Malate initiates a proton-sensing pathway essential for pH regulation of inflammation, 2024, Chen et al.

[jnmaciuch]

Malate initiates a proton-sensing pathway essential for pH regulation of inflammation

Yu-jia-nan Chen, Rong-chen Shi, Yuan-cai Xiang, Li Fan, Hong Tang, Gang He, Mei Zhou, Xin-zhe Feng, Jin-dong Tan, Pan Huang, Xiao Ye, Kun Zhao, Wen-yu Fu, Liu-li Li, Xu-ting Bian, Huan Chen, Feng Wang, Teng Wang, Chen-ke Zhang, Bing-hua Zhou, Wan Chen, Tao-tao Liang, Jing-tong Lv, Xia Kang, …Kang-lai Tang

Abstract

Metabolites can double as a signaling modality that initiates physiological adaptations. Metabolism, a chemical language encoding biological information, has been recognized as a powerful principle directing inflammatory responses. Cytosolic pH is a regulator of inflammatory response in macrophages.

Here, we found that L-malate exerts anti-inflammatory effect via BiP-IRF2BP2 signaling, which is a sensor of cytosolic pH in macrophages. First, L-malate, a TCA intermediate upregulated in pro-inflammatory macrophages, was identified as a potent anti-inflammatory metabolite through initial screening. Subsequent screening with DARTS and MS led to the isolation of L-malate-BiP binding.

Further screening through protein‒protein interaction microarrays identified a L-malate-restrained coupling of BiP with IRF2BP2, a known anti-inflammatory protein. Interestingly, pH reduction, which promotes carboxyl protonation of L-malate, facilitates L-malate and carboxylate analogues such as succinate to bind BiP, and disrupt BiP-IRF2BP2 interaction in a carboxyl-dependent manner.

Both L-malate and acidification inhibit BiP-IRF2BP2 interaction, and protect IRF2BP2 from BiP-driven degradation in macrophages. Furthermore, both in vitro and in vivo, BiP-IRF2BP2 signal is required for effects of both L-malate and pH on inflammatory responses.

These findings reveal a previously unrecognized, proton/carboxylate dual sensing pathway wherein pH and L-malate regulate inflammatory responses, indicating the role of certain carboxylate metabolites as adaptors in the proton biosensing by interactions between macromolecules.

Link | PDF

 

[jnmaciuch]

Explain like I'm brain-foggy:

Malate is a TCA cycle metabolite critical for ATP production through oxidative phosphorylation. Its role in cellular metabolism is well characterized.

However, this paper shows that it also exerts an immunomodulatory effect on macrophages that is independent from its role in cellular metabolism.

This happens through L-malate binding to BiP. IRF2BP2 is a protein that normally interrupts interferon production.

Under homeostatic stress conditions, BiP is a protein that binds to and inhibits IRF2BP2, allowing interferon production to ramp up. However, L-malate can bind to BiP and prevent it from binding to IRF2BP2.

pH changes in the cell can make L-malate (and other TCA cycle metabolites) even more effective at this.

The cumulative effect is that L-malate blocks the thing that normally blocks the thing that blocks interferon production...which is all to say that L-malate --> decreased interferon production (where the --> is a little Rube Goldberg machine of its own)

 

[jnmaciuch]

Some interesting tidbits on temporal dynamics:

The abundance of IRF2BP2 protein, the anti-inflammatory downstream effector of BiP-IRF2BP2 signaling, are downregulated in the early stage (8 h) and are recovered in the late stage (24 h) of LPS-induced macrophages activation (Fig. 5h, i), which can be explained by that the proteasome activation mediated by LPS-TLR4 signaling74,75 counterbalances the tendency of BiP-IRF2BP2 signaling to stabilize IRF2BP2 protein in early stage of TLR4 activation and thereby delayed the activation and anti-inflammatory regulation of the BiP-IRF2BP2 pathway. During later stage of LPS stimulation, levels of intracellular L-malate whose anti-inflammatory effect was structure-dependent but metabolism-independent (Supplementary Fig. 6a–i), were further increased (Supplementary Fig. 5i). And consistently, IRF2BP2 deficiency drastically upregulated the levels of IL-1β in 24 h LPS stimulation (Fig. 4p, q and Fig. 8b, d), but during 16 h LPS stimulation, only a moderate increase the levels of IL-1β were detected (Fig. 4m–o, Fig. 8k, l).

 

[SNT Gatchaman]

In this study, we demonstrated that L-malate, a TCA cycle intermediate, directly binds binding immunoglobulin protein (BiP), a heat shock protein 70 (Hsp70) family member that senses endoplasmic reticulum (ER) stress, disrupts the direct binding of BiP with interferon regulatory factor 2-binding protein 2 (IRF2BP2), a known anti-inflammatory protein.

@jnmaciuch you're hypothesising about an impaired malate-aspartate shuttle. Does that mean increased malate generation in the cytosol not transferring across to mitochondria? And might that be bad for other cell types.

Ie could this also relate to the muscle BiP and WASF3 findings if ↑malate → ↓bindable BiP? They showed decreased BiP in ME/CFS when they expected increased, along with PERK following ER stress UPR. If BiP is bound to L-malate would it register as decreased on immunoblot (figure 5B in below paper) or would that not have any effect on that assay?

WASF3 disrupts mitochondrial respiration and may mediate exercise intolerance in myalgic encephalomyelitis/chronic fatigue syndrome (2023, PNAS) —

WASF3 has been reported to be regulated by BiP (GRP78), an endoplasmic reticulum (ER) chaperone for protein quality control whose defective response can cause ER stress and metabolic disorders. Given the importance of the interaction between the ER and mitochondria for muscle function, we reasoned that ER stress, also reported to play a role in rheumatic diseases which often feature fatigue, may regulate WASF3 in muscle cells. Indeed, the ER stress marker PERK was significantly higher while BiP was lower in the ME/CFS muscle samples. This discordance between PERK and BiP levels in ME/CFS samples suggested impairment of the canonical ER stress pathway, termed “ER Stress Response Failure,”

 

[jnmaciuch]

@jnmaciuch you're hypothesising about an impaired malate-aspartate shuttle. Does that mean increased malate generation in the cytosol not transferring across to mitochondria? And might that be bad for other cell types.

It depends on where exactly the malate-aspartate shuttle is being inhibited (if it is at all). If it's being inhibited upstream at succinate dehydrogenase (a target of itaconate), then you would have increased succinate but less fumarate and malate. If it's at another point (e.g. GOT2), then you'd have a situation where malate crosses into the mitochondria once, but can never get back out (after converting to other forms), so it would need to be constantly replenished by another source. In both cases, you're probably ending up with less or "normal" (if adequately replenished by some backup mechanism) cytosolic malate levels.

The findings of paulendat's paper seemed to suggest that, if itaconate is involved, the primary way it leads to increased interferon production is via PRDX5. This might just be another potential way in which itaconate would result in increased interferon production, though it would be dependent on ER stress.

Interestingly, the text noted that succinate and fumarate were also capable of binding to BiP, but I don't think it did any quantification on whether there might be differences between the metabolites.

Ie could this also relate to the muscle BiP and WASF3 findings if ↑malate → ↓bindable BiP? They showed decreased BiP in ME/CFS when they expected increased, along with PERK following ER stress UPR. If BiP is bound to L-malate would it register as decreased on immunoblot (figure 5B in below paper) or would that not have any effect on that assay?

My intuition tells me that L-malate binding would only affect BiP detection if the antibody for BiP directly recognized the binding site with L-malate or any of those other metabolites. In practice I've never come across a situation where that happened (though I'm hardly a western blot expert). Without knowing the exact site of antibody binding on BiP, I can't be sure though.

 

[Creekside]

Would ingested malic acid (apples, sour candies) affect immune activity via this mechanism? I'm intolerant of malic acid, I assume due to my ME.

 

[jnmaciuch]

Would ingested malic acid (apples, sour candies) affect immune activity via this mechanism? I'm intolerant of malic acid, I assume due to my ME.

That’s fascinating—have you happened to see my posts about a malic acid supplement? It was a bit of a miraculous supplement for my ME.

However, only certain sources of malic acid gave me a benefit (pure malic acid or sumac, not apples/apple juice/other fruits). I assumed that something else usually present in fruits (my guess is certain sugars, but I’m not sure. Malonic acid, which has different properties, is another option) counteracted the benefit for me.

What do you mean specifically by intolerance, and which sources of malic acid have you noticed this effect from?

 

[Creekside]

ME is all about treatments that are miraculous for one person, and having the opposite effect for someone else. Even worse, it changes over time, or you might develop or lose an intolerance to one component of a food while another one takes over. So confusing.

For me, malic acid makes my general ME symptoms worse. I recall apples having that effect, and sour candies (sourness from malic acid). I didn't investigate further; just started checking labels for products to avoid. I can't remember how long it took for the effects to show up.

It's too bad that researchers ignore information from PWME (or other diseases). If there was a data bank for this sort of information, I'd be more willing to experiment to record delay time, whether malic acid affects symptoms 24 hrs after exertion (IFN-g effects), etc. Such reports aren't reliable information, since there's no way to verify that the person has ME, but there might be some valuable information. It's not like researchers are being flooded with useful information from blood tests.

 

[jnmaciuch]

For me, malic acid makes my general ME symptoms worse. I recall apples having that effect, and sour candies (sourness from malic acid). I didn't investigate further; just started checking labels for products to avoid. I can't remember how long it took for the effects to show up.

Have you ever taken a pure malic acid supplement? My main concern is that malic acid derived from fruits (or flavorings trying to mimic fruits) usually comes with a bunch of other metabolites, some of which are known have contradictory effects.

It would be very valuable to know if it was malic acid specifically, but that could only be determined in a pure form. Otherwise, your intolerance is just as likely to come from e.g. Malonic acid instead of malic acid, which have very different cellular effects but are both present in the same dietary sources.

Obviously not asking you to take something that might make you sick, just wondering if you’ve happened to try malic acid alone or something like magnesium malate.

It's too bad that researchers ignore information from PWME (or other diseases). If there was a data bank for this sort of information, I'd be more willing to experiment to record delay time, whether malic acid affects symptoms 24 hrs after exertion (IFN-g effects), etc. Such reports aren't reliable information, since there's no way to verify that the person has ME, but there might be some valuable information. It's not like researchers are being flooded with useful information from blood tests.

It’s not systematic but that’s what I’m trying to do with my thread
https://www.s4me.info/threads/malic-acid-supplement-sumac.42716/

 

[Creekside]

Have you ever taken a pure malic acid supplement?

No, it didn't seem worthwhile. Foods such as sour candies likely use fairly pure industrial ingredients, so that was good enough for me.

If intolerance of something, such as malic acid, was something affecting a majority of PWME, it would be worthwhile testing pure ingredients. For something that affects only a few people--and which has a benefit for others--it's a curiosity rather than useful data.

 

[jnmaciuch]

No, it didn't seem worthwhile. Foods such as sour candies likely use fairly pure industrial ingredients, so that was good enough for me.

I just meant isolated malic acid. As in without any other of the metabolites from apples that could be causing the effect instead of malic acid.

I’m mostly interested because Malonic acid, which is also present in apples and apple flavorings in candies, happens to inhibit the exact enzyme I think is relevant in ME/CFS more broadly, though it might only be a strong enough effect to create an “intolerance” in some.

If you’ve ever taken magnesium malate or something similar that would help determine if it’s actually malic acid specifically causing this effect or something else in apples

 

[Creekside]

No I haven't tried any malate supplements. The sour candies weren't apple flavoured. I also tried a bag of uncommon flavoured jellybeans, so plenty of artificial flavours and colours, with no effect, so I'm not sensitive to those in general. I was hoping that something in that wide assortment of chemicals would have an effect, but none occurred.

Since apple intolerance isn't commonly reported by PWME, I think malonic acid is unlikely to be a common factor in ME.

 

[jnmaciuch]

Also interesting to note that IRF2BP2 is enriched in skeletal muscle:
https://faseb.onlinelibrary.wiley.com/doi/abs/10.1096/fj.10.167049

 

[Snow Leopard]

I don't quite understand the biochemistry of the original study, Bip is a HSP/chaperone that does other useful things and BiP agonising IRF2BP2 seems kind of random. I can't find other IRF2BP2 studies that talk about binding BiP at all, nor the converse.

 

[jnmaciuch]

I can't find other IRF2BP2 studies that talk about binding BiP at all, nor the converse.

I assume that’s because they did screens to identify L-malate in the first place, and then screens to identify binding partners, which ultimately led them to IRF2BP2. I would expect this to be the first mention if no one else was suspecting this interaction

I don't quite understand the biochemistry of the original study, Bip is a HSP/chaperone that does other useful things and BiP agonising IRF2BP2 seems kind of random.

Do you mean you don’t trust the methodology, or you don’t understand the “logic” of that binding interaction?

If the latter, I agree it seems kind of random on its face. But also I can see the utility of using interferon to suppress IL-1B under conditions of pH stress in other cell types, or simply as a countermeasure to make sure the IL-1B response does not get too out of control. Even just looking within the IRFs there are plenty of seemingly counterintertuitive relationships, like IRF2 blocking IRF1 binding but only in specific contexts.

 

[Snow Leopard]

Do you mean you don’t trust the methodology, or you don’t understand the “logic” of that binding interaction?

A bit of both but you are correct in suggesting it is mostly the latter. It just seems confusing to me that it would be an important interaction.

 

[jnmaciuch]

A bit of both but you are correct in suggesting it is mostly the latter. It just seems confusing to me that it would be an important interaction.

My sense is that it is relevant in this context specifically because of BiP’s role as a pH sensor, and the fact that pH is a big part of regulating phenotypic shifts in macrophages where this interaction was found. [edit: I suspect this has to do at least partially with upregulation of itaconate leading to increased cytosolic concentrations of acidic TCA cycle metabolites, as well as lactate from a shift to glycolysis]

Per the intro:

The acidic pH in microenvironment is a hallmark of many inflammatory tissues.32,33 In macrophage, TLR4 activation impairs recovery of cytosolic pH in the acidic inflammatory milieu.34 CO2-induced cytosolic acidification has been found to inhibit the inflammatory cytokines TNF and IL-1 in response to LPS.29 Furthermore, the intracellular alkalizers, a Na+/H+ antiporter, promotes IL-1β production in LPS-activated human monocytes.35 These phenomena indicate that pH fluctuations may regulate inflammation and innate inflammatory cytokine through mechanism sensing cytosolic pH, which remains unclear.

It seems like something that would be beneficial in macrophages and probably wouldn’t do much in other cell types that aren’t expected to induce IRF2 highly anyways. Except, perhaps, if you end up with an aberrant parenchymal interferon response in a tissue where ion flux is a big part of cellular function….(playful speculation on that last part)