Proline, P5C and 4-hydroxyglutamate in ME

What do

• Pre-eclampsia
• The inborn error of metabolism: Primary Hyperoxaluria Type 3
• Myalgic encephalomyelitis (ME)

have in common?

Increased levels of 4-hydroxyglutamate (1, 2, 3).

Germain et al's (2020) new metabolomic study found increased level of plasma 4-hydroxyglutamate  in ME patients compared to controls (3).

From figure S1 in reference 3. 4-hydroxyglutamate. Box plot distribution of logged values from table 2 in reference 3. Controls are shown in red and ME patients in blue. The yellow diamond represents the mean. https://www.mdpi.com/2218-1989/10/1/34


Proline and its metabolite hydroxyproline (OH-proline) are amino acids. They constitute one-third of the amino acids in collagen proteins.

High levels of 4-hydroxyglutamate could result from increased collagen turnover and the release of proline and 4-hydroxyproline. The latter is metabolized to 4-hydroxyglutamate. Proline is re-used and 4-hydroxyproline is broken down and excreted in this pathway (4):



Figure 1 from Riedel et al, reference 4: Metabolism of 4-hydroxyproline and glyoxylate.

Four mitochondrial enzymes are responsible for 4-hydroxproline (4-Hyp) breakdown: hydroxyproline oxidase (HPOX), Δ1-pyrroline-5-carboxylate dehydrogenase (1P5CDH), aspartate aminotransferase (AspAT), and 4-hydroxy-2-oxoglutarate aldolase (HOGA). The terminal HOGA reaction cleaves 4-hydroxy-2-oxoglutarate (HOG) into pyruvate and glyoxylate. Glyoxylate is metabolized either to glycolate by glyoxylate reductase (GR) in the mitochondria and cytoplasm or to glycine by peroxisomal alanine-glyoxylate aminotransferase (AGT). AGT and GR are mutated within primary hyperoxaluria (type 1 and 2, respectively) patients resulting in the buildup of glyoxylate and its conversion by lactate dehydrogenase (LDH) to oxalate, a key component of kidney stones.

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026021

Naviaux et al showed increased plasma level of hydroxyproline in female ME patients (5).

Primary hyperoxaluria type 3 with increased urine 4-hydroxyglutamate is caused by mutations in the HOGA1 gene (4, 6).

Increasd levels of 4-hydroxyglutamate could also result from decreased activity of glutamic-oxaloacetic transaminase 2 (GOT2 = AspAT in figure 1 from ref 4) (1).

Schutzer et al showed decreased level of GOT2 precursor in cerebrospinal fluid from ME patients. Number of unique peptides in cerebrospinal fluid (table S1 in ref. 7): 
1) Controls: 6
2) ME patients: 1
3) Post treatment Lyme patients: 8

Why is 4-hydroxyglutamate increased in ME?

Is the proline-P5C cycle dysregulated in ME?

Naviaux et al showed increased plasma level of 1-pyrroline-5-carboxylic acid  (P5C) in ME patients (5).

A unique aspect of proline metabolism is the cycling of proline and P5C to maintain redox homeostasis between the cytosol and mitochondria. Proline biosynthesis, catabolism, and cycling, known as "the proline-P5C cycle" have been implicated as metabolic pathways selectively altered in cancer cells providing ATP, macromolecules, and redox cofactors (8).

The proline metabolism plays an important role in metabolic reprogramming, not only in cancer but also in related fields such as aging, senescence, and development (9).

The proline-P5C cycle: 

Figure 3 from reference 9. Hypothesis for proline cycle revised.

The cycle has been revised according to locations of the enzymes. The colored areas are for emphasis and do not represent specific locations. The dotted arrows represent putative shuttle systems, for example, malate/aspartate shuttle. PYCR1/2/L, pyrroline-5-carboxylate reductase 1/2/L.

https://www.liebertpub.com/doi/10.1089/ars.2017.7350

Quote from reference 9: 

"The enzyme that oxidizes proline to P5C is tightly bound to mitochondrial inner membranes (30, 37, 38) and is linked to site II of the mitochondrial electron transport chain (30, 61) with a flavine adenine dinucleotide at the active site, which transfers electrons from proline to coenzyme Q (30, 95); at site III, proline-derived electrons have two dispositions. They can be transferred to cytochrome c, which is oxidized at complex IV with electrons transferred to O2 to form H2O. On the contrary, proline-derived electrons can directly reduce dissolved oxygen at complex III to form superoxide (23, 30). Since complex III has access to both the matrix space and the intermembrane space, ROS can evolve in the mitochondrial matrix or in the intermembrane space to be transferred into the cytosol as a putative redox signal. "

Is the collagen being broken down in ME patients to provide electrons for ATP synthesis?

If so, is AMPK involved in the process? And when/if AMPK "gives up", do ME cells go into senescence?

Figure 2 from reference 9. PRODH/POX-mediated signaling. 

AMPK, AMP-activated protein kinase; ETC, electron transport chain; MYC, myelocytomatosis oncogene cellular homologue; PPARγ, peroxisome proliferator-activated receptor gamma. PRODH/POX, proline dehydrogenase/proline oxidase; ROS, reactive oxygen species.

https://www.liebertpub.com/doi/10.1089/ars.2017.7350


A concern for a dysregulated proline-P5C cycle is the potential for cells to accumulate P5C, which was recently listed among the top 30 damage-prone endogenous metabolites. (8, 10).



References: 

1) Sovio et al: 4-Hydroxyglutamate Is a Novel Predictor of Pre-Eclampsia.
PMID: 31098639 DOI: 10.1093/ije/dyz098

2) Pitt et al: 4-hydroxyglutamate Is a Biomarker for Primary Hyperoxaluria Type 3
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4270872/
PMID: 24563386 PMCID: PMC4270872 DOI: 10.1007/8904_2013_291

3) Arnaud Germain , Dinesh K Barupal , Susan M Levine , Maureen R Hanson:
Comprehensive Circulatory Metabolomics in ME/CFS Reveals Disrupted Metabolism of Acyl Lipids and Steroids. Metabolites 2020, 10(1), 4; https://doi.org/10.3390/metabo10010034
https://www.mdpi.com/2218-1989/10/1/34

4) Travis J Riedel , Lynnette C Johnson, John Knight, Roy R Hantgan, Ross P Holmes, W Todd Lowther: Structural and Biochemical Studies of Human 4-hydroxy-2-oxoglutarate Aldolase: Implications for Hydroxyproline Metabolism in Primary Hyperoxaluria.
Plos One https://doi.org/10.1371/journal.pone.0026021https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0026021

5) Naviaux RK, Naviaux JC, Li K, Bright AT, Alaynick WA, Wang L, Baxter A, Nathan N et al (2016) Metabolic features of chronic fatigue syndrome. Proc Natl Acad Sci U S A 113:E5472–E5480. https://doi.org/10.1073/pnas.1607571113

6) Greed, L., Willis, F., Johnstone, L. et al. Metabolite diagnosis of primary hyperoxaluria type 3. Pediatr Nephrol 33, 1443–1446 (2018). https://doi.org/10.1007/s00467-018-3967-6
https://link.springer.com/article/10.1007/s00467-018-3967-6

7) Schutzer et al: Distinct Cerebrospinal Fluid Proteomes Differentiate Post- Treatment Lyme Disease from Chronic Fatigue Syndrome. PLOS One February 2011, volume 6, Issuehttps://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017287

8) John J Tanner, Sarah-Maria Fendt , Donald F Becker , John J Tanner, Sarah-Maria Fendt , Donald F Becker: The Proline Cycle As a Potential Cancer Therapy Target
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6026536/
https://pubs.acs.org/doi/10.1021/acs.biochem.8b00215

9) Phang: Proline Metabolism in Cell Regulation and Cancer Biology: Recent Advances and Hypotheses. Antioxid Redox Signal, 30 (4), 635-649 2019 Feb 1
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338564/

10) Claudia Lerma-Ortiz et al: Nothing of Chemistry Disappears in Biology': The Top 30 Damage-Prone Endogenous Metabolites.
PMID: 27284066 DOI: 10.1042/BST20160073 Biochem Soc Trans (2016) 44 (3): 961–971.
https://doi.org/10.1042/BST20160073