Nature-Translational Psychiatry: Different protein expression in saliva of people with/ without CFS.

[RogerBlack]

In these kinds of systems, it's sort of like a mass production facility. Think of the protein as a machine in a factory. Putting a label on a bottle for example. If you had enough labels, enough power, enough bottles, enough labour, enough space, enough waste management, and efficient transportation for your labels and bottles to get from and to your machine,... Than sure, twice as fast.

But as this is different in CFS, it is likely various of the above is not true, and though more protein has been put in to do a task, it may not even reach the normal efficiency.
There are a few scenarios.
The machinery has correctly determined that the rate of what the protein is doing is too low, and what it's doing in adding it helps, though does not fix the situation.
The machinery does not directly regulate the level of protein, and it's a symptom of something else going wrong, but doesn't do much.
Something is triggering activation of the protein, when it should not be, and its increased level is hurting.

Working out if the increased level of any protein 'means' something clinically significant is hard, and requires deep understanding of the biochemistry and what is the limiting factor. Even quite large changes in some levels may be clinically insignificant.

 

[sdmcvicar]

I find it a little odd that the non-CFS twin questionnaire results weren't included, as this might be more useful than the comparison of the twin to other CFS controls.

Thank you.

Does the 2.2 fold mean that 2.2 times more phosphate is transfered from GTP/ITP to AMP in the patient?

No, it just means that there was 2.2 times as much of that enzyme in the CFS twin. The actual conversion rate is dependent on the concentrations of reactants and products in the chemical reaction(s) that the enzyme catalyzes. The rate might be faster, but it could also mean that a difference in metabolites has caused the mitochondria of the CFS twin to produce twice this much enzyme in order to keep conversion rates similar to that of the non-CFS twin.

 

[A.B.]

The authors proposed that the changes may reflect an attempt by the cells to compensate for some problem in energy production.

 

[lansbergen]

Thanks sdmcvicar and RogerBlack

 

[Snow Leopard]

One of the authors is "A Sickmann".

Seriously though, this study is more interesting than I first thought, I'll post some thoughts when I'm feeling a bit less foggy.

Previous study by these authors -
https://www.ncbi.nlm.nih.gov/pubmed/24088505
http://forums.phoenixrising.me/inde...ic-twins-show-potential-saliva-markers.25638/
The "discovery" phase from platelets of two pairs of monozygotic twins, which was then "validated" in the current study.

 

[M Paine]

So we now have proteomic evidence for downregulation of the metabolism of NADH. In terms of papers related to taking NADH supplementation, it seems controversial at best in terms of the efficacy of taking NADH. I think some of that comes down to difficulties in classifying patients, the sub-group issue, the lack of biomarkers, etc.

But in any event, assuming that metabolism of NADH is being downregulated in some patients, it begs the question, is NADH supplementation a good treatment strategy?

On one hand we have a fair amount of anecdotal evidence of patients who claim it helps their symptoms. On the other hand, we have Metabolomics experts cautioning that this downregulation may be part of an immune defence mechanism, protective against yet unknown aetiology.

It's difficult to know when intervention is helpful, or may prolong illness. Anyone else feeling cautious about NADH? At the same time, I'm eager to try it, in the event it is helpful and not overtly harmful.

 

[GreyOwl]

I'm not qualified to ask these questions, so if I'm totally off course, please be polite.

Is downregulation of NADH a metabolic adaptation to reduce the generation of ROS in order to prevent what the body believes is further damage? Why does this persist so long, and what finally stops it (if it stops at all)? Is there something like a glyoxalate cycle in humans which is used as compensatory/alternate pathway when downregulation of NADH (hypometabolism) is required/initiated?

 

[M Paine]

Is there something like a glyoxalate cycle in humans which is used as compensatory/alternate pathway when downregulation of NADH (hypometabolism) is required/initiated

In terms of ATP production, substrate-level phosphorylation is one way in which ATP can be produced which is not reliant on NADH/FADH2. These reactions add a phosphate back to ADP using a kinase and a substrate donor (phosphate donor) as opposed to oxidative phosphorylation using the electron transport chain with NADH/FADH2 as electron donors to add free floating inorganic phosphate mechanically to ADP (via ATP Synthase).

I suppose oxidative phosphorylation is not strictly NADH dependant either... as FADH2 can serve as an electron donor

 

[M Paine]

"The selected proteins were as follows: aconitate hydratase (ACON), ATP synthase subunit beta (ATPB) and malate dehydrogenase (MDHM)."

As I understand it, the three proteins chosen were chosen from a subset of the ones they could have looked at, as these three proteins were thought likely to be an issue.
it may be that adding more proteins would help in discrimination, or finding disparate patient populations with variants of cfs.

Here's the full list of proteins...

 

[alex3619]

In these kinds of systems, it's sort of like a mass production facility. Think of the protein as a machine in a factory. Putting a label on a bottle for example. If you had enough labels, enough power, enough bottles, enough labour, enough space, enough waste management, and efficient transportation for your labels and bottles to get from and to your machine,... Than sure, twice as fast.

An increase in protein at some point in the system might indicate a deficiency of the entire system, and an attempt to compensate, and sometimes that works. If it does not then in this analogy it could be like the factory manager not knowing where the problem is but deciding to buy more bottles or print more labels in the hope this would fix things.

 

[alex3619]

requires deep understanding of the biochemistry

You need to know the rate of the chemical reactions, not just how much of each substrate or enzyme is present.

 

[alex3619]

The rate might be faster, but it could also mean that a difference in metabolites has caused the mitochondria of the CFS twin to produce twice this much enzyme in order to keep conversion rates similar to that of the non-CFS twin.

Another possibility that I have discussed since maybe 2000 or so it that the proteins may be misfolded and so not working right. If the mitochondria or cell (for the case of cellular proteins imported into the mitochondria) detects this problem then it might over-produce the protein to try to compensate. High ROS and low reduced-glutathione might be responsible.

 

[RogerBlack]

If the mitochondria or cell (for the case of cellular proteins imported into the mitochondria) detects this problem then it might over-produce the protein to try to compensate. High ROS and low reduced-glutathione might be responsible.

Another thought is muscle biopsies + TOF-mass-spec to do a proteome, both when baseline and bad.
Brain biopsies would be better, but for obvious reasons those aren't a thing.

I'm unsure how you would detect misfolded protein.
Attempting to crystalise the proteins, and then x-ray crystallography ? Seems doubtful.

Replacing the mitochondria would be a nice test, but I can only imagine 'star trek' methods with little foundation in current reality like engineered intracellular bacteria, or a virus which encodes the mitochondria as well as something to kill off the hosts ones.

 

[alex3619]

I wonder if you could isolate a sample of the protein without denaturing it, whether you could visually check the protein under an electron microscope. I am unsure of current methods so I do not know what alternatives might exist to identify misfolded proteins. There might be a way to do that with a specially designed antibody test, as they will bind differently to unfolded and folded proteins.

 

[RogerBlack]

I wonder if you could isolate a sample of the protein without denaturing it, whether you could visually check the protein under an electron microscope. I am unsure of current methods so I do not know what alternatives might exist to identify misfolded proteins. There might be a way to do that with a specially designed antibody test, as they will bind differently to unfolded and folded proteins.

Unfortunately, as I understand it, cryo-EM which might otherwise sort-of-work for this - isn't really up to the job for most proteins. Resolution is 3nm or so at best.

This is a whole Zika virus particle, with a resolution of about 3nm.
There are protiens involved in metabolism that are this large - NADH reductase, for example.
https://books.google.co.uk/books?id...=nadh dehydrogenase structure nm long&f=false - has a 2.2nm resolution image, and is about 30nm across.

 

[lansbergen]

Another possibility that I have discussed since maybe 2000 or so it that the proteins may be misfolded and so not working right. If the mitochondria or cell (for the case of cellular proteins imported into the mitochondria) detects this problem then it might over-produce the protein to try to compensate. High ROS and low reduced-glutathione might be responsible.

Yes. I wonder why SOD2 was high in the twin.

Anybody know high SOD2 was found in the later study?

 

[RogerBlack]

Yes. I wonder why SOD2 was high in the twin.

Anybody know high SOD2 was found in the later study?

They only tested three proteins in saliva, not including SOD2.

The initial mitochondrial test method was considerably more expensive, but did more proteins.

 

[M Paine]

I suppose it's worth pointing out that the down-regulated proteins they identified which are involved in NADH metabolism (isocitrate dehydrogenase IDH3A/IDH3B and malate dehydrogenase MDH2), form only part of the citric acid cycle in which NAD+ is reduced. It's not really clear from this paper if NADH is functionally deficient. If anyone knows of other papers that have looked at this in detail, I'd be interested to read them.

*Edit: I made a mistake here, these proteins are up regulated. Same for my post near the top of this page.

 

[M Paine]

I was just looking at the Naviaux metabolomics data deposition, to see what metabolites related to the citric acid cycle.

I've picked out Pyruvic acid, Citric acid, cis-aconitic acid, Fumaric acid, Malic acid, and Oxaloacetic acid

F1 = CFS patients, F2 = Controls. All values represent males (like the twins)

It's interesting to look at that metabolites related to ACON (aconitate hydratase), Citric Acid and cis-aconitic acid (isocitric acid was not listed in the data). Citric acid is low, and cis-aconitic acid is high (quite noticeably so). This seems to correlate with the observation in the twins, and patients that ACON is up-regulated.

Similarly with MDHM (malate dehydrogenase), Malic Acid takes a drop, but in the case of Oxaloacetic acid, we see no increase. I wonder why?

I think this is really interesting, and remarkable to be able to relate this paper to the metabolomics data.

 

[M Paine]

In some earlier posts on this page, I mistakenly said that down regulation was occurring of ACON/MDH2 and by proxy leading to reduced metabolism of NADH. I don't think that's correct, it's the opposite. They are up regulated. In any case, I can't edit one of the posts for some reason. I hope that won't lead to confusion for anyone.