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Regulation of Selective autophagy by sulfide through persulfidation of protein targets.

Periodic Reporting for period 2 - SSHelectPhagy (Regulation of Selective autophagy by sulfide through persulfidation of protein targets.)

Reporting period: 2020-06-15 to 2021-06-14

The overall aim of this project was to shed light to the role of sulfide in the regulation of selective autophagy of ER and Mitophagy, through persulfidation, and to broaden the range of plant targets for reticulophagy and mitophagy that are signaled by sulfide. ATG18a was found to be persulfidated in Arabidopsis under physiological conditions but the role of its persulfidation in autophagy has not been studied. We therefore hypothesized that persulfidation of ATG18a regulates ER stress–induced autophagy in Arabidopsis.
To test this hypothesis, we proposed the following objectives:
O1.1. Study the role of sulfide in ER stress. Functional analysis of ATG18a in vitro.
O1.2. Study of the physiological effects of sulfide under ER stress conditions in Arabidopsis.
O1.3. Functional analysis of persulfidation of ATG18a in ER stress.
The overall aim of the second part of this project was to shed light to the regulation of mitophagy by sulfide and identify new receptors regulated by persulfidation. We therefore hypothesized that sulfide regulates mitophagy in Arabidopsis. To test this hypothesis, we proposed the following objectives:
O2.1 Determine the role of sulfide treatments in mitophagy regulation
O2.2 Isolation and identification of Persulfidated targets during mitophagy.
O2.3. Functional characterization of persulfidated targets.

Autophagy is a conserved degradative process in eucaryotes that serves as a recycling process for misfolded proteins or unfunctional organelles. The research carried out in this project has allowed us to advance in the knowledge of the regulatory mechanisms of autophagy under ER stress. Hydrogen sulfide has been proposed as a regulatory molecule of autophagy, but these results are pioneer in deciphering its way of action through the modification of ATG18a by persulfidation, an autophagy core machinery protein. It has been also demonstrated the effect of sulfide as a negative regulator of mitophagy.
The research developed may have an impact on improvement of crop productivity and adaptation mechanisms of plants against adverse environmental conditions.
Most of the tasks described in the proposal have been accomplished. As a summary, the role of sulfide in the regulation of ATG18a by persulfidation has been deciphered (milestone 1, M-1) and the effect of sulfide in autophagy under ER stress has been determined (M-2). Results obtained have been disseminated in international workshops and other meetings such as outreach activities, and multimedia releases (D-1.2 D-2.2) described in detail in this report. This report includes a description of the identification of cysteine residue modified by persulfidation and the effect of this modification on ATG18a activity (Deliverable1.1 D-1.1) and the physiological effect of sulfide in autophagy during ER stress (D-2.1). The functional effect of persulfidation on ATG18a in ER stress in vivo (M-3) was deciphered during the second part of the project (D-3.1). Besides, results have been published in a peer-reviewed journal with high impact (D-3.2).
The regulation of mitophagy by sulfide (M-4) has been deciphered and results have been included in this report (D-4.1) and other outreach activities (D-4.2) described in detail below. The persulfidated targets involved in mitophagy are still being identified and a list has been achieved (D-5.1 and M-5), which will be published in a manuscript of high impact together with a report of the role of persulfidation of selected targets in mitophagy.
Due to pandemic concerns, milestone-6 and Deliverables-6.1 and D-6.2 have been delayed, which is also described in this report. Nevertheless, part of the results from this project has been disseminated in international workshops fulfilling D-4.2 and also, they were disseminated in international workshops which were postponed until next July 2021 (oral presentation in SEBBM congress) and September 2021 (oral presentation in S-Bio Glucosinolates Workshop), fulfilling the D-6.2.
A total of 6 publications of high impact have been released related with this project:
https://faseb.onlinelibrary.wiley.com/doi/abs/10.1096/fasebj.2020.34.s1.04178
https://doi.org/10.3390/antiox9070621
https://www.pnas.org/content/118/20/e2023604118/tab-article-info
https://academic.oup.com/jxb/advance-article/doi/10.1093/jxb/erab239/6291138
https://www.tandfonline.com/doi/full/10.1080/15548627.2021.1936357
https://academic.oup.com/plcell/article/32/12/3902/6118616
Results have been also disseminated in several talks in workshops,and some of them published in youtube, and posted in twitter. I also participate in other outreach activities such as "La noche europea de los investigadores" (https://lanochedelosinvestigadores.fundaciondescubre.es/actividades/la-importancia-del-reciclaje-celular-y-su-regulacion/ ) and press release (https://www.eldiario.es/andalucia/la-cuadratura-del-circulo/celulas-reciclan_132_6224206.html.)
Sulfide has been proposed in several publications to regulate autophagy in mammals, while in plants it has been recently suggested to be involved in autophagy regulation. Nevertheless, no previous experiments have been performed to reveal this regulation through persulfidation. Besides, no previous research has been focused on regulation of selective autophagy through sulfide.
We will highlight for the first time in plant systems the regulation of selective autophagy, reticulophagy and mitophagy, by sulfide as a signaling molecule.
The front-line knowledge generated in SSHelectPhagy will be used for further research via horizontal transfer, since this innovative research can be of great importance in other eukaryotic systems such as mammals, where this regulation will determine new targets for further studies, including several important human diseases, such as neurodegenerative diseases and cancer.
SSHelectPhagy will contribute to advancing our knowledge of autophagy signaling in response to external stresses, which will allow improving crop yields, which may potentially save billions of Euros worldwide. Overall, the proposed project will create new knowledge leading to a great advancement of this field and will enhance the attractiveness of this signaling mechanism to the scientific community.
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