S-acylation of p62 promotes p62 droplet recruitment into autophagosomes in mammalian autophagy.

p62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal of ubiquitinated proteins by forming p62-liquid droplets. However, it remains unclear how autophagosomes efficiently sequester p62 droplets. Herein, we report that p62 undergoes reversible S-acylation in multiple human-, rat-, and mouse-derived cell lines, catalyzed by zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and deacylated by acyl protein thioesterase 1 (APT1). S-acylation of p62 enhances the affinity of p62 for microtubule-associated protein 1 light chain 3 (LC3)-positive membranes and promotes autophagic membrane localization of p62 droplets, thereby leading to the production of small LC3-positive p62 droplets and efficient autophagic degradation of p62-cargo complexes. Specifically, increasing p62 acylation by upregulating ZDHHC19 or by genetic knockout of APT1 accelerates p62 degradation and p62-mediated autophagic clearance of ubiquitinated proteins. Thus, the protein S-acylation-deacylation cycle regulates p62 droplet recruitment to the autophagic membrane and selective autophagic flux, thereby contributing to the control of selective autophagic clearance of ubiquitinated proteins.

Tethering ATG16L1 or LC3 induces targeted autophagic degradation of protein aggregates and mitochondria.

Proteolysis-targeting chimeras (PROTACs) based on the ubiquitin-proteasome system have made great progress in the field of drug discovery. There is mounting evidence that the accumulation of aggregation-prone proteins or malfunctioning organelles is associated with the occurrence of various age-related neurodegenerative disorders and cancers. However, PROTACs are inefficient for the degradation of such large targets due to the narrow entrance channel of the proteasome. Macroautophagy (hereafter referred to as autophagy) is known as a self-degradative process involved in the degradation of bulk cytoplasmic components or specific cargoes that are sequestered into autophagosomes. In the present study, we report the development of a generalizable strategy for the targeted degradation of large targets. Our results suggested that tethering large target models to phagophore-associated ATG16L1 or LC3 induced targeted autophagic degradation of the large target models. Furthermore, we successfully applied this autophagy-targeting degradation strategy to the targeted degradation of HTT65Q aggregates and mitochondria. Specifically, chimeras consisting of polyQ-binding peptide 1 (QBP) and ATG16L1-binding peptide (ABP) or LC3-interacting region (LIR) induced targeted autophagic degradation of pathogenic HTT65Q aggregates; and the chimeras consisting of mitochondria-targeting sequence (MTS) and ABP or LIR promoted targeted autophagic degradation of dysfunctional mitochondria, hence ameliorating mitochondrial dysfunction in a Parkinson disease cell model and protecting cells from apoptosis induced by the mitochondrial stress agent FCCP. Therefore, this study provides a new strategy for the selective proteolysis of large targets and enrich the toolkit for autophagy-targeting degradation.Abbreviations: ABP: ATG16L1-binding peptide; ATG16L1: autophagy related 16 like 1; ATTEC: autophagy-tethering compound; AUTAC: autophagy-targeting chimera; AUTOTAC: autophagy-targeting chimera; Baf A1: bafilomycin A1; BCL2: BCL2 apoptosis regulator; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CPP: cell-penetrating peptide; CQ: chloroquine phosphate; DAPI: 4',6-diamidino-2-phenylindole; DCM: dichloromethane; DMF: N,N-dimethylformamide; DMSO: dimethyl sulfoxide; EBSS: Earle's balanced salt solution; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; FITC: fluorescein-5-isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HEK293: human embryonic kidney 293; HEK293T: human embryonic kidney 293T; HPLC: high-performance liquid chromatography; HRP: horseradish peroxidase; HTT: huntingtin; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFF: mitochondrial fission factor; MTS: mitochondria-targeting sequence; NBR1: NBR1 autophagy cargo receptor; NLRX1: NLR family member X1; OPTN: optineurin; P2A: self-cleaving 2A peptide; PB1: Phox and Bem1p; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; PROTACs: proteolysis-targeting chimeras; QBP: polyQ-binding peptide 1; SBP: streptavidin-binding peptide; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SPATA33: spermatogenesis associated 33; TIMM23: translocase of inner mitochondrial membrane 23; TMEM59: transmembrane protein 59; TOMM20: translocase of outer mitochondrial membrane 20; UBA: ubiquitin-associated; WT: wild type.

Mechanism of ER stress-mediated ER-phagy by CdTe-QDs in yeast cells.

Endoplasmic reticulum autophagy (ER-phagy) is an important strategy for cells against ER stress and maintain ER homeostasis. ER stress is considered as a potential toxicity of nanoparticles, but only a few studies have explored whether the nanoparticles-induced ER stress can trigger ER-phagy, and the precise molecular mechanism of ER-phagy mediated by nanoparticle-induced ER stress is still poorly understood. Therefore, our study focuses on the relationship between ER stress and ER-phagy caused by emerging nanoparticles CdTe-QDs and its molecular mechanism. The results showed that the accumulation of ROS and ER stress induced by CdTe-QDs contributed to the activation of autophagy and ER-phagy. Importantly, our study unraveled that CdTe-QDs activate autophagy by up-regulating the transcription of core autophagy machinery. It was found that the induced ER-phagy was mediated by Atg11/Atg40/Lst1-Sec23 instead of the autophagy machinery genes. We speculated that the ER-phagy caused by CdTe-QDs may include micro-ER-phagy and macro-ER-phagy. Collectively, this work provided valuable information for the application of CdTe-QDs in the field of biology and a theoretical basis for further understanding of ER-phagy.

Induction of autophagy and endoplasmic reticulum autophagy caused by cadmium telluride quantum dots are protective mechanisms of yeast cell.

Quantum dots (QDs), with unique and tunable optical properties, have been widely used in many fields closely related to our daily lives, such as biomedical application and electronic products. Therefore, the potential toxicity of QDs on the human health should be understood. Autophagy plays an important role in cell survival and death. Endoplasmic reticulum autophagy (ER-phagy), a selective autophagy that degrades ER, responds to the accumulation of misfolded proteins and ER stress. Although many reports have revealed that autophagy can be disturbed by cadmium telluride (CdTe)-QDs and other nanomaterials, there are still lack more detailed researches to illustrate the function of autophagy in CdTe-QDs-treated cells, and the function of ER-phagy in CdTe-QDs-treated cells remains to be illustrated. On the basis of transcriptome analysis, we explored the effect of CdTe-QDs on Saccharomyces cerevisiae and first illustrated that both of autophagy and ER-phagy were protective mechanisms in CdTe-QDs-treated cells. It was found that CdTe-QDs inhibited the proliferation of yeast cells, disrupted homeostasis of cells, membrane integrity, and metabolism process. All of these can be reasons of the reduction of cell viability. The abolishment of autophagy and ER-phagy reduce the cell survival, indicating both of them are cell protective mechanisms against CdTe-QDs toxicity in yeast cells. Therefore, our data are significant for the application of CdTe-QDs and provide precious information for understanding of nanomaterials-related ER-phagy.

The role of membrane contact sites at the bacteria-host interface.

Membrane contact sites (MCSs) refer to the areas of close proximity between heterologous membranes. A growing body of evidence indicates that MCSs are involved in important cellular functions, such as cellular material transfer, organelle biogenesis, and cell growth. Importantly, the study of MCSs at the bacteria-host interface is an emerging popular research topic. Intracellular bacterial pathogens have evolved a variety of fascinating strategies to interfere with MCSs by injecting effectors into infected host cells. Bacteria-containing vacuoles establish direct physical contact with organelles within the host, ensuring vacuolar membrane integrity and energy supply from host organelles and protecting the vacuoles from the host endocytic pathway and lysosomal degradation. An increasing number of bacterial effectors from various bacterial pathogens hijack components of host MCSs to form the vacuole-organelle MCSs for material exchange. MCS-related events have been identified as new mechanisms of microbial pathogenesis to greatly improve bacterial survival and replication within host cells. In this review, we will discuss the recent advances in MCSs at the bacteria-host interface, focussing on the roles of MCSs mediated by bacterial effectors in microbial pathogenesis.

Quantitative Analysis of Salmonella typhimurium Based on Elemental-Tags Laser-Induced Breakdown Spectroscopy.

Current rapid bacterial detection methods are dedicated to the classification and identification of bacteria. However, there is still a lack of a method for specific quantitative analysis of certain bacteria. In this work, a method based on elemental-tags laser-induced breakdown spectroscopy (ETLIBS) was developed for the rapid and specific quantitative analysis of Salmonella typhimurium (S. ty). Elemental tags were first synthesized by assembling copper nanoparticles (CuNPs) with poly(thymine) (poly-T) template that linked with the aptamer sequence. Under the specific recognition of the aptamer, S. ty can be fully combined with the elemental tags within 30 min to achieve labeling. Afterward, the silicon nanowires (SiNWs) array modified with Au@Ag nanoparticles (SiNWs-Au@Ag) was employed to capture S. ty in 30 min. Attributed to the rapid analysis superiority of ETLIBS mapping, 100 spectra of SiNWs-Au@Ag/S. ty/CuNPs can be obtained in 5 min. It was found that the peak area of the Cu(I) atomic emission line at 324.75 nm fitted by the Voigt profile was linearly related to the bacterial concentration in the range of 102-106 CFU/mL(R2 = 0.978). Furthermore, ETLIBS mapping achieved a low limit of detection (LOD) of 61 CFU/mL and showed good selectivity to S. ty compared with other bacteria. Besides, the method exhibited preeminent detection performance in spiked samples with the recoveries of 87-113%. With the advantages of rapidity, high efficiency, and specificity, the proposed method is expected to be a powerful tool for bacterial detection.

Crosstalk between Autophagy and Nanomaterials: Internalization, Activation, Termination.

Nanomaterials (NMs) are comprehensively applied in biomedicine due to their unique physical and chemical properties. Autophagy, as an evolutionarily conserved cellular quality control process, is closely associated with the effect of NMs on cells. In this review, the recent advances in NM-induced/inhibited autophagy (NM-phagy) are summarized, with an aim to present a comprehensive description of the mechanisms of NM-phagy from the perspective of internalization, activation, and termination, thereby bridging autophagy and nanomaterials. Several possible mechanisms are extensively reviewed including the endocytosis pathway of NMs and the related cross components (clathrin and adaptor protein 2 (AP-2), adenosine diphosphate (ADP)-ribosylation factor 6 (Arf6), Rab, UV radiation resistance associated gene (UVRAG)), three main stress mechanisms (oxidative stress, damaged organelles stress, and toxicity stress), and several signal pathway-related molecules. The mechanistic insight is beneficial to understand the autophagic response to NMs or NMs' regulation of autophagy. The challenges currently encountered and research trend in the field of NM-phagy are also highlighted. It is hoped that the NM-phagy discussion in this review with the focus on the mechanistic aspects may serve as a guideline for future research in this field.

Combining autophagy-inducing peptides and brefeldin A delivered by perinuclear-localized mesoporous silica nanoparticles: a manipulation strategy for ER-phagy.

Autophagic degradation of the endoplasmic reticulum (ER-phagy) has been found to play a critical role in human sensory neuropathy. So far, however, specific and efficient intervention means for ER-phagy remain unexplored. Herein, brefeldin A (BFA), a blocking agent on protein transport between the ER and Golgi, was screened from ER stress inducers. BFA was then delivered to the perinuclear area co-localized with the ER by a mesoporous silica nanoparticle-based drug-carrier functionalized with autophagy-inducing peptides of TAT-beclin 1 (MSNs-BFA), to evoke a perturbation of ER-phagy. The molecular mechanism of ER-phagy regulated by BFA was explored by biochemical evaluation including time-lapse live-cell fluorescence imaging. We found that MSNs-BFA treatment caused a lower mRNA/protein expression level of FAM134b even under a compensation of autophagic flux in U2OS cells, and resulted in ER-expansion. The fragmentation of the ER was blocked as a response to ER stress mediated by inactivation of the AKT/TSC/mTOR pathway. Our work developed an efficient external manipulation strategy to regulate ER-phagy and may contribute to the therapeutic application of autophagy-related major human diseases.

Optical Imaging Paves the Way for Autophagy Research.

Autophagy is a degradation process in eukaryotic cells that recycles cellular components for nutrition supply under environmental stress and plays a double-edged role in development of major human diseases. Noninvasive optical imaging enables us to clearly visualize various classes of structures involved in autophagy at macroscopic and microscopic dynamic levels. In this review, we discuss important trends of emerging optical imaging technologies used to explore autophagy and provide insights into the mechanistic investigation and structural study of autophagy in mammalian cells. Some exciting new prospects and future research directions regarding optical imaging techniques in this field are also highlighted.

Fluorescent aptasensor for antibiotic detection using magnetic bead composites coated with gold nanoparticles and a nicking enzyme.

Antibiotic abuse has been bringing serious pollution in water, which is closely related to human health. It is desirable to develop a new strategy for antibiotic detection. To address this problem, a sensitive fluorescent aptasensor for antibiotic detection was developed by utilizing gold nanoparticles modified magnetic bead composites (AuNPs/MBs) and nicking enzyme. AuNPs/MBs were synthesized with the help of polyethylenimine (PEI). The prepared AuNPs/MBs acted as dual-functional scaffolds that owned excellent magnetic separation capacity and strong covalent bio-conjugation. The non-specifically absorbed aptamers in AuNPs/MBs were less than that in MBs. Hence, the fluorescent aptasensor based on AuNPs/MBs show a better signal to background ratio than that based on carboxyl modified magnetic beads (MBs). In this work, ampicillin was employed as a model analyte. In the presence of ampicillin, the specific binding between ampicillin and aptamer induced structure-switching that led to the release of partial complementary DNA (cDNA) of aptamer. Then, the released cDNA initiated the cycle of nicking enzyme assisted signal amplification (NEASA). Therefore, a large amount of taqman probes were cleaved and fluorescence signal was amplified. The prepared fluorescent aptasensor bring sensitive detection in range of 0.1-100 ng mL-1 with the limit of detection of 0.07 ng mL-1. Furthermore, this aptasensor was also successfully applied in real sample detection with acceptable accuracy. The fluorescent aptasensor provides a promising method for efficient, rapid and sensitive antibiotic detection.

New findings of silica nanoparticles induced ER autophagy in human colon cancer cell.

Nanoparticle-induced autophagy has been extensively studied, however, real time information about the endoplasmic reticulum involved autophagic process (ER autophagy) induced by nanomaterials remains unknown. In this work, silica nanoparticles (SNPs) were synthesized with characteristics of low toxicity, good biocompatibility and excellent water dispersibility to treat cells. Results show that either low concentration (10 µg/mL) or high concentration (200 µg/mL) of SNPs could increase the quantity of processing from microtubule-associated protein 1-light chain 3-I (LC3-I) to the other variant of LC3 (LC3-II). Interestingly, the level of autophagy induced by the SNPs is associated with the treated time but not the concentrations of SNPs. Importantly, for the first time, SNP accumulation in ER was discovered through co-localization analysis, which incurs ER autophagy. These new findings about SNPs-induced ER autophagy could open an effective way for securely designing silica-based nanoparticles and enable us to know more about ER autophagy.