scholarly journals Spermidine and Rapamycin Reveal Distinct Autophagy Flux Response and Cargo Receptor Clearance Profile

Cells ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 95
Author(s):  
Sholto de Wet ◽  
Andre Du Toit ◽  
Ben Loos
Keyword(s):  
Brca1 Gene ◽  
Specific Protein ◽  
Receptor Protein ◽  
Autophagy Flux ◽  
Sequestosome 1 ◽  
Total Pool ◽  
Major Attention ◽  
Flux Response ◽  
Protein Recruitment ◽  
Autophagy Activity

Autophagy flux is the rate at which cytoplasmic components are degraded through the entire autophagy pathway and is often measured by monitoring the clearance rate of autophagosomes. The specific means by which autophagy targets specific cargo has recently gained major attention due to the role of autophagy in human pathologies, where specific proteinaceous cargo is insufficiently recruited to the autophagosome compartment, albeit functional autophagy activity. In this context, the dynamic interplay between receptor proteins such as p62/Sequestosome-1 and neighbour of BRCA1 gene 1 (NBR1) has gained attention. However, the extent of receptor protein recruitment and subsequent clearance alongside autophagosomes under different autophagy activities remains unclear. Here, we dissect the concentration-dependent and temporal impact of rapamycin and spermidine exposure on receptor recruitment, clearance and autophagosome turnover over time, employing micropatterning. Our results reveal a distinct autophagy activity response profile, where the extent of autophagosome and receptor co-localisation does not involve the total pool of either entities and does not operate in similar fashion. These results suggest that autophagosome turnover and specific cargo clearance are distinct entities with inherent properties, distinctively contributing towards total functional autophagy activity. These findings are of significance for future studies where disease specific protein aggregates require clearance to preserve cellular proteostasis and viability and highlight the need of discerning and better tuning autophagy machinery activity and cargo clearance.

scholarly journals NBR1-mediated p62-liquid droplets enhance the Keap1-Nrf2 system

2019 ◽  
Author(s):  
Pablo Sánchez-Martín ◽  
Yu-shin Sou ◽  
Shun Kageyama ◽  
Masaaki Komatsu
Keyword(s):  
Oxidative Stress ◽  
Liquid Droplet ◽  
Brca1 Gene ◽  
Droplet Formation ◽  
Receptor Protein ◽  
Liquid Droplets ◽  
Nrf2 Pathway ◽  
Selective Degradation ◽  
System A ◽  
Nrf2 Activation

Abstractp62/SQSTM1 is a multivalent protein that has an ability to cause a liquid-liquid phase separation and serves as a receptor protein that participates in cargo isolation during selective autophagy. This protein is also involved in the non-canonical activation of the Keap1-Nrf2 system, a major oxidative stress response pathway. Here we show a role of Neighbor of BRCA1 gene 1 (NBR1), an autophagy receptor structurally similar to p62/SQSTM1, in the p62-liquid droplet formation and the Keap1-Nrf2 pathway. The overexpression of NBR1 blocked selective degradation of p62/SQSTM1 through autophagy and promoted the accumulation and phosphorylation of p62/SQSTM1 in liquid-like bodies, which is required for the activation of Nrf2. NBR1 was induced in response to oxidative stress, and then the p62-mediated Nrf2 activation was up-regulated. Conversely, loss of Nbr1 suppresses not only the formation of p62/SQSTM1-liquid droplets but also p62-dependent Nrf2 activation during oxidative stress. Taken together, our results show that NBR1 mediates p62/SQSTM1-liquid droplet formation to activate the Keap1-Nrf2 pathway.

Upregulation of miR-133a by adiponectin inhibits pyroptosis pathway and rescues acute aortic dissection

2020 ◽  
Vol 52 (9) ◽  
pp. 988-997
Author(s):  
Haizhen Duan ◽  
Xiaojun Zhang ◽  
Renjie Song ◽  
Tongying Liu ◽  
Yuanyuan Zhang ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Aortic Dissection ◽  
Acute Aortic Dissection ◽  
Smooth Muscle Actin ◽  
Middle Layer ◽  
Specific Protein ◽  
Receptor Protein ◽  
Mice Model

Abstract Acute aortic dissection (AAD) is a cardiovascular emergency caused by the formation of hematoma in the middle layer of the aortic wall. Adiponectin (APN) is an adipose tissue-specific protein that has anti-inflammation and anti-atherosclerosis functions. Pyroptosis, as an inflammatory cell death, depends on the activation of caspase1, while nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) is a typical representative of the pyroptosis pathway. In this study, we aimed to find whether APN affects the AAD process. The results showed that APN overexpression (OE) inhibited the AAD development and the levels of glucose, triglyceride, and total cholesterol in mice model. In addition, APN OE inhibited the productions of gasdermin D (GSDMD), NLRP3, caspase1, interleukin-1β (IL-1β), IL-18, and osteopontin (OPN), as well as α-smooth muscle actin (α-SMA) downregulation in vitro and in vivo. In addition, NLRP3 was found to be a target gene of miR-133a and miR-133a OE showed similar effects to APN OE in attenuating the LPS-induced productions of GSDMD, NLRP3, caspase1, IL-1β, IL-18, and OPN, as well as α-SMA downregulation in vascular smooth muscle cells (vSMCs). Moreover, the beneficial effects of APN OE were abolished by miR-133a knockdown in vSMCs. In conclusion, our present results indicated that the upregulation of miR-133a by APN inhibits pyroptosis pathway, which potentially rescues AAD.

scholarly journals Autophagy precedes apoptosis during degeneration of the Kölliker’s organ in the development of rat cochlea

2019 ◽  
Vol 63 (2) ◽  
Author(s):  
Shule Hou ◽  
Jiarui Chen ◽  
Jun Yang
Keyword(s):  
Postnatal Development ◽  
Supporting Cells ◽  
Autophagy Flux ◽  
Early Postnatal Development ◽  
Sequestosome 1 ◽  
Transmission Electron ◽  
Cellular Apoptosis ◽  
Epithelial Structure ◽  
Apoptosis Markers

The Kölliker’s organ is a transient epithelial structure during cochlea development that gradually degenerates and disappears at postnatal 12-14 days (P12-14). While apoptosis has been shown to play an essential role in the degeneration of the Kölliker’s organ, the role of another programmed cell death, autophagy, remains unclear. In our study, autophagy markers including microtubule associated protein light chain 3-II (LC3-II), sequestosome 1 (SQSTM1/p62) and Beclin1 were detected in the supporting cells of the Kölliker’s organ through immunohistochemistry staining. In addition, Western blot and real-time PCR revealed a gradually decreased expression of LC3-II and an increased expression of p62 during early postnatal development. Compared to apoptosis markers that peaks between P7 and P10, autophagy flux peaked earlier at P1 and decreased from P1 to P14. By transmission electron microscopy, we observed representative autophagosome and autolysosome that packaged various organelles in the supporting cells of the Kölliker’s organ. During the degeneration, these organelles were digested via autophagy well ahead of the cellular apoptosis. These results suggest that autophagy plays an important role in transition and degeneration of the Kölliker’s organ prior to apoptosis during the early postnatal development.

scholarly journals Proteins of Excitable Membranes

1969 ◽  
Vol 54 (1) ◽  
pp. 187-224 ◽  
Author(s):  
David Nachmansohn
Keyword(s):  
Electrical Activity ◽  
Essential Role ◽  
Enzyme Inhibitors ◽  
Specific Protein ◽  
Receptor Protein ◽  
Excitable Membranes ◽  
Protein Properties ◽  
Ion Movements ◽  
Hydrolysis Of

Excitable membranes have the special ability of changing rapidly and reversibly their permeability to ions, thereby controlling the ion movements that carry the electric currents propagating nerve impulses. Acetylcholine (ACh) is the specific signal which is released by excitation and is recognized by a specific protein, the ACh-receptor; it induces a conformational change, triggering off a sequence of reactions resulting in increased permeability. The hydrolysis of ACh by ACh-esterase restores the barrier to ions. The enzymes hydrolyzing and forming ACh and the receptor protein are present in the various types of excitable membranes. Properties of the two proteins directly associated with electrical activity, receptor and esterase, will be described in this and subsequent lectures. ACh-esterase has been shown to be located within the excitable membranes. Potent enzyme inhibitors block electrical activity demonstrating the essential role in this function. The enzyme has been recently crystallized and some protein properties will be described. The monocellular electroplax preparation offers a uniquely favorable material for analyzing the properties of the ACh-receptor and its relation to function. The essential role of the receptor in electrical activity has been demonstrated with specific receptor inhibitors. Recent data show the basically similar role of ACh in the axonal and junctional membranes; the differences of electrical events and pharmacological actions are due to variations of shape, structural organization, and environment.

Identification of PTPRσ-interacting proteins by proximity-labelling assay

2020 ◽  
Author(s):  
Yuanhao Gong ◽  
Shaniya Abudureyimu ◽  
Kenji Kadomatsu ◽  
Kazuma Sakamoto
Keyword(s):  
Axonal Regeneration ◽  
Chondroitin Sulphate ◽  
Protein Tyrosine Phosphatases ◽  
Receptor Protein ◽  
Specific Substrate ◽  
Type I ◽  
Interacting Proteins ◽  
Comprehensive Understanding ◽  
Autophagy Flux ◽  
Receptor Protein Tyrosine Phosphatases

Abstract Receptor protein tyrosine phosphatases (RPTPs) are type-I transmembrane proteins and involved in various biological and pathological processes. Their functions are supposed to be exerted through tyrosine dephosphorylation of their specific substrates. However, our comprehensive understanding of specific substrates or interacting proteins for RPTPs is poor. PTPRσ belongs to class 2a RPTP family, dephosphorylates cortactin, and leads to autophagy flux disruption and axonal regeneration inhibition in response to its ligand chondroitin sulphate. Here, we applied proximity-dependent biotin identification (BioID) assay, a proximity-labelling assay, to PTPRσ and reproducibly identified the 99 candidates as interactors for PTPRσ including already-known interactors such as Liprin-α and Trio. Of note, cortactin was also listed up in our assay. Our results suggest that the BioID assay is a powerful and reliable tool to identify RPTP-interacting proteins including its specific substrate.

scholarly journals NIMA-related kinase 9–mediated phosphorylation of the microtubule-associated LC3B protein at Thr-50 suppresses selective autophagy of p62/sequestosome 1

2019 ◽  
Vol 295 (5) ◽  
pp. 1240-1260 ◽  
Author(s):  
Birendra Kumar Shrestha ◽  
Mads Skytte Rasmussen ◽  
Yakubu Princely Abudu ◽  
Jack-Ansgar Bruun ◽  
Kenneth Bowitz Larsen ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Brca1 Gene ◽  
Effector Proteins ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Selective Autophagy ◽  
Sequestosome 1 ◽  
Interaction Partners ◽  
Impaired Binding

Human ATG8 family proteins (ATG8s) are active in all steps of the macroautophagy pathway, and their lipidation is essential for autophagosome formation. Lipidated ATG8s anchored to the outer surface of the phagophore serve as scaffolds for binding of other core autophagy proteins and various effector proteins involved in trafficking or fusion events, whereas those at the inner surface are needed for assembly of selective autophagy substrates. Their scaffolding role depends on specific interactions between the LC3-interacting region (LIR) docking site (LDS) in ATG8s and LIR motifs in various interaction partners. LC3B is phosphorylated at Thr-50 within the LDS by serine/threonine kinase (STK) 3 and STK4. Here, we identified LIR motifs in STK3 and atypical protein kinase Cζ (PKCζ) and never in mitosis A (NIMA)-related kinase 9 (NEK9). All three kinases phosphorylated LC3B Thr-50 in vitro. A phospho-mimicking substitution of Thr-50 impaired binding of several LIR-containing proteins, such as ATG4B, FYVE, and coiled-coil domain-containing 1 (FYCO1), and autophagy cargo receptors p62/sequestosome 1 (SQSTM1) and neighbor of BRCA1 gene (NBR1). NEK9 knockdown or knockout enhanced degradation of the autophagy receptor and substrate p62. Of note, the suppression of p62 degradation was mediated by NEK9-mediated phosphorylation of LC3B Thr-50. Consistently, reconstitution of LC3B-KO cells with the phospho-mimicking T50E variant inhibited autophagic p62 degradation. PKCζ knockdown did not affect autophagic p62 degradation, whereas STK3/4 knockouts inhibited autophagic p62 degradation independently of LC3B Thr-50 phosphorylation. Our findings suggest that NEK9 suppresses LC3B-mediated autophagy of p62 by phosphorylating Thr-50 within the LDS of LC3B.

scholarly journals Cardamonin Reduces Acetaminophen-Induced Acute Liver Injury in Mice via Activating Autophagy and NFE2L2 Signaling

2020 ◽  
Vol 11 ◽  
Author(s):  
Qiushi Xu ◽  
Yunhui Fan ◽  
Juan J. Loor ◽  
Yusheng Liang ◽  
Xudong Sun ◽  
...  
Keyword(s):  
Liver Injury ◽  
Acute Liver Injury ◽  
High Mobility ◽  
Hepatoprotective Effect ◽  
Receptor Protein ◽  
Related Factor ◽  
Protective Effects ◽  
Toll Like Receptor 4 ◽  
Sequestosome 1 ◽  
Underlying Mechanisms

Cardamonin (CD), a naturally occurring chalcone derived from the Alpinia species, has been shown to exert antioxidant and anti-inflammatory activity, but its role in the prevention of acetaminophen- (APAP-) induced hepatotoxicity remains elusive. The objective of this study was to determine the protective effects of CD against APAP-induced acute liver injury (ALI) and the underlying mechanisms. Wild-type or transcription factor nuclear factor erythroid 2-related factor 2- (NFE2L2-) deficient mice were treated with CD (50 or 100 mg/kg, i.p.) or vehicle for 24 h. Subsequently, these mice were challenged with APAP (400 mg/kg, i.p.) for 6 h. Liver and blood samples were collected to evaluate liver injury and protein abundance. Treatment with CD significantly reduced APAP-induced hepatotoxicity. Furthermore, CD effectively reduced APAP-induced inflammation by inhibiting high mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), and NOD-like receptor protein 3 (NLRP3) signaling. In addition, CD induced activation of sequestosome 1 (p62) and NFE2L2 signaling and facilitated autophagy. By applying autophagy inhibitor 3-methyladenine (3-MA; 20 mg/kg, i.p.), further mechanistic exploration revealed that NFE2L2 deficiency promoted autophagic activity induced by CD treatment, which was conducive to the hepatoprotective effect of CD against APAP-induced hepatoxicity in NFE2L2−/− mice. Overall, data suggest that CD has hepatoprotective effect against APAP-induced ALI, which might contribute to the activation of NFE2L2 and autophagy.

scholarly journals Morphine induces dysfunction of PINK1/Parkin-mediated mitophagy in spinal cord neurons implying involvement in antinociceptive tolerance

2019 ◽  
Vol 11 (12) ◽  
pp. 1056-1068 ◽  
Author(s):  
Hong Kong ◽  
Chun-Yi Jiang ◽  
Liang Hu ◽  
Peng Teng ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Chronic Pain ◽  
Chronic Administration ◽  
Morphine Tolerance ◽  
Ros Production ◽  
Autophagy Flux ◽  
Analgesic Tolerance ◽  
Spinal Cord Neurons ◽  
Sequestosome 1 ◽  
Clinical Challenge

Abstract The development of opioid-induced analgesic tolerance is a clinical challenge in long-term use for managing chronic pain. The mechanisms of morphine tolerance are poorly understood. Mitochondria-derived reactive oxygen species (ROS) is a crucial signal inducing analgesic tolerance and pain. Chronic administration of morphine leads to robust ROS production and accumulation of damaged mitochondria, which are immediately removed by mitophagy. Here, we show that morphine inhibits mitochondria damage-induced accumulation of PTEN-induced putative kinase 1 (PINK1) in neurons. It interrupts the recruitment of Parkin to the impaired mitochondria and inhibits the ubiquitination of mitochondrial proteins catalyzed by Parkin. Consequently, morphine suppresses the recognition of autophagosomes to the damaged mitochondria mediated by LC3 and sequestosome-1 (SQSTM1/p62). Thus, morphine inhibits autophagy flux and leads to the accumulation of SQSTM1/p62. Finally, the impaired mitochondria cannot be delivered to lysosomes for degradation and ultimately induces robust ROS production and morphine tolerance. Our findings suggest that the dysfunction of mitophagy is involved in morphine tolerance. The deficiency of PINK1/Parkin-mediated clearance of damaged mitochondria is crucial for the generation of excessive ROS and important to the development of analgesic tolerance. These findings suggest that the compounds capable of stabilizing PINK1 or restoring mitophagy may be utilized to prevent or reduce opioid tolerance during chronic pain management.

scholarly journals BAG3 and SYNPO (synaptopodin) facilitate phospho-MAPT/Tau degradation via autophagy in neuronal processes

2019 ◽  
Author(s):  
Changyi Ji ◽  
Maoping Tang ◽  
Claudia Zeidler ◽  
Jörg Höhfeld ◽  
Gail VW Johnson
Keyword(s):  
Receptor Protein ◽  
Autophagic Flux ◽  
Catabolic Pathway ◽  
Protein Tau ◽  
Sequestosome 1 ◽  
Cytoskeleton Protein ◽  
Neuronal Processes ◽  
Mature Neurons ◽  
First Time

AbstractA major cellular catabolic pathway in neurons is macroautophagy/autophagy, through which misfolded or aggregation-prone proteins are sequestered into autophagosomes that fuse with lysosomes, and are subsequently degraded. MAPT (microtubule associated protein tau) is one of the protein clients of autophagy. Given that accumulation of hyperphosphorylated MAPT contributes to the pathogenesis of Alzheimer disease and other tauopathies, decreasing endogenous MAPT levels has been shown to be beneficial to neuronal health in models of these diseases. A previous study demonstrated that the HSPA/HSP70 co-chaperone BAG3 (BCL2 associated athanogene 3) facilitates endogenous MAPT clearance through autophagy. These findings prompted us to further investigate the mechanisms underlying BAG3-mediated autophagy in the degradation of endogenous MAPT. Here we demonstrate for the first time that BAG3 plays an important role in autophagic flux in the neuritic processes of mature neurons (20-24 days in vitro [DIV]) through interaction with the post-synaptic cytoskeleton protein SYNPO (synaptopodin). Loss of either BAG3 or SYNPO impeded the fusion of autophagosomes and lysosomes predominantly in the post-synaptic compartment. A block of autophagy leads to accumulation of the autophagic receptor protein SQSTM1/p62 (sequestosome 1) as well as MAPT phosphorylated at Ser262 (p-Ser262). Furthermore, p-Ser262 appears to accumulate in autophagosomes at post-synaptic densities. Overall these data provide evidence of a novel role for the co-chaperone BAG3 in synapses. In cooperation with SYNPO, it functions as part of a surveillance complex that facilitates the autophagic clearance of MAPT p-Ser262, and possibly other MAPT species at the post-synapse. This appears to be crucial for the maintenance of a healthy, functional synapse.

scholarly journals Low Expression of Mas1 Receptor in Pancreas Related with Acute Pancreatitis

2020 ◽  
Author(s):  
Tianyu Cui ◽  
Xiaozheng Yu ◽  
Chunyun Li ◽  
Tianhe Li ◽  
Ruixia Liu ◽  
...  
Keyword(s):  
Acute Pancreatitis ◽  
Gastrointestinal Disease ◽  
Receptor Protein ◽  
Human Pancreas ◽  
Trypsinogen Activation ◽  
Common Causes ◽  
Ar42j Cell ◽  
Autophagy Activity

Abstract Background：Acute pancreatitis (AP) continues to be one of the most common causes of hospitalization among all gastrointestinal disease. There is a lack of therapies directed to its molecular pathogenesis. The aim of this study was to investigate the role and major mechanisms of Mas1 receptor in AP. Methods: AP was induced in C57BL/6 mice by administration of caerulein and lipopolysaccharide. The effects of intervening Mas1 receptor on the severity of AP, trypsinogen activation peptide (TAP), zymogens distribution and autophagy activity were detected in vivo. Then Mas1-lentivirus transfected AR42J cell and incubated with caerulein. TAP, autophagy activity and co-location of trypsin and autophagic were further detected in vitro. Human pancreas tissue from patients with AP and without AP were also used for Mas1 receptor protein and mRNA expression levels.Results: Mas1 receptor proteins were down-regulated in AP mice pancreas tissue as well as in human patients with AP. Compared with Mas1 receptor inhibited in AP mice, AP mice with Mas1 receptor activated decreased severe pathological changes in pancreata, lower levels of trypsinogen activation concomitant with zymogens basolateral distribution, and autophagy down-regulation. Mas1 receptor knockdown and over-expression further verified those results in vitro and showed Mas1 receptor knockdown in AR42J cells had an increased colocalization of LC3II with trypsin.Conclusions: Mas1 receptor decreased expresssion in pancreas may promote zymogens premature activation relating to AP.

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