B12 and Nrf2

Keap1 and Nrf2 have been introduced in the ozone therapy post. After revisiting a previous post this post will take a deep dive into what controls the transcription of the NFE2L2 gene that codes for the transcription factor protein Nrf2. The region upstream of protein coding part of the human NRF2L2 gene, the promoter, has many single nucleotide polymorphisms (SNPs) associated with disease states. The post was originally going to focus on a study postulating that decreased Nrf2 expression is at the root of messed up B12/cobalamin species and thiol status in autism. The ins and outs of the NFE2L2 promoter are examined. This post will conclude that increasing production of the Nrf2 protein will help more humans than just those with autism.

Nrf2 response to oxidative stress revisited

Nrf2 is an abbreviation for Nuclear factor erythroid 2-related factor 2 the protein product of the NFE2L2 gene. In the featured publication mRNA quantities are referred to by the gene name rather than the protein name. Not shown in this cartoon are the many reduced thiols of KEAP1 that are sensitive to reactive oxygen species. When these thiols are reduced NRF2 is targeted to ubiquitination that targets it for degradation by proteosomes. Oxidation of Keap1 thiols releases NRF2 to translocate to the nucleus to promote the transcription of genes associated with the oxidative stress response.

Note that a lot of cartoons revealed by an image search reveal MAF binding slightly downstream, closer to the protein coding part of the gene, than Nrf2. MAF binds to the anti-oxidant response element ARE site as well as many more sites listed MAF in UniProt.org.

Make careful note of Mrf2 and its partner MAF binding to the Anti-oxidant response element (ARE) of the Nrf2 gne NFE2L2. We are about to learn that there are other transcription factors and response elements of this same gene.

The Nrf2 gene NFE2L2 [1]

If NFE2L2 single nucleotide polymorphisms (SNPs) are important in disease states, what controls the transcription of the protein coding parts of the NFE2L2 gene? A 2007 study revealed a functional polymorphisms in the promoter of the NFE2L2 gene in humans increase the risk of acute lung injury. The nice thing about this paper is that the authors listed transcription factor binding sites. The also found a SNP at position -617. This position-617 lines up close to some hyper methylation sites, see below. In modern terminology:

• position -653 (A is wildtype) in NFE2L2 in MZF1, now known as rs35652124:
• position -617(C is wildtype) in NFE2L2 in ARE, now known as rs6721961,

SNPs and promoter activity

This image combines data from two related studies [1,2]. Luciferase is an enzyme in the hind organ of fire flies. One can measure the strength of the promoter by light generation by lucifierase.

It should be noted that Nrf-2 binds to its own promoter. In other works, Nrf-2 can increase transcription of its own gene in times of oxidative stress. Also note that these SNPs at position -617 can decrease promoter activity. UniProt does not have a lot to say about myeloid zinc finger 1. AP-1 is a hetero dimer transcription factor that binds to the AP-1 site. Data from a more recent 2016 vitiligo study [2] have been added to this figure. Song and coauthors were interested in SNPs in the NFE2L2 gene that might predispose a patient for loss of skin pigmentation later in life. [2]

We might have the same exact sequence of our Nrf2 protein, but slight differences in the promoter can change how our bodies produce this anti-oxidant transcription factor.

rs35652124, a popular NFE2L2 SNP

This is a table of PbMed searches. The exact variant is not always specified. Some SNPs are protective, some are the opposite.

Year	country	condition	comment	PMID
2007	Japan	acute lung injury after major trauma	(-617 C/A) decreased the binding affinity (P<0.001) vs wild type (-617 CC).	17384144
2020	UK	adverse drug reaction frailty	G allele (AG/GG) had a significantly lower frailty score compared homozygous A	31102514
2013	USA	forearm vasodilator response	– 617A higher FVR at baseline response to bradykinin or sodium nitroprusside vs wild-type (P < 0.05 for each	PMC3599320
2013	Portugal	Hashimoto’s Thyroiditis	not in isolation	PMC6760180
2023	Solvenia	Alzheimer’s Disease	cognitive test score rs35652124 (p = 0.030).	PMC9952323
2016	China	vitiligo	C protective T less transcription.	PMC5020638
2021	Egypt	vtiligo	can be significant depends on other allele.	PMC8594532
2019	Portugal	hormone dependent breast cancer	none of the NFE2L2 SNPs mattered that much.	30941643
2014	multi Europe	Parkinson’s	rs35652124 A > G, -1.1 years per allele, p = 0.045;	PMC4335439
2017	Sweden	early onset Parkinson’s	recessive model (AA + GA v GG) and dominant model (AA v GA + GG), association under a dominant model (p = 0.013) causality between the absence of G alleles and EO PD	PMC5433415
2014	Japan	hypertension	males, cholinesterase high, and HDL cholesterol low in (AG+GG) carriers. female, diastolic BP was low in (AG+GG) carriers.	PMC4345725
1014	Japan	hypertension	AA higher cardiovascular mortality than (GG+GA)	PMC4045792
2023	Russian USA	autism	ASD children with NRF2 rs35652124 CC higher DNA damage marker H2AX	PMC10048604
2020	India	diabetic foot ulcer	TT var significant risk T2DM , p = 0.01 DFU p < 0.0001	PMC7448237
2021	China	type A aortic dissection	CC higher brain ischemia (9% vs. 0%, p = 0.045).	PMC8313362
2022	Mexico	kidney disease, hypertension..	A allele decreased risk CKD	35881855
2019	Brazil	alcoholic liver disease	A risk factor, G protects	PMC6678089
2016		COPD	KEAP1 and other gene SNP required	30641209

Mzf1 binds to the rs35652124 region and is expressed in the brain

A PubMed search on Mzf1 was not particularly informative as to the regulation and function under well defined environmental conditions. Protein Atlas was consulted for the expression. Because Atlas Antibodies has performed histology, we have an idea of protein expression as well as mRNA transcripts.

Mzf1 seems to be preferential expressed in glia. A PubMed search on Mrf1 and glia reveals a report that a dopamine D1/S2 receptor agonists induces the transcription of the fibroblast grow factor 2 in cultured astrocytes. This article also looked at possible post translational modification of Znf1 elected by D1/D2 receptor activation. The promoter of the FGF2 gene was found to have a binding site for Mzf1. Mzf1 was shown to regulate he transcription of FGF2. We may speculate that an astrocyte stimulated with dopamine might also want to ramp up its anti-oxidant defense by expressing more Nrf2. In this context, the Mzf1 site on the NFE2L2 promoter might be important.

This seems so very crazy and overwhelming. The NFE2L2 gene is unquestionably very important in our response to ROS. The scientific community is so aghast in one SNP region of a response element of a virtually unknown transcription factor.

Decreased cortical Nrf2 gene expression in autism [3]

This section is a continuation of the B12 and glutathione post. This work also came out of Richard C Deth’s laboratory. In their introduction Schrier and coauthors reviewed the interaction between glutathione and B12/Cbl covering similar information in the B12 and glutathione post on this site.

CblD (TMMADHC) associates with CblC (MMACHC). We are reminded that this complesx covalently binds Cbl(II) via a cysteine residue. The two chaperones deliver cobalamin to methionine synthase (MS) or to mitochondrial methylmalonyl-CoA mutase (MUT). Mitochondrial cobalamin is converted to AdoCbl(III), the cofactor for which converts methylmalonyl-CoA (MMA-CoA) to succinyl CoA.

Gene expression

• CBS Cystathionine beta Synthase catalyzing the first step of the transsulfuration pathway, where the hydroxyl group of L-serine is displaced by L-homocysteine in a beta-replacement reaction to form L-cystathionine, the precursor of L-cysteine.
• CTH, Cystathionase aka Cystathionine gamma lyase
• slca1a, Excitatory Amino Acid Transporter 3 Sodium-dependent, high-affinity amino acid transporter that mediates the uptake of L-glutamate and also L-aspartate and D-aspartate. Can also transport cysteine

Cysteine and other excitatory amino acids may not be taken up as well in ASD as in the control. .

• GS glutathione synthase
• GSR, glutathione reductase
• NFE2L2 Nuclear Factor (Erythroid- Derived 2)-like 2, Like NRF2 it is normally ubiquitinated and degraded in the cytoplasm by the BCR(KEAP1) complex. Under oxidative stress conditions it is released and binds to the ARE element of promoters.

With the NFE2L2 mRNA that codes of the Nrf2 transcription factor we are getting to the meat of the argument: Maybe low NFE2L2 mRNA is a causative factor in low Cbl.

Correlation between NLE2L2 and Cbl species

• MeCbl. active form panel a, positive correlation
• AdoCbl, an active form, panel b, no correlation
• OHCbl,hydroxocobalamin(III),panel c, not yet active form. Note the strong inverse correlation
• GSCbl, glutathionylcobalamin(III), panel d, also an intermediate form. slight positive correlation.
• CNCbl cyanocobalamin(III), panel e, no correlation… as if in ASD Cbl is stuck in an intermediate form.
• total -cobalamin, panel f, strong positive correlation
• % OHCbl, There is a very strong inverse correlation with NFW2L2 mRNA and this intermediate form of Cbl. Note that the controls have very little OHCbl.

Does Nrf2 related gene expression correlate with Cbl?

Some tissues may be naturally exposed to more oxidative stress and hence need more NRF2. Do they also have more enzymes needed to process and maintain Cbl levels?

Panel B examines Nrf2 genes rather than total protein levels. Nrf2 and NfW2L2 are both kept by Keap1. Does NFE expression correlate with Cbl? Does NFW2L2 expression correlate with Nrf2 genes?

The model proposed in the Schrier study is as follows:

Methionine synthase reductase MSR Key enzyme in methionine and folate homeostasis responsible for the reactivation of methionine synthase (MTR/MS) activity by catalyzing the reductive methylation of MTR-bound cob(II)alamin. ASD is proposed to be a combination of

• an innate deficit in NFE2L2 transcripts coupled to
• an increased rate of Nrf2 protein turnover followed by
• decreased antioxidant capacity that
• promotes oxidative conditions.

Kicking off R groups like -OH requires GS^–

R-Cob(III)alamin + GS^– → GSR + Cob(I)alamin → Cob(I) +e^– → Cob(II)alamin

This equation is from the Banerjee review covered in the previous post on Cbl and GSH. This post went down the rs35652124, the SNP that occurs in many disease sates. This Deth lab model may apply to multiple disease states.

“During the MS catalytic cycle, cobalamin alternates between fully-reduced Cbl(I), without an upper axial ligand, and a Cbl(III) species covalently bound by a methyl group – MeCbl(III). Cbl(I) periodically oxidizes to Cbl(II) under normal conditions, inactivating the enzyme. Reductive methylation with the assistance of MSR, re-forms Cbl(I) penultimately to MeCbl(III). However, greater presence of ROS in Nrf2-deficient cells, leads to a higher probability that oxidation of Cbl(I) to Cbl(II), will out compete MSR-mediated reactivation. MS will increasingly be inactive in an antioxidant-deficient, pro-oxidative environment. Homocysteine will preferentially be processed by the transsulfuration pathway, and the methionine cycle, along with methylation capacity, will be downregulated as a consequence. This carries the potential for maladaptive epigenetic effects, resulting in altered neurodevelopment.”

The Deth lab is building a very strong case for a causitive relationship between a transcript and active forms of Cbl, or perhaps avoidance of being stuck with an intermediate form like OHCbl. These authors did not stop with a NFE2L2 mRNA that may or may not get translated into active Nrg2 transcription factor.

Pulling the reigns on the NFE2L2 promoter, back to B12/Cbl

It is truly hard to write anything on this promoter. An attempt was made to review an autism study that compared lymphocytes with children with ASD with those from normal adults. The authors mentioned the Nrf-2 agonist dimethlyl fumarate but didn’t really say how this compound was used to stimulate lymphocytes. The homozygous, heterozygous, and haploid statuses seem to be a rabbit hole that this post just cannot explore. Perhaps we simply need to think of Cbl/B12 co vitamin induces of Nrf2 such as alpha lipoic acid and maybe low dose ozone therapy. The role of agonists of AMP/PKA and PKC systems controlling anything with an MZF1 element in its promoter [4] is compelling. Whatever SNP glitches in the response elements of our NFE2L2 promoters might be compensated by activating other transcription factors.

References

1. Marzec JM, Christie JD, Reddy SP, Jedlicka AE, Vuong H, Lanken PN, Aplenc R, Yamamoto T, Yamamoto M, Cho HY, Kleeberger SR. Functional polymorphisms in the transcription factor NRF2 in humans increase the risk of acute lung injury. FASEB J. 2007 Jul;21(9):2237-46. Sci-Hub free paper
2. Song P, Li K, Liu L, Wang X, Jian Z, Zhang W, Wang G, Li C, Gao T. Genetic polymorphism of the Nrf2 promoter region is associated with vitiligo risk in Han Chinese populations. J Cell Mol Med. 2016 Oct;20(10):1840-50. PMC free article
3. Schrier MS, Zhang Y, TrTXNivedi MS, Deth RC. Decreased cortical Nrf2 gene expression in autism and its relationship to thiol and cobalamin status. Biochimie. 2022 Jan;192:1-12. free paper
4. Luo X, Zhang X, Shao W, Yin Y, Zhou J. Crucial roles of MZF-1 in the transcriptional regulation of apomorphine-induced modulation of FGF-2 expression in astrocytic cultures. J Neurochem. 2009 Feb;108(4):952-61. Sci-Hub free article