Cuproptosis

Where this post is going…

The featured image says it all. A lipoic acid binding component of the pyruvate dehydrogenase complex, DLAT, binds Cu²⁺ and causes the oligomerization of the DLAT subunit in such a way as to cause “regulated cell death.” There is a lot of controversy and talk as to which enzyme is most important in this new version of RCD.

Regulated cell death… so many means to choose from

It all started with apoptosis, a form of programed cell death. The image below came largely from the public access 2019 Tang review. [1] I would say just skim through this list just enough to realize that the whole concept that death is a red hot regulated event.

• Apoptosis is characterized by the mitochondria self destructing and activation of proteases called caspases. A caspase is like a PacMan chewing up proteins.
• Necroptosis is a “prgrammed” form of inflammatory cell death with the programing part delivered by RIP kinase… of course!
• Lysosomal cell death is when lysosomes, that function as intracellular stomachs, start leaking their cathepsin digestive enzymes.
• Pyroptosis is driven by the IL-1 and so on producing inflammasomes that we’ve discussed on this site. The “pyro” part must come from the fevers that come along with cytokines produced by cytokines like IL-1
• Netotic cell death has been covered on this site: macrophage and their secreted nets that kills pathogens and maybe bystander cells
• Ferroptosis is, in simple terms. unbound iron generating reactive oxygen species. The rust nail is a reminder that iron reacts with oxygen.
• Entosis is when one cell kills another by endocytosis, we are assuming that this is by non immune cells. .
• Immunological cell death is when dying cells are cleared by phagocytosis
• Parthanatos results from excessive activation PARP and loss of ATP and NAD⁺ PARP can bind to dmaged DNA leading to repair or apoptosis…
• Alkaliptosis is mediated by mediated by intracellular increase in pH or alkalinity.
• Autosis is a lysosome thing when the lysosome intracellular cellular stomach organelles fuse with autoendosomes.
• Oxeiptosis is a novel oxygen radical-induced pathways.

Confusing? Of course it is!

This is just the context of our next story about how Cu²⁺ might contribute to the death of melanoma cells. Sometimes a cell must activate programs because the good of the many cells in an organism outweigh the good of the one.

The discovery, from Boston and Baltimore

Ths studly came out of many prestigious cancer focused institutes in Boston Baltimore. Elesclomol “is a small-molecule bis(thio-hydrazide amide) with oxidative stress induction, pro-apoptotic, and potential antineoplastic activities. Elesclomol induces oxidative stress, creating high levels of reactive oxygen species (ROS), such as hydrogen peroxide, in both cancer cells and normal cells. Because tumor cells have elevated levels of ROS compared to normal cells, the increase in oxidative stress beyond baseline levels elevates ROS beyond sustainable levels, exhausting tumor cell antioxidant capacity, which may result in the induction of the mitochondrial apoptosis pathway. Normal cells are spared because the increase in the level of oxidative stress induced by this agent is below the threshold at which apoptosis is induced” according to PubChem. Elesclomol is evidentially also an ionophore for Cu and other metal ions. The authors were not really indicating the oxidation state of these metals.

The rest of Tsvetkov Figure 1 concerned programmed cell death pathways we are considering too complicated for this post. Note that while Fe can redox cycle, it is not killing cells when chelated to Elesclomol. Note that this is Cu²⁺ that appears to be doing the killing.

Tsvetkov and coauthors presented experiments in Figure 2 that led them to conclude that Eleschomol-Cu was not directly targeting the electron transport chain but rather the TCA cycle. These investigators knocke out seven genes they thought might rescue cell killing by elachlomol-Cu²⁺

These are the suspect enzymes that were knocked out the cell culture system. ϴ denotes enzymes that are needed for cuproptosis.

1. FDX1 (a reductase known to reduce Cu²⁺ to “its more toxic form, Cu1⁺ ” Accroding to GenCards FDX1 is a gene that codes for mitochondrial adrenodoxin a small iron-sulfur protein that transfers electrons from NADPH through ferredoxin reductase to mitochondrial cytochrome P450, involved in steroid, vitamin D, and bile acid metabolism
2. , LIPT1, ϴ mitochondrial lipoyl transferase 1 Catalyzes the transfer of the lipoyl group from lipoyl-AMP to the specific lysine residue of lipoyl domains of lipoate-dependent enzymes
3. LIAS ϴ mitochondrial lipoyl synthase This protein has Fe-S centers shown as
4. DLD ϴ D-lactate dehydrogenase R-lactate + 2 Fe(III)-[cytochrome c] = 2 Fe(II)-[cytochrome c] + 2 H⁺ + pyruvate
5. DLAT ϴ Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 3, ATP + L-seryl-[pyruvate dehydrogenase E1 alpha subunit] = ADP + H⁺ + O-phospho-L-seryl-[pyruvate dehydrogenase E1 alpha subunit
6. PDHA1 Pyruvate dehydrogenase E1 component subunit alpha, (R)-N₆-lipoyl-L-lysyl-[dihydrolipoyllysine-residue acetyltransferase] + H⁺ + pyruvate = (R)-N₆-(S₈-acetyldihydrolipoyl)-L-lysyl-[dihydrolipoyllysine-residue acetyltransferase] + CO₂
7. PDHB Pyruvate dehydrogenase E1 component subunit beta, R)-N₆-lipoyl-L-lysyl-[dihydrolipoyllysine-residue acetyltransferase] + H⁺ + pyruvate = (R)-N₆-(S₈-acetyldihydrolipoyl)-L-lysyl-[dihydrolipoyllysine-residue acetyltransferase] + CO₂

Figure 4 made the case that FDX1 expression tends to correlate with the abundance of lipoic acid. [1] Cancer cell line data backed this up. Wikipedia authors made the case for lipoic acid being important for TCA function. The case for Cu involvement. The authors used affinity resigns that bind metal ions and whatever proteins might bind those metal ions. To elute the metal ion binding protein, one simply elutes with excess metal ion. Figure 5 continued with some clever microbiology that also suggested Cu induced oligomerization.

Note that the working hypothesis has nothing to say about Cu⁺ chaperone proteins. The elesclomol is a very unnatural way to introduce Cu into the cell in a very unnatural oxidation state. It has further not been verified whether Cu⁺ or Cu²⁺ is more reactive with Fe-S centers.

What all of this means

The landmark Tsvetkov cuproptosis paper cites many enzymes in glycine cleavage system (GCS) dual function pathway, one of which is feeding the amino acid glycine into the TCA cycle. The GCS can can also be used for making glycine. The glycine cleavage system, GCS, consists of four enzymes [3]:

1. glycine decarboxylase (P protein)
2. aminomethyl-transferase (T protein)
3. dihydrolipoyl dehydrogenase (L protein)
4. and a carrier protein (H protein)

According to Tsvetkov 2022, many enzymes in this complicated pathway are important for cuproptosis, but it is DLAT that oligomerizes in response to Cu. [2] In DLAT of panel D there are three forms of lipoic acid (1) two SH groups (2) one SH group and the other bound to an acetyl group (3) an internal (white) S-S form.

Mining melanoma databases for cuproptosis

This is a big data study that probably is not that informative. It came out just a few months after the landmark Click here to proceed to a discussion on DLAT and Cu(I), Haozhen Lv and coauthors conducted a data mining experiment of mRNA transcripts of skin cutaneous melanomas from 470 patients. It is recommended that the impatient lay reader only parse through this section. The take home is that cuproptosis is red hot and poorly thought out. There were some normal controls in here too.

• MTF1 … is a Zn²⁺ transcription factor that binds to the metal response element ( MRE) to activate transcription of genes that code for metalothionein. Also regulates the expression of metalloproteases in response to intracellular zinc and functions as a catabolic regulator of cartilages (By similarity)
• GLS..glutaminase, liver isoform, Plays an important role in the regulation of glutamine catabolism. Promotes mitochondrial respiration and increases ATP generation in cells by catalyzing the synthesis of glutamate and alpha-ketoglutarate. Increases cellular anti-oxidant function via NADH and glutathione production. May play a role in preventing tumor proliferation
• CDKN2A is a gene that codes for a protein that is a negative regulator of cylins that control the cell cycle and hence proliferation.
• SLC31A1, aka Ctr1
• ATP7B the Cu⁺ efflux pump

Simplifying Lv 2022 confusing Fig 5

Much of the Lv [4] publication shifted to high expression of LIPT1 expression being correlated with survival. The data presented in Lv 2022 seemed excessive. The PMC link [4] was used to obtain a full size image that was edited to show only significant increases or decreases in the CIBERSORT score.

Panel 5A PD-L1 expression as a function LIPT1 expression

Much of the Lv 2022 that is not being presented in this post discussed that how more LIPT1 is expressed in melanomas than in normal tissue, patients whose melanomas have a light level of LIPT1 expression have better survival than patients whose melanomas have lower expression. [4] Then they show data the more expression the more expression of PD-L1. PD-L1 or prgramed death receptor ligand 1, is a protein on the surface of tumor cells that binds to the programmed death receptor PD-1 found on activated T cells. When the two bind, the activated T cell is turned off. Wikipedia authors discuss how antibodies against PD-L1 are used to treat cancers that include melanomas! This is confusing and makes absolutely no sense whatsoever.

Panel 5B, individual immune cells

Lv did not go into great detail on how they used the CIBERSORT score. For now we will assume that they are looking at mRNA markers of various immune cells in the tumors of 470 melanoma patients with low or high levels of expression of LIPT1. The differential infiltration of some immune cells was found between LIPT1-high and LIPT1-low groups.

• CD4⁺ memory T cells resting ↑
• CD8⁺ T cells ↓
• regulatory T cells (Tregs)↓
• activated NK cells ↓
• M0 macrophages ↓
• neutrophils ↓

To summarize Lv and coauthors found that LIPT1 was upregulated in melanoma by analyzing the data downloaded from the TCGA and GEO databases, respectively. They also found that LIPT1 could be an indicator of the favorable prognosis of melanoma. They just assumed that up regulated LIPT1 might inhibit tumorigenesis by disrupting the TCA cycle. [4] This is what they claimed. LIPT1 actually supports the TCA cycle. In general it is better that tumors have an active TCA cycle and electron transport chain because to do otherwise would force them into the glycolytic Warburg Effect. The high lactic acid tumor microenvironment tends to be immuno suppressant.

Targeting DLAT

Recall that DLAT is the pyruvate dehydrogenase complex component that binds to lipoic acid. It also oligomerizes in response to high copper. [2] D-lipoic acid has been explored as a means of chelating Cu(I) from Cu(I) binding proteins in Wilson’s Disease. [5] The C₅₀ concentration needed to chelate 50% of the Cu(I)Cox17 was 0.498 ± 0.098 mM. The Kd (affinity) was 8.05 × 10⁻¹⁷ M, 10 to 100x than some of the other chelators examined as possible Wilson’s Disease therapeutics for this copper overload disorder. [5] Note, 0.498 mM lipoic acid is not going to happen.

References

1. Tang D, Kang R, Berghe TV, Vandenabeele P, Kroemer G. (2019) The molecular machinery of regulated cell death. Cell Res. 2019 May;29(5):347-364. PMC free article
2. Tsvetkov P, Coy S, Petrova B, Dreishpoon M, Verma A, Abdusamad M, Rossen J, Joesch-Cohen L, Humeidi R, Spangler RD, Eaton JK, Frenkel E, Kocak M, Corsello SM, Lutsenko S, Kanarek N, Santagata S, Golub TR. Copper induces cell death by targeting lipoylated TCA cycle proteins. Science. 2022 Mar 18;375(6586):1254-1261. PMC free article
3. Bernardinelli E, Costa R, Scantamburlo G, To J, Morabito R, Nofziger C, Doerrier C, Krumschnabel G, Paulmichl M, Dossena S. (2017) Mis-targeting of the mitochondrial protein LIPT2 leads to apoptotic cell death. PLoS One. 2017 Jun 19;12(6):e0179591. PMC free article
4. Lv H, Liu X, Zeng X, Liu Y, Zhang C, Zhang Q, Xu J. (2022) Comprehensive Analysis of Cuproptosis-Related Genes in Immune Infiltration and Prognosis in Melanoma. Front Pharmacol. 2022 Jun 28;13:930041. PMC free article
5. Smirnova J, Kabin E, Järving I, Bragina O, Tõugu V, Plitz T, Palumaa P. Copper(I)-binding properties of de-coppering drugs for the treatment of Wilson disease. α-Lipoic acid as a potential anti-copper agent. Sci Rep. 2018 Jan 23;8(1):1463. PMC free article

0.498 ± 0.098

8.05 × 10−17