications

(214 reference statements)

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“…Since the establishment of the complex as an E3 ligase, discoveries of putative or in vitro confirmed ubiquitination targets of the mammalian complex have come to light, such as transcription factor HBP1, nuclear matrix protein Lamin B2, energy regulator AMPK, glycolysis enzymes PKM2 and LDHA, and its own subunit muskelin [ 16 , 22 , 93 , 94 , 95 ]. These findings have implicated the GID/CTLH complex in a number of critical functions in different organisms, such as zygote development in D. melanogaster , nodule organogenesis in Lotus japonicus plants, organismal lifespan in Caenorhabditis elegans , neurodevelopment in X. laevis , and erythrocyte differentiation in mammals ( Figure 5 ) [ 45 , 47 , 52 , 53 , 93 , 94 , 96 , 97 , 98 ].…”

Section: Functions and Ubiquitination Targets Of The Ctlh Complex Fro...mentioning

confidence: 99%

“…Despite the overall conservation between the yeast and mammalian complexes, the mammalian (human or mouse) complex does not regulate gluconeogenesis, and does not ubiquitinate human Fbp1, likely because, as already mentioned, the degrons are not the same [ 16 , 134 , 135 ]. Instead, the human complex has been demonstrated to inhibit the opposite pathway, glycolysis, by regulating the ubiquitination of enzymes PKM and LDHA [ 95 ]. Instead of degradation, however, PKM and LDHA activities were increased in RanBP9-deficient cells, and global proteomic and ubiquitinome analyses suggested that non-degradative ubiquitination by the complex may be prevalent [ 95 ].…”

Section: Functions and Ubiquitination Targets Of The Ctlh Complex Fro...mentioning

confidence: 99%

“…Instead, the human complex has been demonstrated to inhibit the opposite pathway, glycolysis, by regulating the ubiquitination of enzymes PKM and LDHA [ 95 ]. Instead of degradation, however, PKM and LDHA activities were increased in RanBP9-deficient cells, and global proteomic and ubiquitinome analyses suggested that non-degradative ubiquitination by the complex may be prevalent [ 95 ]. A corresponding increased glycolytic flux and altered metabolism was observed in RanBP9-deficient HeLa cells [ 95 ], a hallmark of cancer cells which enables them to survive as highly proliferating cells [ 136 ].…”

Section: Functions and Ubiquitination Targets Of The Ctlh Complex Fro...mentioning

confidence: 99%

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Structural and Functional Insights into GID/CTLH E3 Ligase Complexes

Maitland

Lajoie

Shaw

et al. 2022

IJMS

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Multi-subunit E3 ligases facilitate ubiquitin transfer by coordinating various substrate receptor subunits with a single catalytic center. Small molecules inducing targeted protein degradation have exploited such complexes, proving successful as therapeutics against previously undruggable targets. The C-terminal to LisH (CTLH) complex, also called the glucose-induced degradation deficient (GID) complex, is a multi-subunit E3 ligase complex highly conserved from Saccharomyces cerevisiae to humans, with roles in fundamental pathways controlling homeostasis and development in several species. However, we are only beginning to understand its mechanistic basis. Here, we review the literature of the CTLH complex from all organisms and place previous findings on individual subunits into context with recent breakthroughs on its structure and function.

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Section: Functions and Ubiquitination Targets Of The Ctlh Complex Fro...mentioning

confidence: 99%

Section: Functions and Ubiquitination Targets Of The Ctlh Complex Fro...mentioning

confidence: 99%

Section: Functions and Ubiquitination Targets Of The Ctlh Complex Fro...mentioning

confidence: 99%

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Structural and Functional Insights into GID/CTLH E3 Ligase Complexes

Maitland

Lajoie

Shaw

et al. 2022

IJMS

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“…However, the regulation of PKM2 by ubiquitination-related processes is more diverse. For instance, U-box E3 ligase CHIP reduces aerobic glycolysis by ubiquitylating and degrading PKM2 in ovarian carcinoma (OV) cells, while CTLH ubiquitylates PKM2 and lactate dehydrogenase A (LDHA) in a non-degradative pathway, thereby inhibiting glycolysis ( 19 ). On the contrary, USP7/USP20 increases the stability of PKM2 proteins by directly interacting with PKM2 ( 20 ).…”

Section: Discussionmentioning

confidence: 99%

DNAAF5 promotes hepatocellular carcinoma malignant progression by recruiting USP39 to improve PFKL protein stability

Liu

Sun

et al. 2022

Front. Oncol.

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PurposesDynein axonemal assembly factor 5 (DNAAF5) is the transcription factor of regulating the cytoskeleton and hydrodynamic protein complex assembly, however, it was not well elucidated in the malignant progression of hepatocellular carcinoma (HCC).MethodsWe investigated the role of DNAAF5 in hepatocellular carcinoma by using multiple groups of clinical tissues combined with data from the TCGA database. Then we overexpressed DNAAF5 in hepatocellular carcinoma tumor tissues, which correlates with poor patient survival outcomes. Furthermore, we constructed stable cell lines of HCC cells to confirm the cancer-promoting effects of DNAAF5 in hepatocellular carcinoma. To explore the mechanisms of DNAAF5, transcriptome sequencing combined with mass spectrometry was also performed, which showed that DNAAF5 affects its downstream signaling pathway by interacting with PFKL and that DNAAF5 regulates PFKL protein stability by recruiting the deubiquitination protein, USP39. To corroborate these findings, the same series of tissue microarrays were used to confirm correlations between DNAAF5 and PFKL expressions. In animal experiments, DNAAF5 also promoted the proliferation of HCC cells.ResultsWe found that DNAAF5 expressions were markedly higher in HCC tissues, compared to the adjacent normal tissues. Increased levels of DNAAF5 were associated with significantly worse prognostic outcomes for HCC patients. Cell function experiments showed that HCC cells of overexpressing DNAAF5 exhibited faster proliferation rates, stronger clone formation abilities and higher drug resistance rates. However, tumor cell proliferation rates and colony formation were significantly decreased after DNAAF5 knockout, accompanied by an increase in sensitivity to sorafenib. In addition, the results of our study showed that DNAAF5 accelerates PFKL protein deubiquitination by recruiting USP39 in HCC cells. Furthermore, The overexpression of DNAAF5 could promote HCC cell proliferation in vivo and in vitro, whereas USP39 knockdown inhibited this effect. Overall, DNAAF5 serves as a scaffold protein to recruit USP39 to form a ternary complex by directly binding the PFKL protein, thereby improving the stability of the latter, which promotes the malignant process of hepatocellular carcinoma.ConclusionsThese findings revealed DNAAF5 was negatively correlated with the prognosis of patients with hepatocellular carcinoma. It underlying mechanism showed that DNAAF5 directly binds PFKL and recruits the deubiquitinated protein (USP39) to improve the stability of the PFKL protein, thus enhancing abnormal glycolysis in HCC cells.

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“…Samples for western blots were prepared as described above, with 35 μg of protein loaded per well to 10% SDS-PAGE gels. Samples for mass spectrometry analysis for global proteomics were prepared exactly as described previously, but following the digestion and acidification, peptides were desalted using Pierce TM C18 Spin Tips (Cat# 84850)[67]. Samples were then dried in a Speed vacuum, resuspended in 0.1% formic acid, and quantified by BCA assay.…”

Section: Methodsmentioning

confidence: 99%

Noncannonical functions of Ku may underlie essentiality in human cells

Kelly

Parmar

Bayat

et al. 2022

Preprint

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The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair but has also been involved in other cellular functions like telomere regulation and maintenance, in which Ku’s role is not fully characterized. It was previously reported that knockout of Ku80 in a human cell line results in lethality, but the underlying cause of Ku essentiality in human cells has yet to be fully explored. Here, we established conditional Ku70 knockout cells to study the essentiality of Ku70 function. Endogenous Ku70 knockout was achieved using CRISPR/Cas9 editing in cells where Ku70 expression was maintained through integration of an HA-tagged Ku70 cDNA under the control of a doxycycline-inducible promoter. Ku70 conditional knockout cell lines were identified via western blotting, and edits were validated by Sanger sequencing. We visually observed cell death in Ku70 knockout cells 8-10 days post Ku70-HA depletion, and loss of viability following Ku depletion was quantified using crystal violet assays. Interestingly, assessment of telomere length in Ku70 knockout cells using telomere restriction fragment analyses did not reveal any changes in average telomere length following Ku70-HA depletion. Immunofluorescence analysis used to assess γH2AX foci accumulation as a measure of double-stranded DNA breaks following Ku70-HA depletion allowed us to conclude that increased DNA damage is not the driving cause of loss of cell viability. Finally, quantitative proteome analysis of Ku70 knockout cells following Ku70-HA depletion identified a number of pathways and proteins that are significantly dysregulated following the loss of Ku70, including processes which Ku function has been previously associated with such as cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Overall, this conditional Ku70 knockout system reveals that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in other cellular processes beyond DNA repair and telomere maintenance to maintain cell viability.Author SummaryThe Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair, where it acts as a scaffold for other repair factors needed to process double-stranded DNA breaks. Ku has also been involved in other cellular functions like telomere regulation and maintenance, in which Ku’s role is not fully characterized. Previous data suggest that while loss of Ku70/80 can be tolerated in other species, Ku is essential to humans. We have established a conditional Ku70 knockout in HEK293 cells to evaluate the basis of Ku essentiality in human cells. While we observed loss of cell viability upon Ku depletion, we did not observe significant changes in telomere length nor did we record lethal levels of DNA damage upon loss of Ku, suggesting that the reasons for the loss of viability is not linked to the functions of Ku in DNA repair or at telomeres. Analysis of global proteome changes following Ku70 depletion revealed dysregulations of several cellular pathways including cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Our study reveals that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in cellular processes beyond DNA repair and telomere maintenance to maintain cell viability.

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Proteomic analysis of ubiquitination substrates reveals a CTLH E3 ligase complex‐dependent regulation of glycolysis

Abstract: This is an open access article under the terms of the Creat ive Commo ns Attri butio n-NonCo mmerc ial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Cited by 26 publications

References 59 publications

Structural and Functional Insights into GID/CTLH E3 Ligase Complexes

Structural and Functional Insights into GID/CTLH E3 Ligase Complexes

DNAAF5 promotes hepatocellular carcinoma malignant progression by recruiting USP39 to improve PFKL protein stability

Noncannonical functions of Ku may underlie essentiality in human cells

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