Biogenesis of autophagosomes from the ERGIC membrane system

Introduction Macroautophagy(hereafter referred as autophagy)is a process of cellular self-degradation.In response to nutrient deprivation or other stimuli,a nascent double-membrane autophagosome,encapsulating intracellular materials or damaged organelles,is generated.The autophagosome is transported toward and eventually fuses with the lysosome(or the vacuole in yeast and plant cells).

Protective effect of Liangxue Huayu decoction on human retinal pigment epithelial cell(ARPE-19)injury induced by hypoxia through autophagy pathway

Background:Exploring the protective mechanism of the Liangxue Huayu(LXHY)decoction on human retinal pigment epithelial(RPE)cells induced by hypoxia through the autophagy pathway.Methods:The appropriate LXHY decoction concentration was determined by CCK-8.ARPE-19 cells were divided into the normal control group(A group),CoCl_(2)group(B group),3-Methyladenine(3-MA)group(treated with 3-MA(the inhibition of autophagy pathway))(C group),blank serum(BS)group(D group),LXHY drug-contained serum(DCS)group(E group),and Rapamycin(RAP)group[treated with LXHY drug-contained serum combined with rapamycin group(the activation of autophagy pathway)](F group).Counting the number of autophagosomes and autolysosomes in each group of cells under transmission electron microscopy.After infection of cells in each group by mRFP-GFP-LC3 fusion protein adenovirus,the strength of autophagic flux was detected.The mRNA expression levels of LC3 and Beclin-1 were detected by Q-PCR.Results:CCK-8 assay results showed that LXHY DCS could inhibit the cell proliferation of ARPE-19 under hypoxia(all P<0.05).As the transmission electron microscopy assay result showed,compared with the normal control group,the number of autolysosomes was significantly increased in the CoCl_(2)group(P<0.05).Compared with CoCl_(2)group,the number of autolysosomes was significantly reduced the 3-Methyladenine group,blank serum group and LXHY drug-contained serum group(all P<0.001).As autophagic flux assay result showed,compared with the normal control group,the level of autophagosomes and autolysosomes were significantly risen in CoCl_(2)group(all P<0.001).Compared with the CoCl_(2)group,the level of autophagosomes and autolysosomes were significantly fell down in 3-Methyladenine group,blank serum group and LXHY drug-contained serum group(all P<0.05).The level of autolysosomes in the LXHY drug-contained serum group was lower than in the blank serum group(P<0.05).Compared with the LXHY drug-contained serum group,the levels of autophagosomes and autolysosomes were significantly risen in the LXHY drug-contained serum combined with the rapamycin group(all P<0.05).As the Q-PCR result showed,compared with the normal control group,the expression of LC3 and Beclin-1 mRNA were significantly reduced in the CoCl_(2)group(all P<0.001).Compared with the CoCl_(2)group,the expression of LC3 mRNA were significantly increased in the 3-Methyladenine group,blank serum group and LXHY drug-contained serum group(all P<0.001).Beclin-1 mRNA expression was increased significantly(all P<0.001)in the blank serum group and the LXHY drug-contained serum group.And Beclin-1 mRNA expression in the LXHY drug-contained serum group was statistically significant increased than blank serum group(P<0.001).In the LXHY drug-contained serum combined with the rapamycin group,the LC3 and Beclin-1 mRNA expression was reduced significantly compared with the LXHY drug-contained serum group(all P<0.001).Conclusion:The LXHY DCS has the ability to protect the human retinal pigment epithelial cell(ARPE-19)damage under hypoxia through the autophagy pathway.

Membrane dynamics of ATG4B and LC3 in autophagosome formation

The biogenesis of autophagosomes provides the basis for macroautophagy to capture and degrade intracellular cargoes.Binding of the autophagy-related protein ATG8/LC3 to autophagic membranes is essential to autophagosome formation,which involves the specific and dynamic processing of ATG8/LC3 by cysteine protease ATG4.However,to date,the mechanism whereby ATG4 is recruited to the membranes,the interaction of ATG4 and ATG8/LC3 on the membranes,and its role in the growth of phagophore are not completely understood.Here,we used fluorescence recovery after photobleaching to monitor the turnover of GFP-tagged ATG4B and LC3B in living animal cells.The data show that ATG4B localizes to early autophagic membranes in an LC3B-dependent manner.During autophagy,ATG4B and LC3B undergo rapid cytosol/isolation membrane exchange but not at the cytosol/completed autophagosome.In addition,ATG4B activity controls the efficiency of autophagosome formation by impacting the membrane binding/dissociation of LC3B.These data suggest that ATG4 and LC3 play interdependent roles in the formation of autophagosomes.

Autophagy in mammalian cells

Autophagy is a regulated process for the degradation of cellular components that has been well conserved in eukaryotic cells. The discovery of autophagy-regulating proteins in yeast has been important in understanding this process. Although many parallels exist between fungi and mammals in the regulation and execution of autophagy, there are some important differences. The preautophagosomal structure found in yeast has not been identified in mammals, and it seems that there may be multiple origins for autophagosomes, including endoplasmic reticulum, plasma membrane and mitochondrial outer membrane. The maturation of the phagophore is largely dependent on 5'-AMP activated protein kinase and other factors that lead to the dephosphorylation of mammalian target of rapamycin. Once the process is initiated, the mammalian phagophore elongates and matures into an autophagosome by processes that are similar to those in yeast. Cargo selection is dependent on the ubiquitin conjugation of protein aggregates and organelles and recognition of these conjugates by autophagosomal receptors. Lysosomal degradation of cargo produces metabolites that can be recycled during stress. Autophagy is an impor-tant cellular safeguard during starvation in all eukaryotes; however, it may have more complicated, tissue specific roles in mammals. With certain exceptions, autophagy seems to be cytoprotective, and defects in the process have been associated with human disease.

Schwann cells differentiated from skin-derived precursors provide neuroprotection via autophagy inhibition in a cellular model of Parkinson’s disease

Autophagy has been shown to play an important role in Parkinson’s disease.We hypothesized that skin-derived precursor cells exhibit neuroprotective effects in Parkinson’s disease through affecting autophagy.In this study,6-hydroxydopamine-damaged SH-SY5Y cells were pretreated with a culture medium containing skin-derived precursors differentiated into Schwann cells(SKP-SCs).The results showed that the SKP-SC culture medium remarkably enhanced the activity of SH-SY5Y cells damaged by 6-hydroxydopamine,reduced excessive autophagy,increased tyrosine hydroxylase expression,reducedα-synuclein expression,reduced the autophagosome number,and activated the PI3K/AKT/mTOR pathway.Autophagy activator rapamycin inhibited the effects of SKP-SCs,and autophagy inhibitor 3-methyladenine had the opposite effect.These findings confirm that SKP-SCs modulate the PI3K/AKT/mTOR pathway to inhibit autophagy,thereby exhibiting a neuroprotective effect in a cellular model of Parkinson’s disease.This study was approved by the Animal Ethics Committee of Laboratory Animal Center of Nantong University(approval No.S20181009-205)on October 9,2018.

Effect of the Vacuolation of Helicobacter Pylori

Cytotoxic test in vitro combined with cytochemical stain, fluorescent stain, transmission electronmicrograph was used to study the vacuolated effect by helicobacter pylori (H.pylori) (Toxin+) and its pathological mechanism. 78..26 % patients with peptic ulcer associated with H.pylori was infected with H.pylori (Toxin+), while 42.86 % patients with gastritis was infected with H.pylori (Toxin+). It was positive in vacuole with acridine orange and acid phosphatase stain. Transmission electronmicrograph of vacuole revealed the presence of abounding membrane. There was a closed relationship between infection with H.pylori (Toxin+) and peptic ulcer disease. The vacuole induced by H.pylori (Toxin+) was autophagosome, which was pathological phenomenon induced by toxin.

Homeostatic cell cycle and the origin of autophagosomal vesicles

The autophagosomes were identified in the viable cycloheximide (CHX)-treated cells which had an incapacitated translational process and thus disabled synthesis of endoplasmic reticulum (ER)-derived vesicular transporters. They were found devoid of the proteins transported from ER to cell organelles, were unable to fuse with ER, Golgi or mitochondria, and displayed affinity with lysosomes. The analysis of autophagosomes, derived from the CHX cell organelles, revealed that their lipid composition resemble that of the maternal organelle. Thus, the ER-derived autophagosomes were marked with the presence of phosphatidylinositol (PI), Golgi-derived vesicles contained sphingomyelin (SM) and glycosphingolipids (GLL), and the mitochondria-derived autophagosomes contained phosphatidylglycerol (PG) and cardiolipin (CL). The incubation of the vesicles with intact lysosomes afforded their and the lysosome membrane lipids degradation. The analysis of the products derived from incubation of lysosomes and autophagosomes with radiolabeled SM, in the presence and the absence of TritonX100, allowed us to conclude that during autophagosome degradation the lysosomal enzymes are not released to cytosol, and that only lysosomes contain the enzymes degrading membrane lipids. In summary, our findings allowed us to authenticate the vesicles generated in the CHX-treated cells as organelle-specific autophagosomes and to determine that complete cycle of cell restitution and debridement includes intralysosomal degradation of the lysosomal membrane engulfing the autophagosomes vesicles.

Dual Roles of CD38 in Autophagy

CD38 is a versatile, ubiquitously expressed protein that was identified as a multifunctional enzyme. Recently, cumulating evidence has suggested that CD38 is involved in autophagy, which is an evolutionarily conserved lysosomal degradation and recycling system. Acting as a enzyme, CD38 utilizes nicotinamide adenine dinucleotide phosphate (NADP) to synthesize nicotinic acid adenine dinucleotide phosphate (NAADP), which acts as a key messenger for Ca2+-mobilizing in lysosome by targeting two-pore channels (TPCs) or transient receptor potential mucolipins (TRPMLs). Multiple studies have indicated that CD38 is involved in autophagy by modulating intracellular Ca2+ signaling. However, the control of autophagy by CD38 signaling is the subject of two contrary views. The autophagosomes trafficking and fusion with lysosomes to form autolysosomes are crucial steps in autophagy. On the one hand, the avail-able evidence indicates that lysosome trafficking and fusion to autophagosomes is positively modulated by CD38. On the other hand, overexpression of TPC2, which is positively modulated by CD38, was shown to promote the accumulation of autophagosomes, thus suppress autophagy. This review will reveal the interesting contrary dual roles of CD38 in autophagy, and critical insight into the molecular mechanisms of CD38 in autophagy regulation.

Stay in touch with the endoplasmic reticulum

The endoplasmic reticulum(ER),which is composed of a continuous network of tubules and sheets,forms the most widely distributed membrane system in eukaryotic cells.As a result,it engages a variety of organelles by establishing membrane contact sites(MCSs).These contacts regulate organelle positioning and remodeling,including fusion and fission,facilitate precise lipid exchange,and couple vital signaling events.Here,we systematically review recent advances and converging themes on ER-involved organellar contact.The molecular basis,cellular influence,and potential physiological functions for ER/nuclear envelope contacts with mitochondria,Golgi,endosomes,lysosomes,lipid droplets,autophagosomes,and plasma membrane are summarized.

Hepatic COX1 loss leads to impaired autophagic flux and exacerbates nonalcoholic steatohepatitis

The mechanisms underlying autophagic defects in nonalcoholic steatohepatitis(NASH)remain largely unknown.We aimed to elucidate the roles of hepatic cyclooxygenase 1(COX1)in autophagy and the pathogenesis of diet-induced steatohepatitis in mice.Human nonalcoholic fatty liver disease(NAFLD)liver samples were used to examine the protein expression of COX1 and the level of autophagy.Cox1^(Δhepa)mice and their wildtype littermates were generated and fed with 3 different NASH models.We found that hepatic COX1 expression was increased in patients with NASH and diet induced NASH mice models accompanied by impaired autophagy.COX1 was required for basal autophagy in hepatocytes and liver specific COX1 deletion exacerbated steatohepatitis by inhibiting autophagy.Mechanistically,COX1 directly interacted with WD repeat domain,phosphoinositide interacting 2(WIPI2),which was crucial for autophagosome maturation.Adeno-associated virus(AAV)-mediated rescue of WIPI2 reversed the impaired autophagic flux and improved NASH phenotypes in Cox1^(Δhepa)mice,indicating that COX1 deletion-mediated steatohepatitis was partially dependent on WIPI2-mediated autophagy.In conclusion,we demonstrated a novel role of COX1 in hepatic autophagy that protected against NASH by interacting with WIPI2.Targeting the COX1 WIPI2 axis may be a novel therapeutic strategy for NASH.

Cytoplasmic YAP1-mediated ESCRT-III assembly promotes autophagic cell death and is ubiquitinated by NEDD4L in breast cancer

Background:Nuclear Yes1-associated transcriptional regulator(YAP1)promotes tumor progression.However,the function of cytoplasmic YAP1 in breast cancer cells and its impact on the survival of breast cancer patients remain unclear.Our research aimed to explore the biological function of cytoplasmic YAP1 in breast cancer cells and the possibility of cytoplasmic YAP1 as a predictive marker of breast cancer survival.Methods:We constructed cell mutant models,including NLS-YAP15SA(nuclear localized),YAP1S94A(incapable of binding to the TEA domain transcription factor family)and YAP1S127D(cytoplasmic localized),and used Cell Counting Kit-8(CCK-8)assays,5-ethynyl-2’-deoxyuridine(EdU)incorporation assays,and Western blotting(WB)analysis to detect cell proliferation and apoptosis.The specific mechanism of cytoplasmic YAP1-mediated endosomal sorting complexes required for transport III(ESCRT-III)assembly was studied by co-immunoprecipitation,immunofluorescence staining,and WB analysis.Epigallocatechin gallate(EGCG)was used to simulate YAP1 retention in the cytoplasm in in vitro and in vivo experiments to study the function of cytoplasmic YAP1.YAP1 binding to NEDD4-like E3 ubiquitin protein ligase(NEDD4L)was identified using mass spectrometry and was verified in vitro.Breast tissue microarrays were used to analyze the relationship between cytoplasmic YAP1 expression and the survival of breast cancer patients.Results:YAP1 was mainly expressed in the cytoplasm in breast cancer cells.Cytoplasmic YAP1 promoted autophagic death of breast cancer cells.Cytoplasmic YAP1 bound to the ESCRT-III complex subunits charged multivesicular body protein 2B(CHMP2B)and vacuolar protein sorting 4 homolog B(VPS4B),promoting assembly of CHMP2B-VPS4B and activating autophagosome formation.EGCG retained YAP1 in the cytoplasm,promoting the assembly of CHMP2B-VPS4B to promote autophagic death of breast cancer cells.YAP1 bound to NEDD4L,and NEDD4L mediated ubiquitination and degradation of YAP1.Breast tissue microarrays revealed that high levels of cytoplasmic YAP1 were beneficial to the survival of breast cancer patients.Conclusions:Cytoplasmic YAP1 mediated autophagic death of breast cancer cells by promoting assembly of the ESCRT-III complex;furthermore,we established a new breast cancer survival prediction model based on cytoplasmic YAP1 expression.

The late stage of autophagy: cellular events and molecular regulation

Autophagy is an intracellular degradation system that delivers cytoplasmic contents to the lysosome for degradation.It is a“self-eating”process and plays a“house-cleaner”role in cells.The complex process consists of several sequential steps-induction,autophagosome formation,fusion of lysosome and autophagosome,degradation,efflux transportation of degradation products,and autophagic lysosome reformation.In this review,the cellular and molecular regulations of late stage of autophagy,including cellular events after fusion step,are summarized.

Diverse cellular strategies for the export of leaderless proteins

Unconventional protein export/secretion(UPE/UPS),in contrast to the classical ER-Golgi-dependent export/secretion of proteins with a leader sequence(signal peptide),employs multiple means to release leaderless cargoes(and in some special cases,cargoes with a leader sequence)to the extracellular space.By far,two major types of UPE have been classified,vesicle-independent UPE and vesicle-dependent UPE.In the former,UPE cargoes can directly translocate across the plasma membrane from the cytoplasm without the assistance of a vesicle carrier.In the latter,UPE cargoes translocate into the lumen of a vesicle which then delivers them out of the cell through membrane trafficking.Both types of UPE require multiple unconventional solutions to complete secretion.Here,we briefly discuss the multiple strategies for a UPE cargo release,focusing on two key steps of leaderless cargoes release in UPE:protein translocation and membrane trafficking.

Organelle aging:Lessons from model organisms

Most cellular processes descend into failure during aging. While a large collection of longevity pathways has been identified in the past decades, the mechanism for age-related decline of cellular homeostasis and organelle function remains largely unsolved. It is known that many organelles undergo structural and functional changes during normal aging, which significantly contributes to the decline of tissue function at old ages. Since recent studies have revealed an emerging role of organelles as regulatory hubs in maintaining cellular homeostasis, understanding of organelle aging will provide important insights into the cellular basis of organismal aging. Here we review current progress on the characterization of age-dependent structural and functional alterations in the more well-studied organelles, as well as the known mechanisms governing organelle aging in model organisms, with a special focus on the fruit fly Drosophila melanogaster.

Visualization of reticulophagy in living cells using an endoplasmic reticulum-targeted p62 mutant

Reticulophagy is a type of selective autophagy in which protein aggregate-containing and/or damaged endoplasmic reticulum(ER)fragments are engulfed for lysosomal degradation, which is important for ER homeostasis. Several chemical drugs and mutant proteins that promote protein aggregate formation within the ER lumen can efficiently induce reticulophagy in mammalian cells.However, the exact mechanism and cellular localization of reticulophagy remain unclear. In this report, we took advantage of the self-oligomerization property of p62/SQSTM1, an adaptor for selective autophagy, and developed a novel reticulophagy system based on an ER-targeted p62 mutant to investigate the process of reticulophagy in living cells. LC3 conversion analysis via western blot suggested that p62 mutant aggregate-induced ER stress triggered a cellular autophagic response. Confocal imaging showed that in cells with moderate aggregation conditions, the aggregates of ER-targeted p62 mutants were efficiently sequestered by autophagosomes, which was characterized by colocalization with the autophagosome precursor marker ATG16L1, the omegasome marker DFCP1, and the late autophagosomal marker LC3/GATE-16. Moreover, time-lapse imaging data demonstrated that the LC3-or DFCP1-positive protein aggregates are tightly associated with the reticular structures of the ER, thereby suggesting that reticulophagy occurs at the ER and that omegasomes may be involved in this process.

Prostate cancer cells at a therapeutic gunpoint of the autophagy process

In a normal prostate,the process of controling cell death is essential to maintain tissue homeostasis and its inhibition may lead to the development of cancer.Androgen receptor signaling plays pivotal roles in the prostate development and homeostasis as well as in the progression of prostate cancer.The main treatment for prostate cancer is a combination of androgen deprivation therapy(ADT)using anti-androgens and docetaxil administration.However,ADT eventually fails due to a pathological unbalance of cell death processes,in particular apoptosis and autophagy.As a result prostate tumors may re-grow and progress into the castration resistant stage.The role of autophagy in tumorigenesis is complex and it could be a double-edged sword process,as autophagy defects promote cancer progression in association with various dangerous cellular processes,while functional autophagy enables cancer cell survival under stress and likely contributes to the resistance of treatment.Autophagy is often impaired in prostate cancer,due to either activation of the Akt/mTOR pathway,which normally inhibits autophagy,or through allelic loss of Beclin-1(BECN1),an essential autophagy gene.In particular,elucidating the interplay between autophagy and tumor cell metabolism will provide unique opportunities to identify new therapeutic targets and to develop synthetically lethal treatment strategies that preferentially target cancer cells,while sparing normal tissues.

Regulation of autophagy:a promising therapeutic target for the treatment of hearing loss

Autophagy,a ubiquitous cellular biological behavior that features a lysosome-dependent degradation pathway,is an important mechanism for cellular self-protection in eukaryotes.Autophagy plays essential roles in cell survival,renewal,material reuse and the maintenance of homeostasis.This paper reviews recent advances in understanding the physiological function of autophagy and its possible roles in auditory diseases.We focused our review on original publications on animal models,drug models,and molecular mechanisms of hearing impairment involved in the dysregulation of autophagy.As research on the mechanisms of autophagy has deepened,it has become obvious that autophagy plays essential roles not only in cell survival,but the occurrence and development of a variety of auditory-related disorder,including aminoglycoside-induced hearing loss,age-related hearing loss,and noise-induced hearing loss.While clinical treatment of such conditions via regulation of the development of autophagy is a novel idea,more time is needed to fully elucidate the specific regulatory pathways and modes of autophagy in auditory diseases.The continued study of the mechanisms and regulation of autophagy in auditory diseases will be of great significance for the future treatment and prevention of these conditions.