Wenwen Luo

Bio: Wenwen Luo is an academic researcher from Chinese Academy of Sciences. The author has contributed to research in topics: TFEB & Autophagy. The author has an hindex of 6, co-authored 7 publications receiving 213 citations.

AMPK regulates autophagy by phosphorylating BECN1 at threonine 388

Abstract: Macroautophagy/autophagy is a conserved catabolic process that recycles cytoplasmic material during low energy conditions. BECN1/Beclin1 (Beclin 1, autophagy related) is an essential protein for function of the class 3 phosphatidylinositol 3-kinase (PtdIns3K) complexes that play a key role in autophagy nucleation and elongation. Here, we show that AMP-activated protein kinase (AMPK) regulates autophagy by phosphorylating BECN1 at Thr388. Phosphorylation of BECN1 is required for autophagy upon glucose withdrawal. BECN1(T388A), a phosphorylation defective mutant, suppresses autophagy through decreasing the interaction between PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3) and ATG14 (autophagy-related 14). The BECN1(T388A) mutant has a higher affinity for BCL2 than its wild-type counterpart; the mutant is more prone to dimer formation. Conversely, a BECN1 phosphorylation mimic mutant, T388D, has stronger binding to PIK3C3 and ATG14, and promotes higher autophagy activity than the wild-type control. These findings uncover a novel mechanism of autophagy regulation.

Phosphorylation of ULK1 affects autophagosome fusion and links chaperone-mediated autophagy to macroautophagy

Abstract: The Unc-51 like autophagy activating kinase 1 (ULK1) complex plays a central role in the initiation stage of autophagy. However, the function of ULK1 in the late stage of autophagy is unknown. Here, we report that ULK1, a central kinase of the ULK1 complex involved in autophagy initiation, promotes autophagosome–lysosome fusion. PKCα phosphorylates ULK1 and prevents autolysosome formation. PKCα phosphorylation of ULK1 does not change its kinase activity; however, it decreases autophagosome–lysosome fusion by reducing the affinity of ULK1 for syntaxin 17 (STX17). Unphosphorylated ULK1 recruited STX17 and increased STX17′s affinity towards synaptosomal-associated protein 29 (SNAP29). Additionally, phosphorylation of ULK1 enhances its interaction with heat shock cognate 70 kDa protein (HSC70) and increases its degradation through chaperone-mediated autophagy (CMA). Our study unearths a key mechanism underlying autolysosome formation, a process in which the kinase activity of PKCα plays an instrumental role, and reveals the significance of the mutual regulation of macroautophagy and CMA in maintaining the balance of autophagy. The ULK complex plays a well-known role in initiating autophagy, to recycle cellular components in response to nutritional stress. Here, the authors demonstrate a late role for ULK in auotophagosome–lysosome fusion and provide a direct link between macroautophagy and chaperone mediated autophagy.

Oxidative Stress Induces Neuronal Apoptosis Through Suppressing Transcription Factor EB Phosphorylation at Ser467.

Abstract: Background/aims This study determined the role and mechanism of action of transcription factor EB (TFEB) in H2O2-induced neuronal apoptosis. Methods SH-SY5Y cells were treated with Akt inhibitor/activator and different concentrations of H2O2. Cell apoptosis was detected by flow cytometric analysis. Akt and TFEB phosphorylation and PARP cleavage were determined by Western blotting. HEK293T cells were transfected with different truncated TFEB mutants and HA-Akt-WT; SH-SY5Y cells were transfected with Flag-vector, Flag-TFEB, Flag-TFEB-S467A or Flag-TFEB-S467D; and TFEB interaction with Akt was determined by co-immunoprecipitation and GST pull-down assays. Results A low concentration of H2O2 induces TFEB phosphorylation at Ser467 and nuclear translocation, facilitating neuronal survival, whereas a high concentration of H2O2 promotes SH-SY5Y cell apoptosis via suppressing TFEB Ser467 phosphorylation and nuclear translocation. The TFEB-S467D mutant is more easily translocated into the nucleus than the non-phosphorylated TFEB-S467A mutant. Further, Akt physically binds to TFEB via its C-terminal tail interaction with the HLH domain of TFEB and phosphorylates TFEB at Ser467. Mutation of TFEB-Ser467 can prevent the phosphorylation of TFEB by Akt, preventing inhibition of oxidative stress-induced apoptosis. Conclusions Oxidative stress induces neuronal apoptosis through suppressing TFEB phosphorylation at Ser467 by Akt, providing a novel therapeutic strategy for neurodegenerative diseases.

The C-ETS2-TFEB Axis Promotes Neuron Survival under Oxidative Stress by Regulating Lysosome Activity

Abstract: Excessive reactive oxygen species/reactive nitrogen species (ROS/RNS) produced as a result of ageing causes damage to macromolecules and organelles or leads to interference of cell signalling pathways, which in turn results in oxidative stress. Oxidative stress occurs in many neurodegenerative diseases (e.g., Parkinson's disease) and contributes to progressive neuronal loss. In this study, we show that cell apoptosis is induced by oxidative stress and that lysosomes play an important role in cell survival under oxidative stress. As a compensatory response to this stress, lysosomal genes were upregulated via induction of transcription factor EB (TFEB). In addition, localization of TFEB to the nucleus was increased by oxidative stress. We also confirmed that TFEB protects cells from oxidative stress both in vitro and in vivo. Finally, we found that C-ETS2 senses oxidative stress, activates TFEB transcription, and mediates the upregulation of lysosomal genes. Our results demonstrate a mechanistic pathway for inducing lysosomal activity during ageing and neurodegeneration.

AMPK: guardian of metabolism and mitochondrial homeostasis.

Abstract: Cells constantly adapt their metabolism to meet their energy needs and respond to nutrient availability. Eukaryotes have evolved a very sophisticated system to sense low cellular ATP levels via the serine/threonine kinase AMP-activated protein kinase (AMPK) complex. Under conditions of low energy, AMPK phosphorylates specific enzymes and growth control nodes to increase ATP generation and decrease ATP consumption. In the past decade, the discovery of numerous new AMPK substrates has led to a more complete understanding of the minimal number of steps required to reprogramme cellular metabolism from anabolism to catabolism. This energy switch controls cell growth and several other cellular processes, including lipid and glucose metabolism and autophagy. Recent studies have revealed that one ancestral function of AMPK is to promote mitochondrial health, and multiple newly discovered targets of AMPK are involved in various aspects of mitochondrial homeostasis, including mitophagy. This Review discusses how AMPK functions as a central mediator of the cellular response to energetic stress and mitochondrial insults and coordinates multiple features of autophagy and mitochondrial biology.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Abstract: In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Cellular adaptation to hypoxia through hypoxia inducible factors and beyond.

Abstract: Molecular oxygen (O2) sustains intracellular bioenergetics and is consumed by numerous biochemical reactions, making it essential for most species on Earth. Accordingly, decreased oxygen concentration (hypoxia) is a major stressor that generally subverts life of aerobic species and is a prominent feature of pathological states encountered in bacterial infection, inflammation, wounds, cardiovascular defects and cancer. Therefore, key adaptive mechanisms to cope with hypoxia have evolved in mammals. Systemically, these adaptations include increased ventilation, cardiac output, blood vessel growth and circulating red blood cell numbers. On a cellular level, ATP-consuming reactions are suppressed, and metabolism is altered until oxygen homeostasis is restored. A critical question is how mammalian cells sense oxygen levels to coordinate diverse biological outputs during hypoxia. The best-studied mechanism of response to hypoxia involves hypoxia inducible factors (HIFs), which are stabilized by low oxygen availability and control the expression of a multitude of genes, including those involved in cell survival, angiogenesis, glycolysis and invasion/metastasis. Importantly, changes in oxygen can also be sensed via other stress pathways as well as changes in metabolite levels and the generation of reactive oxygen species by mitochondria. Collectively, this leads to cellular adaptations of protein synthesis, energy metabolism, mitochondrial respiration, lipid and carbon metabolism as well as nutrient acquisition. These mechanisms are integral inputs into fine-tuning the responses to hypoxic stress.

AMP-activated protein kinase: the current landscape for drug development.

Abstract: Since the discovery of AMP-activated protein kinase (AMPK) as a central regulator of energy homeostasis, many exciting insights into its structure, regulation and physiological roles have been revealed. While exercise, caloric restriction, metformin and many natural products increase AMPK activity and exert a multitude of health benefits, developing direct activators of AMPK to elicit beneficial effects has been challenging. However, in recent years, direct AMPK activators have been identified and tested in preclinical models, and a small number have entered clinical trials. Despite these advances, which disease(s) represent the best indications for therapeutic AMPK activation and the long-term safety of such approaches remain to be established.