Proteolethargy is a pathogenic mechanism in chronic disease, 2024, Dall’Agnese et al

[SNT Gatchaman]

Proteolethargy is a pathogenic mechanism in chronic disease
Alessandra Dall’Agnese; Ming M. Zheng; Shannon Moreno; Jesse M. Platt; An T. Hoang; Deepti Kannan; Giuseppe Dall’Agnese; Kalon J. Overholt; Ido Sagi; Nancy M. Hannett; Hailey Erb; Olivia Corradin; Arup K. Chakraborty; Tong Ihn Lee; Richard A. Young

SUMMARY
The pathogenic mechanisms of many diseases are well understood at the molecular level, but there are prevalent syndromes associated with pathogenic signaling, such as diabetes and chronic inflammation, where our understanding is more limited. Here, we report that pathogenic signaling suppresses the mobility of a spectrum of proteins that play essential roles in cellular functions known to be dysregulated in these chronic diseases. The reduced protein mobility, which we call proteolethargy, was linked to cysteine residues in the affected proteins and signaling-related increases in excess reactive oxygen species. Diverse pathogenic stimuli, including hyperglycemia, dyslipidemia, and inflammation, produce similar reduced protein mobility phenotypes. We propose that proteolethargy is an overlooked cellular mechanism that may account for various pathogenic features of diverse chronic diseases.

HIGHLIGHTS
• Pathogenic signaling leads to reduced mobility of proteins with diverse functions

• Reduced protein mobility (proteolethargy) is linked to dysregulated redox environments

• Diverse pathogenic stimuli associated with chronic diseases cause proteolethargy

• Proteolethargy may account for diverse cellular phenotypes seen in chronic diseases

Link | PDF (Cell) [Paywall]

 

[SNT Gatchaman]

Selected quotes from introduction —

Recently, pathogenic signaling in certain chronic diseases was reported to cause reduced movement of receptor molecules into functional protein assemblies. These findings led us to consider the possibility that dysregulated signaling might cause a more general defect in protein mobility in cells and that reduced protein mobility in and of itself might be a pathogenic mechanism shared across these diseases.

Here, we show that pathogenic signaling reduces the mobility of key proteins involved in diverse cellular processes and that this reduction in protein mobility, which we call proteolethargy, is associated with a dysregulated redox environment that consequently impacts oxidation-sensitive cysteines. Reduced protein mobility may account for the diversity of dysregulated cellular processes evident in chronic disease.

 

[SNT Gatchaman]

Selected quotes from results/1 —

Single-particle tracking (SPT) and fluorescence recovery after photobleaching (FRAP) allow for the measurement of the kinetics of protein mobility in living cells

In fasting patients with insulin resistance, liver cells are subject to continuous high concentrations of insulin (~3 nM), and this chronic high level of insulin no longer fully activates the signaling response. Thus, normal and pathogenic insulin signaling can be modeled in cell culture by treating liver-derived cells with normal or elevated (pathogenic) concentrations of insulin for prolonged periods of time

To test the possibility that pathogenic insulin signaling may alter protein mobility, we treated HepG2 cells with normal or pathogenic concentrations of insulin

SPT analysis revealed that the mobility of IR, MED1, HP1a, and FIB1 was reduced in cells that were treated with pathogenic levels of insulin, whereas that of SRSF2 was unaffected

Given the broad range of proteins whose mobility was affected by pathogenic insulin signaling, we asked whether changes in cellular viscosity or in the chemical environment might be responsible for the observed changes in protein mobility.

We detected a change in cytoplasmic viscosity but no change in nuclear viscosity

Substantial changes in the chemical environment are known features of chronic diseases such as insulin resistance due to high levels of ROS. Here, we hypothesize that if an oxidative environment leads to changes in protein mobility, then treating cells with pathologically relevant concentrations of the oxidizing agent H2O2 should phenocopy the effects observed in cells treated with pathogenic insulin signaling. Indeed, FRAP analysis showed that treatment of cells with H2O2 caused reduced mobility of IR, MED1, HP1a, and FIB1 but not of SRSF2 or nuclear GFP.

If high levels of ROS lead to reductions in protein mobility, then treatment with the antioxidant N-acetyl cysteine (NAC) should restore some degree of protein mobility in cells exposed to pathogenic levels of insulin. As expected, FRAP revealed that treating insulin-resistant cells with 1 mM NAC partially rescued the mobility of IR, MED1, HP1a, and FIB1, but it had little effect on the mobility of SRSF2 and nuclear GFP. These results are consistent with the possibility that elevated levels of ROS cause a decrease in the mobility of certain proteins and suggest that the change in protein behavior is caused by an alteration in the oxidative environment.

 

[SNT Gatchaman]

Selected quotes from results/2 —

The sensitivity of proteins to the oxidative environment suggests that oxidation-sensitive amino acids might influence protein mobility. When we analyzed amino acid content, we found that the proteins whose mobility was affected by pathogenic insulin signaling and H2O2 have cysteines with surface-exposed side chains, whereas this was not the case for the proteins whose mobility was not affected by these pathogenic factors. Surface cysteines create the potential for crosslinking through disulfide bonds, which might reduce the rate of diffusion by diverse mechanisms, including increasing effective protein mass, altering protein conformation, promoting binding to immobile proteins, altering interaction with transporters, and increasing cellular viscosity

Taken together, these results indicate that surface-exposed cysteines can affect protein mobility when cells are exposed to oxidative stress and pathogenic signaling.

Next, we asked whether there are reports of any of the proteins studied here having missense mutations resulting in gaining a cysteine and, if so, whether these might affect protein mobility. A tyrosine-to-cysteine mutation (Y1361C) was reported in the IR. This mutation occurs outside of the structured domain and does not appear to decrease protein stability. Modeling indicates that the cysteine gained through this mutation is surface exposed.

We introduced this mutation into the IR-GFP fusion protein (IR Y1361C-GFP) in both alleles in HepG2 cells. By performing FRAP, we found that the gain-of-cysteine mutation caused a reduction in IR protein mobility in HepG2 cells under normal redox conditions and that treating cells expressing IR Y1361C-GFP with NAC enhanced IR Y1361C protein mobility. Mutating the same amino acid to serine had little to no effect on IR protein mobility.

These results indicate that mutations that add surface cysteines sensitize the IR to physiological levels of ROS, reducing its mobility under normal redox conditions, and that addition of an antioxidant can enhance this receptor’s mobility.

Gain-of-cysteine mutations are among the most pathogenic missense mutations, and their effect on protein mobility may not be limited to IR, but it may extend to other disease-relevant proteins.

Pathogenic stimuli that induce oxidative stress include hyperglycemia, high fat, inflammation, genotoxic stress, endotoxin, and drug toxicity. These stimuli have been shown to increase ROS through diverse mechanisms, which include, but are not limited to, dysregulation of mitochondria, dysregulation of redox homeostasis proteins, ER stress, and eNOS dysregulation.

 

[SNT Gatchaman]

Selected quotes from discussion —

Pathogenic signaling contributes to prevalent diseases characterized by dysregulation of remarkably diverse cellular processes. Consequently, equally diverse pathogenic mechanisms are assumed to cause these phenotypes. However, the findings on protein mobility in healthy and dysregulated cells described here suggest an alternative explanation, namely, that a common mechanism—suppressed mobility, here referred to as proteolethargy—contributes to dysregulation of a range of cellular processes in the setting of diverse pathogenic stimuli.

Proteolethargy, the phenomenon of reduced protein mobility in the setting of pathogenic stimuli, might be caused by any number of mechanisms, but several lines of evidence converge on the effects of excess ROS on protein mobility as a common mechanism that can impact proteins throughout the cell in diverse chronic syndromes. Cells exposed to diverse pathogenic stimuli produce excess ROS through mechanisms that include dysregulation of mitochondria, dysregulation of redox homeostasis proteins, ER stress, and eNOS dysregulation, among others.

We estimate that ~50% of human proteins contain at least one surface-exposed cysteine, so there is potential for half of the proteome to be directly susceptible to proteolethargy in high ROS environments.

Variations of this model are possible, where changes in disulfide bond rates or lifetimes have additional influences on protein mobility through improper complex formation, changing protein conformation, promoting binding to immobile proteins, disrupting associations with transport proteins, or altering cytoplasmic viscosity. It is also likely that the effects of elevated oxidative environments can impact protein mobility more indirectly; for example, changes in plasma membrane fluidity due to altered lipid oxidation and composition have the potential to influence protein mobility, and changes that affect cytoskeleton-associated proteins have been noted to impact cellular fluidity.

The model described here for proteolethargy in disease has implications for the development of therapeutics for certain chronic diseases. Restoring protein mobility might be considered among the therapeutic hypotheses for these chronic diseases. Protein mobility biosensors, such as the one developed for this study, may prove to be valuable for high-throughput screening for drugs that restore normal protein mobility under pathogenic signaling conditions.

 

[SNT Gatchaman]

I wonder if we'll detect any potentially relevant gain of cysteine mutations in DecodeME.

 

[forestglip]

Barely understand, but if the main idea, proteolethargy, is that a large variety of proteins are literally swimming more slowly through the blood, then maybe the body would upregulate all those proteins since it's taking them longer than normal to get to their destination - it would compensate by making more?

One of the PolyBio talks was about widespread protein upregulation in long COVID.

Proteomics: Large numbers of proteins involved in a variety of body systems are upregulated in long COVID.

 

[Jonathan Edwards]

I wonder if we'll detect any potentially relevant gain of cysteine mutations in DecodeME.

I didn't think the paper was suggesting mutations in cysteine coding by DNA?
I cannot yet see quite what they are suggesting but I assume some form of post-translational cysteine modification of disulphide bonds.

 

[Jonathan Edwards]

swimming more slowly through the blood, then maybe the body would upregulate all those proteins since it's taking them longer than normal to get to their destination - it would compensate by making more?

Movement of proteins in blood is 99% due to the heart pumping. I think they are talking about movement of proteins from one side of a cell to another - i.e. inside a cell. That is presumably due to some change in surface residues like cysteine.

 

[forestglip]

Movement of proteins in blood is 99% due to the heart pumping. I think they are talking about movement of proteins from one side of a cell to another - i.e. inside a cell. That is presumably due to some change in surface residues like cysteine.

Oh understood, but might upregulation of these proteins still be a consequence in that case?

 

[Jonathan Edwards]

Oh understood, but might upregulation of these proteins still be a consequence in that case?

I don't see any particular reason why. I am finding it hard to see how this concept of proteolethargy relates to disease if it occurs in both diabetes and inflammation, which present with unrelated symptoms. Much of the time diabetics with functional insulin lack are asymptomatic until complications set in or high sugar produces diuresis.

 

[SNT Gatchaman]

I didn't think the paper was suggesting mutations in cysteine coding by DNA?

I think they are suggesting that the subset of normal proteins that have exposed cysteine may be subject to this pathophysiology, but that other proteins might also be involved in some individuals when they have mutations that produce an otherwise unremarkable change to a cysteine residue. I can see that potentially this could explain a genetic predisposition toward ME/CFS. An example might be if GRP78 could be impaired in its translocation between ER lumen, cell surface and nucleus in response to ER stress. Perhaps then regulation of WASF3 might be affected, which then tends to accumulate and interfere with complex III/IV assembly (per Huang et al).

Next, we asked whether there are reports of any of the proteins studied here having missense mutations resulting in gaining a cysteine and, if so, whether these might affect protein mobility. A tyrosine-to-cysteine mutation (Y1361C) was reported in the [insulin receptor]. This mutation occurs outside of the structured domain and does not appear to decrease protein stability. Modeling indicates that the cysteine gained through this mutation is surface exposed.

They then showed that the proteins that don't have the surface-exposed cysteine residue were not directly subject to intracytoplasmic slowing, although there was a small secondary effect on them.

Proteins without surface-exposed cysteines remain in a monomeric state even at higher levels of ROS. As a result, the average diffusion coefficient of proteins containing surface exposed cysteine decreased more than that of proteins lacking surface-exposed cysteines, owing to dimer and multimer formation. The mobility of proteins lacking cysteines slightly decreased at higher levels of ROS, due to the increase in effective viscosity caused by the crosslinking of the proteins containing cysteines present in the environment.

I am finding it hard to see how this concept of proteolethargy relates to disease if it occurs in both diabetes and inflammation, which present with unrelated symptoms. Much of the time diabetics with functional insulin lack are asymptomatic until complications set in or high sugar produces diuresis

I think they're suggesting that oxidative stress would have an effect on proteins in specific cells (likely more than one cell type) contributing to specific diseases, rather than necessarily the whole explanation. I don't know if it would be all or nothing in terms of which proteins, but it sounds as if potentially it could be all that have the surface cysteine / risk of forming disulfide bonds.

We estimate that ~50% of human proteins contain at least one surface-exposed cysteine, so there is potential for half of the proteome to be directly susceptible to proteolethargy in high ROS environments.

Taken together, these results are consistent with a model in which diverse pathogenic stimuli known to induce oxidative stress cause suppressed protein mobility in multiple disease-relevant cell types.

The cellular processes that have been reported to be dysregulated in chronic syndromes include reduced phosphorylation of substrates, altered gene regulation, and repression of heterochromatic repeats, among others. To confirm that these processes are indeed dysregulated in cells under the conditions studied here, we conducted assays in cells that were treated with normal and with pathogenic insulin. The results showed evidence of dysregulated features noted previously in chronic syndromes. Phosphorylation of IRS1 was reduced, genes occupied by the mediator coactivator subunit MED1 were expressed at lower levels, and there was elevated expression of heterochromatic repeats. These results are consistent with a model where reduced protein mobility can contribute to the diversity of dysregulated processes that are evident in chronic disease.

Proteolethargy would be expected to adversely impact diverse functions in cells. In healthy cells, proteins with prominent roles in diverse cellular processes are highly mobile and thus able to transit a space equivalent to the diameter of a cell in 2–10 s. In cells subjected to pathogenic signaling, however, the mobility of most proteins studied here was reduced by 20%–35%. Since many biological processes in cells are collision limited, decreases in protein mobility are expected to reduce functional outputs.

The cellular processes that have been reported to be dysregulated in chronic syndromes such as diabetes and inflammatory disorders are diverse and include signaling activity, gene regulation, heterochromatin repression, and metabolic activity. These cellular functions were found to be dysregulated in the cell system studied here. We thus suggest that proteolethargy may account for the diversity of dysregulated cellular functions noted for at least some chronic diseases.

So simplistically ME/CFS might become a stable pathological state if there were initiation with high ROS during infection and then continued reinforcement with mitochondrial dysfunction that keeps the ROS generation high enough?? What then stops that happening in everyone? Double-hit? Some threshold of high-enough ROS generation? (Noting that identical twins can be affected / unaffected)

 

[Jonathan Edwards]

ROS generation in infection is local and as far as I know not a major feature of viral infection.
As an immunologist this quotes all sound to me like stringing together various popular memes without any real understanding of inflammation.

And presumably if people had mutations in gremlin encoding unusual cysteines that led to movement problems that would not need to have anything to do with ROS anyway.

To me it is a bit like trying to explain why someone won a game of bridge using the rules of snap. Nothing is in perspective.

 

[Murph]

This is an unusual but deeply-researched paper, by leading biologists out of MIT in the US. Their work is beautifully explained by @SNT_gatchaman .

If I understand it right they're proposing a whole new hypothesis for why oxidative stress can impair cellular function: simply by slowing the rate at which reactions happen (fewer collisions) and the speed with which proteins move to their destinations.

Their conclusion is consistent with their findings but of course it might not pan out. A new paradigm like this needs to be solidified.

If it is supported, measuring protein speeds could become useful to see how well a proposed treatment is working, in vitro. Have an in vitro model, apply some molecule, see if the proteins that trail a cysteine speed up at all. It's not obvious you could measure it in vivo.

I know I clicked on it because the term proteolethargy is novel and fun, it's not upstream enough to be specific to me/cfs, and the authors don't claim it is. But it certainly could be a downstream factor that is in play, especially in PEM. Oxidative stress is famously one of our problems. And some people claim benefit from NAC.