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Research Paper review on TCA Cycle

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BCMB30011 Written Assignment on Journal Article
As the keystone of cellular respiration, the tricarboxylic acid (TCA) cycle helps to oxidise
acetyl-coenzyme A (CoA) from glycolysis and pyruvate dehydrogenase activity to facilitate
oxidative phosphorylation in mitochondria (Arnold & Finley, 2022). Despite its importance,
mammalian cells continue to exhibit variation in tricarboxylic acid (TCA) cycle activity,
suggesting a possible biochemical alternative to the canonical TCA cycle. Two key enzymes
that are key for understanding the study are ATP citrate lyase (ACL) and aconitase 2 (ACO2),
as these were manipulated through the study. ACLY is a cytosolic enzyme that catalyses the
conversion of citrate and CoA to oxaloacetate and acetyl-CoA (Fig. 1) whilst simultaneously
hydrolysing ATP into ADP and inorganic phosphate (Chypre et al., 2012). ACO2 is a
mitochondrial enzyme involved in the canonical TCA cycle that converts citrate to isocitrate,
another intermediate downstream in the TCA cycle (Arnold & Finley, 2022). The authors
investigated the presence of distinct modules amongst enzymes involved in the TCA cycle,
utilising metabolic gene essentiality scores obtained from analysing CERES gene dependency
values from the DepMap Portal Project Achilles, where genome-wide CRISPR screens were
performed. With genes involved in the same metabolic pathways having similar degrees of
essentiality (Wainberg et al., 2021) , the authors found that TCA-cycle-associated genes formed
two distinct functional modules. Mapping the genes in each cluster to the canonical TCA cycle
revealed a correlation between the enzymes involved in cytosolic citrate metabolism and those
involved in citrate production, ultimately suggesting the presence of a non-canonical TCA
cycle (Fig. 1). On top of investigating into its properties, authors sought to study the role this
alternative cycle plays in determining cell state and fate.
Figure 1: Visualisation of location of canonical and non-canonical TCA cycle intermediates in
mitochondria and cytoplasm of cell, based on Arnold et al. (2022). Enzymes ATP citrate lyase
(ACLY) and Aconitase 2 (ACO2) have been indicated in red
To measure fluxes in non-canonical TCA cycle, the authors utilised isotope tracing, where [U13
C]glucose was traced through its metabolism. After glycolysis and oxidative decarboxylation
of pyruvate, the labelled metabolite formed M+2-labelled (two heavy-labelled carbons) citrate.
Thereafter, formation of M+2-labelled TCA cycle intermediates indicated that the M+2labelled citrate underwent the canonical TCA-cycle. Comparatively, ACL activity would form
unlabelled oxaloacetate and M+2-labelled acetyl-CoA, which would be liberated in the
cytoplasm. Hence, reductions in M+2-labelled TCA-cycle intermediates downstream of citrate
suggested the channelling of M+2-labelled citrate into the non-canonical TCA cycle. In the
non-small cell lung cancer (NSCLC) cell lines utilised for this test, the authors wanted to
confirm that this discrepancy in labelled metabolites downstream of citrate was not due to
glutamine anaplerosis. By inhibiting ACL, they found that all NSCLC lines underwent
increases in the ratio of malate M+2 to citrate M+2 (mal+2/cit+2), a representation of canonical
TCA cycle activity, suggesting the previous loss of labelled intermediates in the TCA cycle to
be due to channelling to ACL activity in the non-canonical TCA cycle.
To compare between the effects of disrupting canonical and non-canonical TCA cycles, the
authors formed genetically edited mouse embryonic stem (ES) cell lines with repressed ACL
or ACO2. Amongst these undifferentiated ES cells, ACL disruption promoted canonical TCA
cycle activity and affected the quantities of cytosolic metabolites associated with citrate
metabolism, while ACO2 disruption not only promoted non-canonical TCA cycle activity, but
also lead to minimal effect on the levels of TCA cycle intermediates. These suggest an
emphasised usage of non-canonical TCA cycle in undifferentiated ES cells. Considering the
self-renewal properties of ES cells in the right conditions, non-canonical TCA cycle could be
used to facilitate the generation of cytosolic acetyl-CoA and thereafter lipids, purposed for cell
membrane expansion during cell division. To confirm the involvement of the mitochondrial
citrate/malate antiporter (SLC25A1), malate dehydrogenase 1 (MDH1) and ACL in the noncanonical TCA cycle, the authors similarly formed ES cell lines with SLC25A1 and MDH1
repressed. In their absence, these cell lines showed reduced non-canonical TCA cycle activity
and reduced replenishment of citrate from cytosolic oxaloacetate, thereby demonstrating their
involvement in the non-canonical TCA cycle.
In comparison, myotubes, which are differentiated multinucleated muscle fibres formed
through the fusion of undifferentiated precursor myoblasts (Lehka et al., 2020), were the
subjects used by the authors to investigate TCA-cycle choice in different cell states. ACL
inhibition lead to more significant increases in mal+2/cit+2 ratios, as well as more significant
increases in citrate levels and decreases in cytosolic metabolites, in myoblasts compared to
myotubes. On the other hand, ACO2 inhibition decreased mal+2/cit+2 ratios more
significantly, and had a greater effect on TCA-cycle metabolites, in myotubes compared to
myoblasts. These results suggest the engagement of canonical TCA cycle in differentiated
myotubes and non-canonical TCA cycle in undifferentiated myoblasts. The use of canonical
TCA cycle in differentiated myotubes could be to facilitate effective ATP synthesis and cellular
respiration, particularly since, as muscle cells, myotubes have high energetic demands. The use
of non-canonical TCA cycle in undifferentiated myoblasts could mirror the biosynthetic
reasons as undifferentiated ES cells.
The authors also tested if changes in the TCA-cycle engaged are required for changes in cell
state. By inducing naïve, undifferentiated ES cells to exit pluripotency and begin
differentiation, the authors studied changes in TCA cycle activity. As the naïve ES cells exited
pluripotency, they observed increased usage of glutamine-derived carbons, a decrease in
mal+2/cit+2 ratios and an increase in citrate levels from cytosolic metabolites. These results
indicate a shift from canonical to non-canonical TCA cycle engagement as the ES cells exit
pluripotency and begin differentiation. Similar to the differentiation observed in myoblasts to
myotubes, the shift towards the non-canonical TCA cycle prioritises maintenance of TCAcycle intermediates and cell viability.
The authors next tested if ACL was required for ES cells to exit pluripotency, and found that
ACL inhibition mitigated the expected reduction in pluripotency reporter (Rex1::GFPd2).
Without functional ACL, the cells had enhanced expression of pluripotency genes Nanog,
Esrrb and Rex1, and ability to form colonies, as well as impaired expression of differentiation
marker Sox1. They repeated this test with inhibitions of SLC25A1 and MDH1, ultimately
finding that ACL, SLC25A1 and MDH1 were all required for the establishment of cellular
metabolic identity. Without which, cells exiting pluripotency have reduced viability.
Overall, by uncovering the flexible and dynamic nature of the TCA cycles, and their
relationship with cell state, the authors have uncovered a new domain of possible metabolic
strategies employed in cells. With regards to application, they have opened up a new avenue
of opportunities to explore in cancer cell research with its close link to ES cell behaviour.
Cancer cells’ reliance on either TCA cycle in different mediums could provide a new
opportunity for intervention in the future.
References
Arnold, P. K., & Finley, L. W. S. (2022). Regulation and function of the mammalian
tricarboxylic acid cycle. Journal of Biological Chemistry, 0(0).
https://doi.org/10.1016/j.jbc.2022.102838
Arnold, P. K., Jackson, B. T., Paras, K. I., Brunner, J. S., Hart, M. L., Newsom, O. J.,
Alibeckoff, S. P., Endress, J., Drill, E., Sullivan, L. B., & Finley, L. W. S. (2022). A
non-canonical tricarboxylic acid cycle underlies cellular identity. Nature, 603(7901),
477–481. https://doi.org/10.1038/s41586-022-04475-w
Chypre, M., Zaidi, N., & Smans, K. (2012). ATP-citrate lyase: A mini-review. Biochemical
and Biophysical Research Communications, 422(1), 1–4.
https://doi.org/10.1016/j.bbrc.2012.04.144
Lehka, L., Topolewska, M., Wojton, D., Karatsai, O., Alvarez-Suarez, P., Pomorski, P., &
Rędowicz, M. J. (2020). Formation of Aberrant Myotubes by Myoblasts Lacking
Myosin VI Is Associated with Alterations in the Cytoskeleton Organization, Myoblast
Adhesion and Fusion. Cells, 9(7), 1673. https://doi.org/10.3390/cells9071673
Wainberg, M., Kamber, R. A., Balsubramani, A., Meyers, R. M., Sinnott-Armstrong, N.,
Hornburg, D., Jiang, L., Chan, J., Jian, R., Gu, M., Shcherbina, A., Dubreuil, M. M.,
Spees, K., Meuleman, W., Snyder, M. P., Bassik, M. C., & Kundaje, A. (2021). A
genome-wide atlas of co-essential modules assigns function to uncharacterized genes.
Nature Genetics, 53(5), 638–649. https://doi.org/10.1038/s41588-021-00840-z
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