THE “THERMOSTAT” OF CELLULAR OXIDATION: DISCOVERED THE MECHANISM OF AQUAPORIN GATING

Each cell of our body is in continuous “dialogue” with the surrounding environment: the exchange of molecules is the most used means to receive and give information. Aquaporins, as you can easily guess from the name, are pores (more precisely, channels) integrated within the cell membrane, which allow the passage of water molecules.

Predicted AQP8 structure, seen from above. Kind courtesy of Dr. Medraño-Fernandez.
Predicted AQP8 structure, seen from above. Kind courtesy of Dr. Medraño-Fernandez.

One of the molecules involved in several cellular processes is hydrogen peroxide (H2O2); normally, H2O2 cannot freely spread through cell membranes, but it needs specific channels that facilitate its entry and exit from the cell. Many studies in the literature have recently demonstrated that members of the aquaporin family also perform this function: to favor the passage of hydrogen peroxide.

Cells, however, need to carefully monitor the entry of this molecule, which is a powerful oxidizing agent. At physiological concentrations, it helps maintaining the oxidative balance of the cell, as high concentrations of H2O2 can alter this balance, thus causing an oxidative stress that produces damage to different intracellular structures. For this reason, it is essential that the cell keeps its intracellular H2O2 concentration under control.

The research we are telling about adds an important piece to the study of the regulation of hydrogen peroxide transport and oxidative stress. The protagonist of this study, published on the prestigious international journal Science Advances, is aquaporin-8 (AQP8), a channel belonging to the aquaporins family.

The main authors of the research are the researchers of the “Protein transport and secretion” Unit headed by Roberto Sitia, Full Professor of Molecular Biology at the Vita-Salute San Raffaele University.

AQUAPORIN GATING MECHANISM

According to Dr. Iria Medraño-Fernandez, among the authors and coordinators of the research: “In two previous studies, we had shown that i) AQP8 was able to transport H2O2 through the plasma membrane, and ii) that this channel was reversibly gated under conditions of cellular stress. In the present study we have gone a step forward, discovering the mechanism responsible for channel closure”.

Three are the key-steps:

  1. AQP8 is located at the plasma membrane, where allows the transport of molecules such as water and hydrogen peroxide. Under physiological conditions, one of the amino acids [the “bricks” that constitute a protein, Editor’s Note] that make up AQP8, cysteine 53 (C53), has a sulfur atom (S) which – due to its chemical nature and strategic position at the entrance to the canal – shows particular reactivity towards the oxidizing agents entering through the aquaporin.
  1. As H2O2 flows through the channel, the reduced cysteine reacts with hydrogen peroxide, creating a chemical form called sulfenic acid (C53-SOH): this oxidation is essential, because it makes C53 more susceptible to further chemical reactions.
  2. When excess H2O2 accumulates, like for instance in stressed cells, an enzyme is activated, cystathionine β-synthase (CBS), which is located nearby AQP8. An important function of CBS is the production of hydrogen sulphide (H2S) that interacts with C53-SOH to form a C53-SSH complex. This modification pushes another very bulky amino acid into the channel, thus causing its closure, consequently preventing the entry of more hydrogen peroxide.
aquaporin-scheme_eng

Dr. Stefano Bestetti, PostDoc at the “Protein transport and secretion” Unit and paper’s first author, explains: “We treated cells in culture with hydrogen sulphide (H2S) (left panel), and after the addition of H2O2 (right panel), only cells expressing AQP8 C53S (a mutant version of the protein, without the key-amino acid for regulation, replaced by a serine) were able to import hydrogen peroxide; on the contrary, those expressing AQP8 wt (“wild-type”, i.e. the “regular” version of the protein) did not import H2O2. Together with other experiments we have conducted, this result has shown that the C53 residue is the main target of H2S and necessary to regulate the entry of H2O2 into cells.

Top: cells expressing the wt version of the protein (left panel): after treatment with H2S, they cannot import H2O2 (compare the central panel with the one on the right). Bottom: cells expressing the mutated C53S protein: even after treatment with H2S, they are able to import H2O2, as it can be seen from the red color that stains cells in the presence of H2O2 (right panel). Kind courtesy of Dr. Stefano Bestetti.
Top: cells expressing the wt version of the protein (left panel): after treatment with H2S, they cannot import H2O2 (compare the central panel with the one on the right). Bottom: cells expressing the mutated C53S protein: even after treatment with H2S, they are able to import H2O2, as it can be seen from the red color that stains cells in the presence of H2O2 (right panel). Kind courtesy of Dr. Stefano Bestetti.

We can think of this system as a sort of “redox-stat”, that is a “thermostat” that controls and regulates the oxidative state of the cell” explains Prof. Sitia.

Once the channel is closed, how is the permeability of AQP8 restored? “This remains an open question”, the researchers reflect; “further studies are needed to identify the molecular players involved in channel reopening”.

Dr. Medraño-Fernandez observes: “Our study has identified a new mechanism through which cells integrate different signals to adapt to changes in the environment. We believe that this is a remarkable result, if we consider that this could be involved in diseases such as cancer and chronic inflammation”.

Finally, Prof. Sitia confirms: “By replacing the C53 with a serine (which, has an oxygen atom in the place of the sulfur atom of a cysteine), the AQP8 is no longer modulable during stress. Of particular interest, tumor cells expressing this mutant grow faster and are more resistant to chemotherapeutics or radiation. These observations make these results particularly relevant for molecular oncology, since most human tumors show alterations in the redox homeostasis that contribute to their aggressiveness. The molecules and chemical reactions that we have identified in this study may therefore represent new therapeutic targets for such pathologies”.

The “Protein transport and secretion” Unit. Prof. Sitia is standing next to the blackboard, wearing a dark green cardigan; Dr. Bestetti is the fifth from the right; on his right, Dr. Medraño-Fernandez.
The “Protein transport and secretion” Unit. Prof. Sitia is standing next to the blackboard, wearing a dark green cardigan; Dr. Bestetti is the fifth from the right; on his right, Dr. Medraño-Fernandez.

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