RESUMO
Dominant variants in WFS1 (wolframin ER transmembrane glycoprotein), the gene coding for a mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) resident protein, have been associated with Wolfram-like syndrome (WLS). In vitro and in vivo, WFS1 loss results in reduced ER to mitochondria calcium (Ca2+) transfer, mitochondrial dysfunction, and enhanced macroautophagy/autophagy and mitophagy. However, in the WLS pathological context, whether the mutant protein triggers the same cellular processes is unknown. Here, we show that in human fibroblasts and murine neuronal cultures the WLS protein WFS1E864K leads to decreases in mitochondria bioenergetics and Ca2+ uptake, deregulation of the mitochondrial quality system mechanisms, and alteration of the autophagic flux. Moreover, in the Wfs1E864K mouse, these alterations are concomitant with a decrease of MAM number. These findings reveal pathophysiological similarities between WS and WLS, highlighting the importance of WFS1 for MAM's integrity and functionality. It may open new treatment perspectives for patients with WLS.Abbreviations: BafA1: bafilomycin A1; ER: endoplasmic reticulum; HSPA9/GRP75: heat shock protein family A (Hsp70) member 9; ITPR/IP3R: inositol 1,4,5-trisphosphate receptor; MAM: mitochondria-associated endoplasmic reticulum membrane; MCU: mitochondrial calcium uniporter; MFN2: mitofusin 2; OCR: oxygen consumption rate; ROS: reactive oxygen species; ROT/AA: rotenone+antimycin A; VDAC1: voltage dependent anion channel 1; WLS: Wolfram-like syndrome; WS: Wolfram syndrome; WT: wild-type.
RESUMO
BACKGROUND: Plant-derived nanovesicles (PDNVs) are a novelty in medical and agrifood environments, with several studies exploring their functions and potential applications. Among fruits, apples (sp. Malus domestica) have great potential as PDNVs source, given their widespread consumption, substantial waste production, and recognized health benefits. Notably, apple-derived nanovesicles (ADNVs) can interact with human cell lines, triggering anti-inflammatory and antioxidant responses. This work is dedicated to the comprehensive biochemical characterization of apple-derived nanovesicles (ADNVs) through proteomic and lipidomic analysis, and small RNAs sequencing. This research also aims to shed light on the underlying mechanism of action (MOA) when ADNVs interface with human cells, through observation of intracellular calcium signalling in human fibroblasts, and to tackles differences in ADNVs content when isolated from fruits derived from integrated and organic production methods cultivars. RESULTS: The ADNVs fraction is mainly composed of exocyst-positive organelles (EXPOs) and MVB-derived exosomes, identified through size and molecular markers (Exo70 and TET-3-like proteins). ADNVs' protein cargo is heterogeneous and exhibits a diverse array of functions, especially in plant's protection (favouring ABA stress-induced signalling, pathogen resistance and Reactive Oxygen Species (ROS) metabolism). Noteworthy plant miRNAs also contribute to phytoprotection. In relation with human cells lines, ADNVs elicit spikes of intracellular Ca2+ levels, utilizing the cation as second messenger, and produce an antioxidant effect. Lastly, organic samples yield a substantial increase in ADNV production and are particularly enriched in bioactive lysophospholipids. CONCLUSIONS: We have conclusively demonstrated that ADNVs confer an antioxidant effect upon human cells, through the initiation of a molecular pathway triggered by Ca2+ signalling. Within ADNVs, a plethora of bioactive proteins, small RNAs, and lipids have been identified, each possessing well-established functions within the realm of plant biology. While ADNVs predominantly function in plants, to safeguard against pathogenic agents and abiotic stressors, it is noteworthy that proteins with antioxidant power might act as antioxidants within human cells.