Metabolomics insights into the potential of encapsulated essential oils as multifunctional food additives.

Growing consumer concern about foodborne disease outbreaks and health risks associated with chemical additives has propelled the usage of essential oils (EOs) as novel food additives, but are limited by instability. In this regard, a series of EOs nano/micro-capsules have been widely used to enhance their stability and improve food quality. However, classical food quality assessment methods are insufficient to fully characterize the effects of encapsulated EOs on food properties, including physical, biochemical, organoleptic, and microbial changes. Recently, the rapid development of high-throughput sequencing is accelerating the application of metabolomics in food safety and quality analysis. This review seeks to present the most recent achievements in the application of non-targeted metabolomics to identify and quantify the overall metabolite profile associated with food quality, which can guide the development of emerging food preservation technologies. The scientific findings confirm that metabolomics opens up exciting prospects for biomarker screening in food preservation and contributes to an in-depth understanding of the mechanisms of action (MoA) of EOs. Future research should focus on constructing food quality assessment criteria based on multi-omics technologies, which will drive the standardization and commercialization of EOs for food industry applications.

Nondegradable ubiquitinated ATG9A organizes Golgi integrity and dynamics upon stresses.

ATG9A is a highly conserved membrane protein required for autophagy initiation. It is trafficked from the trans-Golgi network (TGN) to the phagophore to act as a membrane source for autophagosome expansion. Here, we show that ATG9A is not just a passenger protein in the TGN but rather works in concert with GRASP55, a stacking factor for Golgi structure, to organize Golgi dynamics and integrity. Upon heat stress, the E3 ubiquitin ligase MARCH9 is promoted to ubiquitinate ATG9A in the form of K63 conjugation, and the nondegradable ubiquitinated ATG9A disperses from the Golgi apparatus to the cytoplasm more intensely, accompanied by inhibiting GRASP55 oligomerization, further resulting in Golgi fragmentation. Knockout of ATG9A or MARCH9 largely prevents Golgi fragmentation and protects Golgi functions under heat and other Golgi stresses. Our results reveal a noncanonical function of ATG9A for Golgi dynamics and suggest the pathway for sensing Golgi stress via the MARCH9/ATG9A axis.

Aminoacyl-tRNA synthetase inhibition activates a pathway that branches from the canonical amino acid response in mammalian cells.

Signaling pathways that sense amino acid abundance are integral to tissue homeostasis and cellular defense. Our laboratory has previously shown that halofuginone (HF) inhibits the prolyl-tRNA synthetase catalytic activity of glutamyl-prolyl-tRNA synthetase (EPRS), thereby activating the amino acid response (AAR). We now show that HF treatment selectively inhibits inflammatory responses in diverse cell types and that these therapeutic benefits occur in cells that lack GCN2, the signature effector of the AAR. Depletion of arginine, histidine, or lysine from cultured fibroblast-like synoviocytes recapitulates key aspects of HF treatment, without utilizing GCN2 or mammalian target of rapamycin complex 1 pathway signaling. Like HF, the threonyl-tRNA synthetase inhibitor borrelidin suppresses the induction of tissue remodeling and inflammatory mediators in cytokine-stimulated fibroblast-like synoviocytes without GCN2, but both aminoacyl-tRNA synthetase (aaRS) inhibitors are sensitive to the removal of GCN1. GCN1, an upstream component of the AAR pathway, binds to ribosomes and is required for GCN2 activation. These observations indicate that aaRS inhibitors, like HF, can modulate inflammatory response without the AAR/GCN2 signaling cassette, and that GCN1 has a role that is distinct from its activation of GCN2. We propose that GCN1 participates in a previously unrecognized amino acid sensor pathway that branches from the canonical AAR.

Regulation of mATG9 trafficking by Src- and ULK1-mediated phosphorylation in basal and starvation-induced autophagy.

Autophagy requires diverse membrane sources and involves membrane trafficking of mATG9, the only membrane protein in the ATG family. However, the molecular regulation of mATG9 trafficking for autophagy initiation remains unclear. Here we identified two conserved classic adaptor protein sorting signals within the cytosolic N-terminus of mATG9, which mediate trafficking of mATG9 from the plasma membrane and trans-Golgi network (TGN) via interaction with the AP1/2 complex. Src phosphorylates mATG9 at Tyr8 to maintain its endocytic and constitutive trafficking in unstressed conditions. In response to starvation, phosphorylation of mATG9 at Tyr8 by Src and at Ser14 by ULK1 functionally cooperate to promote interactions between mATG9 and the AP1/2 complex, leading to redistribution of mATG9 from the plasma membrane and juxta-nuclear region to the peripheral pool for autophagy initiation. Our findings uncover novel mechanisms of mATG9 trafficking and suggest a coordination of basal and stress-induced autophagy.

Structural basis for the phosphorylation of FUNDC1 LIR as a molecular switch of mitophagy.

Mitophagy is a fundamental process that determines mitochondrial quality and homeostasis. Several mitophagy receptors, including the newly identified FUNDC1, mediate selective removal of damaged or superfluous mitochondria through their specific interaction with LC3. However, the precise mechanism by which this interaction is regulated to initiate mitophagy is not understood. Here, we report the solution structure of LC3 in complex with a peptide containing the FUNDC1 LC3-interacting region (LIR) motif. The structure reveals a noncanonical LC3-LIR binding conformation, in which the third LIR residue (Val20) is also inserted into the hydrophobic pocket of LC3, together with the conserved residues Tyr18 and Leu21. This enables Tyr18 to be positioned near Asp19 of LC3, and thus phosphorylation of Tyr18 significantly weakens the binding affinity due to electrostatic repulsion. Functional analysis revealed that mitochondrial targeting of the LIR-containing cytosolic portion of FUNDC1 is necessary and sufficient to initiate mitophagy when Tyr18 is unphosphorylated, even in the absence of mitochondrial fragmentation. Thus, we demonstrated that phosphorylation of Tyr18 of FUNDC1 serves as a molecular switch for mitophagy. This may represent a novel target for therapeutic intervention.

Mitochondrial outer-membrane E3 ligase MUL1 ubiquitinates ULK1 and regulates selenite-induced mitophagy.

Mitochondria serve as membrane sources and signaling platforms for regulating autophagy. Accumulating evidence has also shown that damaged mitochondria are removed through both selective mitophagy and general autophagy in response to mitochondrial and oxidative stresses. Protein ubiquitination through mitochondrial E3 ligases plays an integrative role in mitochondrial outer membrane protein degradation, mitochondrial dynamics, and mitophagy. Here we showed that MUL1, a mitochondria-localized E3 ligase, regulates selenite-induced mitophagy in an ATG5 and ULK1-dependent manner. ULK1 partially translocated to mitochondria after selenite treatment and interacted with MUL1. We also demonstrated that ULK1 is a novel substrate of MUL1. These results suggest the association of mitochondria with autophagy regulation and provide a new mechanism for the beneficial effects of selenium as a chemopreventive agent.

Hypoxia regulates Hippo signalling through the SIAH2 ubiquitin E3 ligase.

The Hippo signalling pathway plays important roles in animal development, physiology and tumorigenesis. Understanding how the activity of this pathway is regulated by the cellular microenvironment remains a major challenge. Here we elucidate a molecular mechanism by which hypoxia deactivates Hippo signalling. We demonstrate that the E3 ubiquitin ligase SIAH2 stimulates YAP by destabilizing LATS2, a critical component of the Hippo pathway, in response to hypoxia. Loss of SIAH2 suppresses tumorigenesis in a LATS2-dependent manner in a xenograft mouse model. We further show that YAP complexes with HIF1α and is essential for HIF1α stability and function in tumours in vivo. LATS2 is downregulated in human breast tumours and negatively correlates with SIAH2 expression levels, indicating that the SIAH2-LATS2 pathway may have a role in human cancer. Our data uncover oxygen availability as a microenvironment signal for the Hippo pathway and have implications for understanding the regulation of Hippo signalling in tumorigenesis.

A regulatory signaling loop comprising the PGAM5 phosphatase and CK2 controls receptor-mediated mitophagy.

Mitochondrial autophagy, or mitophagy, is a major mechanism involved in mitochondrial quality control via selectively removing damaged or unwanted mitochondria. Interactions between LC3 and mitophagy receptors such as FUNDC1, which harbors an LC3-interacting region (LIR), are essential for this selective process. However, how mitochondrial stresses are sensed to activate receptor-mediated mitophagy remains poorly defined. Here, we identify that the mitochondrially localized PGAM5 phosphatase interacts with and dephosphorylates FUNDC1 at serine 13 (Ser-13) upon hypoxia or carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) treatment. Dephosphorylation of FUNDC1 catalyzed by PGAM5 enhances its interaction with LC3, which is abrogated following knockdown of PGAM5 or the introduction of a cell-permeable unphosphorylated peptide encompassing the Ser-13 and LIR of FUNDC1. We further observed that CK2 phosphorylates FUNDC1 to reverse the effect of PGAM5 in mitophagy activation. Our results reveal a mechanistic signaling pathway linking mitochondria-damaging signals to the dephosphorylation of FUNDC1 by PGAM5, which ultimately induces mitophagy.

ULK1 translocates to mitochondria and phosphorylates FUNDC1 to regulate mitophagy.

Autophagy eliminates dysfunctional mitochondria in an intricate process known as mitophagy. ULK1 is critical for the induction of autophagy, but its substrate(s) and mechanism of action in mitophagy remain unclear. Here, we show that ULK1 is upregulated and translocates to fragmented mitochondria upon mitophagy induction by either hypoxia or mitochondrial uncouplers. At mitochondria, ULK1 interacts with FUNDC1, phosphorylating it at serine 17, which enhances FUNDC1 binding to LC3. A ULK1-binding-deficient mutant of FUNDC1 prevents ULK1 translocation to mitochondria and inhibits mitophagy. Finally, kinase-active ULK1 and a phospho-mimicking mutant of FUNDC1 rescue mitophagy in ULK1-null cells. Thus, we conclude that FUNDC1 regulates ULK1 recruitment to damaged mitochondria, where FUNDC1 phosphorylation by ULK1 is crucial for mitophagy.