Autophagy reports最新文献

Brain and nervous system functions depend upon maintaining the integrity of synaptic structures over the lifetime. Autophagy, a key homeostatic quality control system, plays a central role not only in neuronal development and survival/cell death, but also in regulating synaptic activity and plasticity. Glutamate is the major excitatory neurotransmitter that activates downstream targets, with a key role in learning and memory. However, an excess of glutamatergic stimulation is pathological in stroke, epilepsy and neurodegeneration, triggering excitotoxic cell death or a sublethal process of excitatory mitochondrial calcium toxicity (EMT) that triggers dendritic retraction. Markers of autophagy and mitophagy are often elevated following excitatory neuronal injuries, with the potential to influence cell death or neurodegenerative outcomes of these injuries. Interestingly, leucine-rich repeat kinase 2 (LRRK2) and PTEN-induced kinase 1 (PINK1), two kinases linked to autophagy, mitophagy and Parkinson disease, play important roles in regulating mitochondrial calcium handling, synaptic density and function, and maturation of dendritic spines. Mutations in LRRK2, PINK1, or proteins linked to Alzheimer's disease perturb mitochondrial calcium handling to sensitize neurons to excitatory injury. While autophagy and mitophagy can play both protective and harmful roles, studies in various excitotoxicity and stroke models often implicate autophagy in a pathogenic role. Understanding the role of autophagic degradation in regulating synaptic loss and cell death following excitatory neuronal injuries has important therapeutic implications for both acute and chronic neurological disorders.

Autophagy is a dynamic process critical in maintaining cellular homoeostasis. Dysregulation of autophagy is linked to many diseases and is emerging as a promising therapeutic target. High-throughput methods to characterise autophagy are essential for accelerating drug discovery and characterising mechanisms of action. In this study, we developed a scalable image-based temporal profiling approach to characterise ~900 morphological features at a single cell level with high temporal resolution. We differentiated drug treatments based on morphological profiles using a random forest classifier with ~90% accuracy and identified the key features that govern classification. Additionally, temporal morphological profiles accurately predicted biologically relevant changes in autophagy after perturbation, such as total cargo degraded. Therefore, this study acts as proof-of-principle for using image-based temporal profiling to differentiate autophagy perturbations in a high-throughput manner and has the potential identify biologically relevant autophagy phenotypes. Ultimately, approaches like image-based temporal profiling can accelerate drug discovery.

LC3-interacting region (LIR) motifs are essential for recruiting proteins onto autophagosomes, the hallmark of autophagy. We recently explored the relevance of the specific position of the LIRs in RavZ and ATG4B (autophagy-related 4B). RavZ's N-terminal LIRs drive substrate recognition and enzymatic activity, while its C-terminal LIR aids membrane localization. In contrast, ATG4B's C-terminal LIR is indispensable for LC3B (microtubule-associated protein 1 light chain 3B)-phosphatidylethanolamine (PE) delipidation on autophagosomes but not required for cytosolic LC3B priming, which is mediated solely by its catalytic domain (CAD). These findings underscore the structural adaptation of LIRs for context-specific functions. This novel nuanced understanding provides a framework for developing therapeutic tools to modulate autophagy by precisely targeting LIRs or their associated processes, offering potential treatment for diseases like neurodegenerative disorders and infections characterized by autophagy dysregulation.

OPTN (optineurin), an amyotrophic lateral sclerosis (ALS)-associated modifier, plays vital roles in autophagy and cellular vesicular transport in mammals. OPTN can associate with RAB8A and the GTPase-activating protein TBC1D17, and facilitate the negative regulation of RAB8A by TBC1D17 (TBC domain family member 17). Recently, we reported the biochemical and structural characterizations of the interactions between OPTN, RAB8A and TBC1D17. We determined the crystal structure of the leucine-zipper domain (LZD) of OPTN with the GTP-bound active RAB8A and uncovered the molecular mechanism underpinning the specific interaction of OPTN with RAB8A. Moreover, we revealed that OPTN LZD and the TBC (Tre-2/Bub2/Cdc16) domain of TBC1D17 competitively bind to active RAB8A, while the central coiled-coil domain of OPTN and the active RAB8A can simultaneously interact with TBC1D17 TBC. In summary, our study provided mechanistic insights into the interaction of OPTN with RAB8A, and revealed the interaction relationship among OPTN, RAB8A and TBC1D17.

Olaparib has been approved as a treatment for metastatic pancreatic ductal adenocarcinoma in patients with BRCA1 (BRCA1 DNA repair associated) or BRCA2 mutations. However, a large portion of pancreatic cancer patients either exhibit inherent resistance or develop resistance over time. Understanding the molecular mechanisms that drive this resistance is crucial to develop more effective targeted therapies. In this study, we found that NLRP4 (NLR family pyrin domain containing 4) upregulation is associated with increased resistance to olaparib in pancreatic cancer. In addition, NLRP4 plays a role in both the DNA damage response (DDR) and autophagy. Specifically, NLRP4 enhances DNA repair capacity and leads to increased reactive oxygen species (ROS) production and autophagy upon olaparib treatment. Notably, NLRP4-generated mitochondrial ROS promote autophagy without directly impacting DNA damage. Inhibition of either mitochondrial ROS production with MitoQ or autophagy with chloroquine (CQ) could sensitize pancreatic cancer cells to olaparib. These findings emphasize NLRP4's role in promoting both autophagy and DNA repair in response to olaparib, suggesting that patients with low NLRP4 expression might respond more favorably to olaparib treatment.

Positive-strand RNA viruses, which are important pathogens of humans, animals and plants, subvert cellular membranes and induce de novo membrane proliferation to generate viral replication organelles (VROs) that support virus replication. Tomato bushy stunt virus (TBSV), an extensively-studied plant virus replicating in yeast model host and plants, hijacks ATG2 (autophagy-related 2), a lipid transfer protein (LTP) that transports lipids between adjacent organelles at membrane contact sites, for the biogenesis of their membranous VROs. Subversion of ATG2 by TBSV is important to enrich VRO membranes with phosphatidylethanolamine (PE), phosphatidylserine (PS) and the phosphoinositide phosphatidylinositol-3-phosphate [PI(3)P], which are all required for viral replication. TBSV replication protein directly interacts with ATG2 leading to recruitment to VRO membranes independently of the autophagy machinery.

Pseudorabies virus (PRV) poses a significant threat to the global swine industry, characterized by high morbidity and a range of sequelae in infected pigs. Mitochondria serve as a crucial platform for innate immunity, playing a pivotal role in a wide array of antiviral responses. In our recent study, we revealed that PRV infection induces mitochondrial disruption, which in turn triggers PINK1/PARKIN-mediated mitophagy. We also show that this process leads to the degradation of the mitochondrial antiviral signaling protein (MAVS) and the inhibition of antiviral interferon production and signaling, ultimately facilitating viral replication.

Chaperone-Mediated Autophagy (CMA) is a major pathway of lysosomal proteolysis critical for cellular homoeostasis and metabolism. While extensively studied in mammals, CMA's existence in fish has only been confirmed recently, offering exciting insights into its role in species facing environmental stress. Here, we shed light on the existence of 2 genes encoding the CMA-limiting factor Lamp2A (lysosomal associated membrane protein 2A) in rainbow trout (RT, Oncorhynchus mykiss), revealing distinct expression patterns across various tissues. Notably, RT lacking the most expressed Lamp2A exhibit profound hepatic proteome disturbances during acute nutritional stress, underscoring its pivotal role as a guardian of hepatic proteostasis. Building upon these findings, we introduce and validate the CMA activation score as a reliable indicator of CMA status, providing a valuable tool for detecting cellular stress in fish under environmental threats. Overall, our study offers new perspectives into understanding CMA from evolutionary and environmental contexts.

Autophagy is a process of cellular self-eating, which allows organisms to eliminate and recycle unwanted components and damaged organelles to maintain cellular homeostasis. It is an important process in the development of eukaryotic organisms. Autophagy plays a critical role in many physiological processes in plants such as nutrient remobilization, cell death, immunity, and abiotic stress responses. Autophagy thus represents an obvious target for generating resilient crops. During plant development, autophagy is also implicated in the differentiation and maturation of various cell types and plant organs, including root cap cells, tracheary elements, gametes, fruits and seeds. Here, we review our current understanding and recent advances of plant autophagy including insight into autophagy regulation and signaling as well as autophagosome membrane biogenesis. In addition, we describe how autophagy contributes to development, metabolism, biotic and abiotic stress tolerance and where the autophagic field is heading in terms of applied research for crop improvement.

Protein ATG8ylation refers to a post-translational modification involving covalent attachment of ubiquitin-like autophagy-related protein ATG8 (LC3/GABARAP) to other cellular proteins, with reversal mediated by ATG4 proteases. While lipid ATG8ylation is important for autophagosome formation and mechanistically well-characterized, little is known about the mechanism of protein ATG8ylation. Here, we investigated the conjugation machinery of protein ATG8ylation in CRISPR/Cas9-engineered knockout human cell lines, utilizing a deconjugation-resistant (Q116P G120) form of MAP1LC3B. We report that protein ATG8ylation requires the E1-like activating enzyme ATG7 and E2-like conjugating enzyme ATG3, in common with ATG8 lipidation. However, in contrast, the E3-like ATG12-ATG5-ATG16L1 complex involved in lipidation is dispensable for protein ATG8ylation, since ATG5 knockout cells can form ATG8ylated protein conjugates. Further, we uncover that ATG7 itself is a target of ATG8ylation. Overall, our work provides crucial insight into the mechanism of protein ATG8ylation, distinguishing it from ATG8 lipidation, which will aid investigating its functional role.