Lipoylation, a pivotal post-translational modification (PTM), plays a critical role in diverse cellular processes. This modification involves the covalent attachment of lipoic acid to proteins, fostering structural and functional alterations. The impact of lipoylation extends across various biological pathways, underscoring its indispensability in cellular homeostasis.
Analytical Methods of Lipoylation
- Mass Spectrometry. Mass spectrometry stands at the forefront of analytical methods for lipoylation research. This high-throughput technique enables the identification and quantification of lipoylated proteins, providing a comprehensive view of the lipoylated proteome. Tandem mass spectrometry (MS/MS) facilitates the sequencing of peptides, allowing precise determination of the lipoylation sites on target proteins. This method not only aids in cataloging lipoylated proteins but also sheds light on the stoichiometry of lipoylation, crucial for understanding the regulatory dynamics of this modification.
- Gel-Based Assays. Gel-based assays, particularly Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), serve as indispensable tools for the separation and visualization of lipoylated species. By exploiting the differential mobility of lipoylated and non-lipoylated proteins under electrophoretic conditions, researchers can assess the extent of lipoylation in a given sample. Complemented by immunoblotting techniques targeting lipoic acid, SDS-PAGE allows for precise detection and characterization of lipoylated proteins.
- Antibody-Based Approaches. Antibody-based methods offer specificity in detecting lipoylated proteins. Utilizing antibodies against lipoic acid or lipoylated lysine residues enables the selective recognition of lipoylated species in complex biological samples. Immunoprecipitation coupled with mass spectrometry enhances the sensitivity and accuracy of identifying lipoylated proteins, facilitating a targeted approach to dissecting the lipoylation landscape within a cellular context.
Application of Lipoylation
- Metabolic Regulation. Lipoylation emerges as a key regulator in metabolic pathways, particularly in the tricarboxylic acid (TCA) cycle and pyruvate dehydrogenase complex. The modification of enzymes involved in these processes influences energy production and metabolic flux.
- Mitochondrial Functionality. The impact of lipoylation extends to mitochondrial function, where lipoylated proteins contribute to the integrity and efficiency of the electron transport chain. This underscores the pivotal role of lipoylation in cellular respiration.
- Neurological Implications. Research indicates a connection between lipoylation and neurological disorders. Investigating the lipoylation status of proteins associated with neurodegenerative diseases offers promising avenues for therapeutic interventions.
Other Important Aspects in Lipoylation Research
- Dynamic Nature of Lipoylation. Lipoylation is a dynamic process, subject to intricate regulatory mechanisms. Unraveling the temporal aspects of lipoylation and understanding how it responds to environmental stimuli enhances our comprehension of its functional implications.
- Cross-talk with Other PTMs. Exploring the cross-talk between lipoylation and other PTMs unravels a complex network of regulatory interactions. Interplays with acetylation, phosphorylation, and ubiquitination highlight the interconnectedness of cellular signaling pathways.
- Therapeutic Implications. As our understanding of lipoylation deepens, its therapeutic potential becomes increasingly apparent. Targeting the lipoylation status of specific proteins may offer innovative therapeutic strategies for metabolic disorders and neurodegenerative diseases.
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In the field of cell biology, lipoylation is a multifaceted player that influences diverse cellular processes. Creative Proteomics follows the advances in analytical technology, coupled with a comprehensive exploration of its applications and complexities, in helping researchers delve into the dynamic landscape of lipoylation, new avenues for therapeutic intervention, and a deeper understanding of the cellular regulation it provides.
Glycine decarboxylase maintains mitochondrial protein lipoylation to support tumor growth
Journal: Cell Metab
Published: 2022
Background
The study focuses on understanding the role of glycine cleavage system (GCS) in hepatocellular carcinoma (HCC). While previous research has explored the importance of serine in supplying one-carbon units for nucleotide biosynthesis in cancer cells, the role of glycine oxidation, particularly through GCS, remains unclear. The study aims to investigate the significance of GCS flux in HCC and its potential impact on nucleotide biosynthesis, protein lipoylation, and mitochondrial activity.
Sample
The researchers examined hepatocellular carcinoma (HCC) cells, specifically HepG2, as well as other cell lines, to investigate the expression and metabolic flux of the glycine cleavage system. The study also involved mouse xenografts with HCC cells to explore the in vivo effects of glycine cleavage inhibition.
Technical Approach
The researchers employed a combination of stable and radioactive isotope tracing techniques along with computational flux decomposition to quantify mitochondrial glycine cleavage system (GCS) flux. They developed a novel method for measuring GCS flux using isotopically labeled serine and glycine and applied this approach to HCC cell lines. Additionally, they conducted genetic silencing experiments and utilized CRISPR-Cas9 knockout to investigate the effects of inhibiting glycine decarboxylase (GLDC), a key enzyme in GCS.
Results
The study revealed high GCS flux in hepatocellular carcinoma (HCC), supporting nucleotide biosynthesis(Figure 1). Surprisingly, GCS was found to play a crucial role in maintaining protein lipoylation and mitochondrial activity in addition to supplying one-carbon units. Genetic silencing of glycine decarboxylase inhibited lipoylation and impaired tumor growth, suggesting GLDC as a potential novel drug target for HCC. The research also highlighted the importance of considering the tissue of origin in understanding tumor-specific metabolic rewiring.
To systematically identify tumors with high glycine cleavage system activity, the authors analyzed the expression levels of four genes encoding GCS subunits (GLDC, AMT, GCSH, and DLD) in The Cancer Genome Atlas (TCGA). Two subunits of the GCS, GLDC and AMT, were found to be significantly overexpressed in hepatocellular carcinoma cells (HCC) and renal carcinoma (Figure 1).
Figure 1
Treatment of HCC cells with glycine revealed an important contribution of GCS flux to endogenous purine and pyrimidine synthesis. The isotopic labeling fraction of folate-derived 1C units in ATP and deoxythymidine triphosphate (dTTP) was higher compared to non-HCC cell lines. In particular, in HepG2 cells, the authors found that approximately 7% of the 1C units in approximately ATP were derived from glycine, suggesting that GCS-derived 1C units flow to purine synthesis at a rate of 0.16 mM/h. Considering that GCS flux in HepG2 cells is 0.3 mM/h, the authors concluded that 1C units produced by GCS flux flow primarily to the purine synthesis pathway (Figure 2).
Figure 2
The results of the study showed that GLDC silencing inhibited the growth of HepG2, Hep3B, and HUH6 cells (Figure 3A), and inhibited the growth of tumors, with a 4-fold reduction in tumor size.
Figure 3
To investigate how GLDC-induced proteolipid acylation affects mitochondrial metabolism, the researchers examined the ability of mitochondria to oxidize a variety of metabolic substrates, and found that the ability to specifically oxidize pyruvate and α-KG, the lipoic acid-dependent complexes PDH and oxoglutarate dehydrogenase (OGDH), was markedly reduced in GLDC-silenced cells. In HepG2 GLDC-silenced cells, for both PDH and OGDH, a significant increase in the substrate-to-product ratio was further found (Figure 4).
Figure 4
Conclusion
The study demonstrated the high expression of glycine cleavage system (GCS) genes and substantial glycine cleavage flux in hepatocellular carcinoma (HCC). While previous research has emphasized the role of glycine in cancer cell proliferation, this study revealed a novel connection between glycine cleavage and key cellular processes in HCC. The inhibition of glycine decarboxylase (GLDC), a crucial enzyme in GCS, resulted in the impairment of cell proliferation, both in vitro and in vivo, highlighting GLDC as a potential therapeutic target for HCC. The findings provided insights into the diverse roles of GCS, including its involvement in nucleotide biosynthesis, protein lipoylation, and mitochondrial metabolism. The study emphasized the importance of considering the tissue of origin in understanding metabolic rewiring in tumors and called for further exploration of GLDC as a potential drug target through the development of novel inhibitors and pre-clinical studies. Additionally, the researchers identified limitations, such as the need for further in vivo isotopic tracing experiments and investigation into the safety and potential resistance mechanisms associated with targeting GLDC in HCC.
Reference
- Mukha D, Fokra M, Feldman A, et al,. Glycine decarboxylase maintains mitochondrial protein lipoylation to support tumor growth. Cell Metab. 2022 May 3;34(5):775-782.e9.
