Comprehensive Profiling Of Amino Acid Response Uncovers Methionine-Deprived Response. Creatine

[Terma]

@Travis
Extremely convenient study. Got around to reading this, backlogged. Caveat: Only 1-2 days long, MCF7 breast cancer cells and PC3 prostate cancer cells.

Comprehensive Profiling of Amino Acid Response Uncovers Unique Methionine-Deprived Response Dependent on Intact Creatine Biosynthesis

Besides being building blocks for protein synthesis, amino acids serve a wide variety of cellular functions, including acting as metabolic intermediates for ATP generation and for redox homeostasis. Upon amino acid deprivation, free uncharged tRNAs trigger GCN2-ATF4 to mediate the well-characterized transcriptional amino acid response (AAR). However, it is not clear whether the deprivation of different individual amino acids triggers identical or distinct AARs. Here, we characterized the global transcriptional response upon deprivation of one amino acid at a time. With the exception of glycine, which was not required for the proliferation of MCF7 cells, we found that the deprivation of most amino acids triggered a shared transcriptional response that included the activation of ATF4, p53 and TXNIP. However, there was also significant heterogeneity among different individual AARs. The most dramatic transcriptional response was triggered by methionine deprivation, which activated an extensive and unique response in different cell types. We uncovered that the specific methionine-deprived transcriptional response required creatine biosynthesis. This dependency on creatine biosynthesis was caused by the consumption of S-Adenosyl-L-methionine (SAM) during creatine biosynthesis that helps to deplete SAM under methionine deprivation and reduces histone methylations. As such, the simultaneous deprivation of methionine and sources of creatine biosynthesis (either arginine or glycine) abolished the reduction of histone methylation and the methionine-specific transcriptional response. Arginine-derived ornithine was also required for the complete induction of the methionine-deprived specific gene response. Collectively, our data identify a previously unknown set of heterogeneous amino acid responses and reveal a distinct methionine-deprived transcriptional response that results from the crosstalk of arginine, glycine and methionine metabolism via arginine/glycine-dependent creatine biosynthesis.

[...]

Based on these data, we propose a model that the intact arginine- and glycine-dependent creatine biosynthesis contributes to two events in the methionine-deprived specific gene response: depletion of SAM for epigenetic changes and maintaining ornithine-mediated signaling.

• Arginine or Glycine restriction abolished effects of MR, primarily through preventing Creatine synthesis from soaking up SAMe
• MR effects depend on lowering Histone methylation (can both activate and repress transcription), but not DNA methylation (over 1-2 days)
• Effects of MR depend on presence of Ornithine ("addition of ornithine with DZNep rescued most of the methionine-deprived specific gene response when the creatine biosynthesis was abrogated by arginine depletion"), but...
• Forcefully inhibiting polyamine synthesis increased the effects of MR; they did not write this verbatim, but presumably MR restriction diverts Ornithine from polyamine synthesis toward other uses ("The addition of ornithine enhanced the induction of some genes in the context of methionine deprivation alone, but ornithine alone was unable to rescue the induction of the methionine-deprived specific genes when the creatine biosynthesis was blocked by arginine depletion")

There are other conjectures I could make but those are enough.

Re: Polyamines (they hypothethized that polyamine pathway was a vehicle for MR effects, and found the opposite):

In MCF7, the MTAP locus is deleted and the methionine salvage pathway is disrupted [32]. Therefore, the supplemented 5'-Methylthioadenosine (MTA) cannot be readily salvaged back to replenish methionine during methionine deprivation. Therefore, MTA could not abolish the methionine-specific response by simply restoring methionine levels. MTA is a byproduct of the polyamine biosynthesis that combines the decarboxylated S-adenosyl-methionine (dcSAM) (from SAM) and putrescine (from ornithine) to synthesize spermidine and spermine (S4A Fig). High levels of MTA inhibit polyamine biosynthesis [33]. Therefore, we reasoned that the supplementation of MTA might abolish the methionine-specific response by inhibiting the polyamine pathway. To test this possibility, we interrupted polyamine synthesis by inhibiting critical enzymes or removing its substrate arginine. We targeted the two key enzymes in the polyamine synthesis: ornithine cyclodeaminase (ODC1 that catalyzes the synthesis of putrescine from ornithine) and spermidine synthase (SRM that catalyzes the synthesis of spermidine from putrescine) to determine methionine-deprived specific responses. Surprisingly, we found that the inhibition of polyamine synthesis by genetically silencing ODC1 (Fig 5A) or SRM (S5A Fig) further enhanced the induction of TEX14, DAPK3 and BAG5 during methionine deprivation. Similarly, the ODC1 inhibitors POB and DFMO also did not abolish the methionine-deprived specific gene responses (S5B Fig).

Other:

deprivation of most amino acids, except glycine, triggered a robust and mostly conserved AAR. Quite unexpectedly, methionine deprivation triggered the most dramatic and extensive gene expression changes.

These results suggested that the level of intracellular methionine needed to drop below a certain threshold to trigger the methionine transcriptional response, and that this threshold varies in different cell types.

First, blocking the urea cycle by nor-NOHA (an arginase inhibitor) did not repress the inductions of methionine specific genes TEX14, DAPK3 and EGR1 (S6A Fig). Second, neither a NO scavenger (c-PTIO) nor inhibition of the nitric oxide synthesis (NOS) by L-NAME affected the methionine-specific gene responses (S6B Fig). These data mostly ruled out the role of the urea cycle and nitric oxide production as mechanisms by which arginine regulates the methionine-specific response.

Correct me on anything I got wrong

 

[LeeLemonoil]

Great find Terma, this is very handy to have to read and refer to.

 

[Terma]

Thanks, but I plan on taking it a little further. I've toyed with the idea of exploiting Arginine for some time; anyone who reads Suppversity has seen it can have benefits, not associated to or in spite of NO production. (eg this, this)

Here we see another theoretically great use, maybe the best I've seen so far (though remember this was in vitro in cancer cells, but I figure liver cells must behave similar). The idea of Arginine benefiting MR fits into popular examples, because vegan diets most associated with MR typically are high in nut consumption which are higher in Arginine. If-I-recall-correctly (and I probably don't), only their beans provide much toward the Lysine side.

Quite simply I figure you could take a strong NOS (particularly iNOS, maybe Methylene Blue) inhibitor together with several grams of Arginine and Glycine supplementation, together with Methionine restriction. You may also have to watch out how you take Lysine because it competes with Arginine for uptake, though you still need it. You could add an Ornithine supplement but I'd expect the extra contribution to be minor if you take enough Arginine. I supposed you could try a mix of Arginine and Ornithine. The Glycine might not need be that high because it appears difficult to starve the cells of it; rather I'd take just enough to make sure it doesn't get drained from other pathways; it was thought to be a minor limiting factor in creatine synthesis; though there's always that posited 10g daily shortfall in one of the other studies.

 

[Terma]

Also to protect brain function, would take significant Magnesium and/or NMDA antagonists, prevent the NOS cascade slightly upstream.

 

[Travis]

What I had gathered from reading previous studies was that spermine strongly binds dNA and induces the Z-dNA configuration, a left-handed helix, and also increases the rate of dNA replication in vitro during PCR. These molecular properties are so reliable and fundamental that I believe they transcend other explanations such as histone or dNA methylation. Another trend that I've found was that spermidine has a preference for CpG islands, and also that all CpG islands are always found on 'housekeeping genes.' From these observations, and a few others, I have concluded that spermine normally and nonspecifically replicates housekeeping genes located at CpG islands. This would make polyamine transcrition completely independent of the inducible transcription factors such as PPARγ and VDR, something like a transcription catalyst which creates background mRNA for primary metabolism.

 

[Terma]

Well this study on the surface I'm not sure outright discounts that the diversion away from polyamine synthesis itself could be a major contributor. It could be in line with the study but I can't analyze this today (yet don't want to divert your attention from other things).

They did a thorough array of tests so might or might not be able to discern from this. They were convinced histone methylation was major and ornithine had a positive influence through a non-polyamine pathway, and the latter is an interesting possibility, but then again they stated their initial "hypothesis" was that polyamine synthesis was driving the MR, so it's hard to trust them.

Personally I always lean toward the combined contribution of multiple simultaneous pathways rather than any single one, but this study doesn't quantify enough to say.

Since intact creatine biosynthesis was required for reduction of histone methylations during methionine deprivation, we examined whether the inhibitors of histone methylation was able to restore the methionine-deprived specific gene response in the context of blocked creatine biosynthesis. Indeed, the general histone methylation inhibitor DZNep (DZN) fully restored the induction of TEX14, ING2 and BAG5 genes when creatine biosynthesis was prevented by co-depleting serine, threonine and glycine (the glycine branch of creatine synthesis) (Fig 7A). However, DZN only partially restored the induction of the methionine-deprived specific genes when the arginine branch of creatine biosynthesis was blocked (Fig 7A). Besides being used for creatine biosynthesis, arginine can be synthesized to ornithine, as arginine deprivation reduced the level of intracellular ornithine (S6G Fig). We hypothesized that ornithine-mediated signaling was also required for the full induction of methionine-deprive specific gene response. Indeed, addition of ornithine with DZNep rescued most of the methionine-deprived specific gene response when the creatine biosynthesis was abrogated by arginine depletion (Fig 7B). We also examined UNC0638 (UNC), a specific inhibitor of methyltransferases G9a and GLP, which are responsible for H3K9 di-methylation. We found that UNC had no significant rescuing effects on the induction of methionine-deprived specific genes (Fig 7B), suggesting that demethylation of H3K9me2 alone may be not sufficient for the observed methionine-deprived transcriptional changes. The addition of ornithine enhanced the induction of some genes in the context of methionine deprivation alone, but ornithine alone was unable to rescue the induction of the methionine-deprived specific genes when the creatine biosynthesis was blocked by arginine depletion (S7A Fig). Creatine had similar enhancement effects as ornithine on the methionine-deprived gene response (S7B Fig). Taken together, our data suggested a model in which an intact arginine- and glycine-dependent creatine biosynthesis contributes to SAM depletion and resulting epigenetic changes. The combination of these epigenetic alterations and ornithine-mediated signaling are required to fully induce a specific methionine-deprived transcriptional response (Fig 7C). Based on these data, we propose a model that the intact arginine- and glycine-dependent creatine biosynthesis contributes to two events in the methionine-deprived specific gene response: depletion of SAM for epigenetic changes and maintaining ornithine-mediated signaling (Fig 7C).

[Now that I think about it this quote is too narrow in scope, I'll have to go over the rest again]