Autophagy is an important biological process in host defense against viral infection. However, many viruses have evolved various strategies to disrupt the host antiviral system. Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus with a large economic impact on the swine industry. At present, studies on the escape mechanism of PRRSV in the autophagy process, especially through chaperone-mediated autophagy (CMA), are limited. This study confirmed that PRRSV glycoprotein 5 (GP5) could disrupt the formation of the GFAP-LAMP2A complex by inhibiting the MTORC2/PHLPP1/GFAP pathway, promoting the dissociation of the pGFAP-EF1α complex, and blocking the K63-linked polyubiquitination of LAMP2A to inhibit the activity of CMA. Further research demonstrated that CMA plays an anti-PRRSV role by antagonizing nonstructural protein 11 (NSP11)-mediated inhibition of type I interferon (IFN-I) signaling. Taken together, these results indicate that PRRSV GP5 inhibits the antiviral effect of CMA by targeting LAMP2A. This research provides new insight into the escape mechanism of immunosuppressive viruses in CMA.
OBJECTIVE: Viruses have evolved sophisticated mechanisms to manipulate autophagy to evade degradation and immune responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus that causes enormous economic losses in the swine industry. However, the mechanism by which PRRSV manipulates autophagy to defend against host antiviral effects remains unclear. In this study, we found that PRRSV GP5 interacts with LAMP2A and disrupts the formation of the GFAP-LAMP2A complex, thus inhibiting the activity of CMA and subsequently enhancing the inhibitory effect of the NSP11-mediated IFN-I signaling pathway, ultimately facilitating PRRSV replication. Our study revealed a novel mechanism by which PRRSV escapes host antiviral effects through CMA, providing a potential host target, LAMP2A, for developing antiviral drugs and contributing to understanding the escape mechanism of immunosuppressive viruses.

Breast cancer (BC) is most commonly diagnosed worldwide. Liquiritigenin is a flavonoid found in various species of the Glycyrrhiza genus, showing anti-tumor activity. This article was to explore the influences of liquiritigenin on the biological behaviors of BC cells and its underlying mechanism. BC cells were treated with liquiritigenin alone or transfected with oe-HSP90 before liquiritigenin treatment. RT-qPCR and Western blotting were employed to examine the levels of HSP90, Snail, E-cadherin, HSC70, and LAMP-2A. Cell viability, proliferation, migration, and invasion were evaluated by performing MTT, colony formation, scratch, and Transwell assays, respectively. Liquiritigenin treatment reduced HSP90 and Snail levels and enhanced E-cadherin expression as well as inhibiting the proliferation, migration, and invasion of BC cells. Moreover, liquiritigenin treatment decreased the expression of HSC70 and LAMP-2A, proteins related to chaperone-mediated autophagy (CMA). HSP90 overexpression promoted the CMA, invasion, and migration of BC cells under liquiritigenin treatment. Liquiritigenin inhibits HSP90-mediated CMA, thereby suppressing BC cell growth.

Human APOE4 (apolipoprotein E4 isoform) is a powerful genetic risk factor for late-onset Alzheimer disease (AD). Many groups have investigated the effect of APOE4 on the degradation of amyloid β (Aβ), the main component of plaques found in the brains of AD patients. However, few studies have focused on the degradation of APOE itself. We investigated the lysosomal trafficking of APOE in cells and found that APOE from the post-Golgi compartment is degraded through an autophagic process requiring the lysosomal membrane protein LAMP2A. We found that APOE4 accumulates in enlarged lysosomes, alters autophagic flux, and changes the proteomic contents of lysosomes following internalization. This dysregulated lysosomal trafficking may represent one of the mechanisms that contributes to AD pathogenesis.

Chaperone-mediated autophagy (CMA) is part of the mammalian cellular proteostasis network that ensures protein quality control, maintenance of proteome homeostasis, and proteome changes required for the adaptation to stress. Loss of proteostasis is one of the hallmarks of aging. CMA decreases with age in multiple rodent tissues and human cell types. A decrease in lysosomal levels of the lysosome-associated membrane protein type 2A (LAMP2A), the CMA receptor, has been identified as a main reason for declined CMA in aging. Here, we report constitutive activation of CMA with calorie restriction (CR), an intervention that extends healthspan, in old rodent livers and in an in vitro model of CR with cultured fibroblasts. We found that CR-mediated upregulation of CMA is due to improved stability of LAMP2A at the lysosome membrane. We also explore the translational value of our observations using calorie-restriction mimetics (CRMs), pharmacologically active substances that reproduce the biochemical and functional effects of CR. We show that acute treatment of old mice with CRMs also robustly activates CMA in several tissues and that this activation is required for the higher resistance to lipid dietary challenges conferred by treatment with CRMs. We conclude that part of the beneficial effects associated with CR/CRMs could be a consequence of the constitutive activation of CMA mediated by these interventions.

Autophagy mediates the degradation of intracellular macromolecules and organelles within lysosomes. There are three types of autophagy: macroautophagy, microautophagy, and chaperone-mediated autophagy. Heat shock protein 70.1 (Hsp70.1) exhibits dual functions as a chaperone protein and a lysosomal membrane stabilizer. Since chaperone-mediated autophagy participates in the recycling of ∼30% cytosolic proteins, its disorder causes cell susceptibility to stress conditions. Cargo proteins destined for degradation such as amyloid precursor protein and tau protein are trafficked by Hsp70.1 from the cytosol into lysosomes. Hsp70.1 is composed of an N-terminal nucleotide-binding domain (NBD) and a C-terminal domain that binds to cargo proteins, termed the substrate-binding domain (SBD). The NBD and SBD are connected by the interdomain linker LL1, which modulates the allosteric structure of Hsp70.1 in response to ADP/ATP binding. After the passage of the Hsp70.1-cargo complex through the lysosomal limiting membrane, high-affinity binding of the positive-charged SBD with negative-charged bis(monoacylglycero)phosphate (BMP) at the internal vesicular membranes activates acid sphingomyelinase to generate ceramide for stabilizing lysosomal membranes. As the integrity of the lysosomal limiting membrane is critical to ensure cargo protein degradation within the acidic lumen, the disintegration of the lysosomal limiting membrane is lethal to cells. After the intake of high-fat diets, however, β-oxidation of fatty acids in the mitochondria generates reactive oxygen species, which enhance the oxidation of membrane linoleic acids to produce 4-hydroxy-2-nonenal (4-HNE). In addition, 4-HNE is produced during the heating of linoleic acid-rich vegetable oils and incorporated into the body via deep-fried foods. This endogenous and exogenous 4-HNE synergically causes an increase in its serum and organ levels to induce carbonylation of Hsp70.1 at Arg469, which facilitates its conformational change and access of activated μ-calpain to LL1. Therefore, the cleavage of Hsp70.1 occurs prior to its influx into the lysosomal lumen, which leads to lysosomal membrane permeabilization/rupture. The resultant leakage of cathepsins is responsible for lysosomal cell death, which would be one of the causative factors of lifestyle-related diseases.

Alzheimer\'s disease (AD) is a progressive brain disorder marked by abnormal protein accumulation and resulting proteotoxicity. This study examines Chaperone-Mediated Autophagy (CMA), particularly substrate translocation into lysosomes, in AD. The study observes: (1) Increased substrate translocation activity into lysosomes, vital for CMA, aligns with AD progression, highlighted by gene upregulation and more efficient substrate delivery. (2) This CMA phase strongly correlates with AD\'s clinical symptoms; more proteotoxicity links to worse dementia, underscoring the need for active degradation. (3) Proteins like GFAP and LAMP2A, when upregulated, almost certainly indicate AD risk, marking this process as a significant AD biomarker. Based on these observations, this study proposes the following hypothesis: As AD progresses, the aggregation of pathogenic proteins increases, the process of substrate entry into lysosomes via CMA becomes active. The genes associated with this process exhibit heightened sensitivity to AD. This conclusion stems from an analysis of over 10,000 genes and 363 patients using two AI methodologies. These methodologies were instrumental in identifying genes highly sensitive to AD and in mapping the molecular networks that respond to the disease, thereby highlighting the significance of this critical phase of CMA.

Obesity is one of the most common metabolic diseases around the world, which is distinguished by the abnormal buildup of triglycerides within adipose cells. Recent research has revealed that autophagy regulates lipid mobilization to maintain energy balance. TIGAR (Trp53 induced glycolysis regulatory phosphatase) has been identified as a glycolysis inhibitor, whether it plays a role in the metabolism of lipids is unknown. Here, we found that TIGAR transgenic (TIGAR+/+) mice exhibited increased fat mass and trended to obesity phenotype. Non-target metabolomics showed that TIGAR caused the dysregulation of the metabolism profile. The quantitative transcriptome sequencing identified an increased levels of LRRK2 and RAB7B in the adipose tissue of TIGAR+/+ mice. It was confirmed in vitro that TIGAR overexpression increased the levels of LRRK2 by inhibiting polyubiquitination degradation, thereby suppressing macroautophagy and chaperone-mediated autophagy (CMA) while increasing lipid accumulation which were reversed by the LRRK2 inhibitor DNL201. Furthermore, TIGAR drove LRRK2 to interact with RAB7B for suppressing lysosomal degradation of lipid droplets, while the increased lipid droplets in adipocytes were blocked by the RAB7B inhibitor ML282. Additionally, fat-specific TIGAR knockdown of TIGAR+/+ mice alleviated the symptoms of obesity, and adipose tissues-targeting superiority DNL201 nano-emulsion counteracted the obesity phenotype in TIGAR+/+ mice. In summary, the current results indicated that TIGAR performed a vital function in the lipid metabolism through LRRK2-mediated negative regulation of macroautophagy and CMA in adipocyte. The findings suggest that TIGAR has the potential to serve as a viable therapeutic target for treating obesity and its associated metabolic dysfunction.

OBJECTIVE: To investigate the effect of chaperone-mediated autophagy (CMA) on the damage of mouse microglial BV2 cells induce by unconjugated bilirubin (UCB).
METHODS: The BV2 cell experiments were divided into two parts. (1) For the CMA activation experiment: control group (treated with an equal volume of dimethyl sulfoxide), QX77 group (treated with 20 μmol/L QX77 for 24 hours), UCB group (treated with 40 μmol/L UCB for 24 hours), and UCB+QX77 group (treated with both 20 μmol/L QX77 and 40 μmol/L UCB for 24 hours). (2) For the cell transfection experiment: LAMP2A silencing control group (treated with an equal volume of dimethyl sulfoxide), LAMP2A silencing control+UCB group (treated with 40 μmol/L UCB for 24 hours), LAMP2A silencing group (treated with an equal volume of dimethyl sulfoxide), and LAMP2A silencing+UCB group (treated with 40 μmol/L UCB for 24 hours). The cell viability was assessed using the modified MTT method. The expression levels of p65, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), and cysteinyl aspartate specific proteinase-1 (caspase-1) were detected by Western blot. The relative mRNA expression levels of the inflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α) were determined by real-time quantitative polymerase chain reaction. Levels of IL-6 and TNF-α in the cell culture supernatant were measured using ELISA. The co-localization of heat shock cognate protein 70 with p65 and NLRP3 was detected by immunofluorescence.
RESULTS: Compared to the UCB group, the cell viability in the UCB+QX77 group increased, and the expression levels of inflammation-related proteins p65, NLRP3, and caspase-1, as well as the mRNA relative expression levels of IL-1β, IL-6, and TNF-α and levels of IL-6 and TNF-α decreased (P<0.05). Compared to the control group, there was co-localization of heat shock cognate protein 70 with p65 and NLRP3 in both the UCB and UCB+QX77 groups. After silencing the LAMP2A gene, compared to the LAMP2A silencing control+UCB group, the LAMP2A silencing+UCB group showed increased expression levels of inflammation-related proteins p65, NLRP3, and caspase-1, as well as increased mRNA relative expression levels of IL-1β, IL-6, and TNF-α and levels of IL-6 and TNF-α (P<0.05).
CONCLUSIONS: CMA is inhibited in UCB-induced BV2 cell damage, and activating CMA may reduce p65 and NLRP3 protein levels, suppress inflammatory responses, and counteract bilirubin neurotoxicity.
目的: 探讨分子伴侣介导的自噬（chaperone-mediated autophagy, CMA）对未结合胆红素（unconjugated bilirubin, UCB）诱导的小鼠小胶质细胞BV2损伤的影响。方法: BV2细胞实验分为两部分。（1）CMA激活实验分为：对照组（等体积二甲基亚砜处理）、QX77组（20 μmol/L QX77处理24 h）、UCB组（40 μmol/L UCB处理24 h）、UCB+QX77组（20 μmol/L QX77和40 μmol/L UCB共处理24 h）。（2）细胞转染实验分为：LAMP2A沉默对照组（等体积二甲基亚砜处理）、LAMP2A沉默对照+UCB组（40 μmol/L UCB处理24 h）、LAMP2A沉默组（等体积二甲基亚砜处理）、LAMP2A沉默+UCB组（40 μmol/L UCB处理24 h）。采用改良MTT法检测细胞存活率，蛋白免疫印迹法检测p65、核苷酸结合寡聚化结构域样受体蛋白3（nucleotide-binding oligomerization domain-like receptor protein 3, NLRP3）、半胱氨酸天冬氨酸蛋白水解酶-1（cysteinyl aspartate specific proteinase-1, caspase-1）蛋白表达水平，实时荧光定量聚合酶链反应法检测炎症因子白细胞介素（interleukin, IL）-1β、IL-6、肿瘤坏死因子-α（tumor necrosis factor-α, TNF-α）mRNA相对表达量，ELISA法检测细胞培养上清液中炎症因子IL-6和TNF-α水平，免疫荧光法检测细胞内p65、NLRP3与热休克同源蛋白70的荧光共定位。结果: 与UCB组相比，UCB+QX77组细胞存活率升高，炎症相关蛋白p65、NLRP3、caspase-1表达水平降低，炎症因子IL-1β、IL-6、TNF-α mRNA相对表达量降低以及IL-6、TNF-α水平降低（P<0.05）。与对照组相比，UCB组和UCB+QX77组热休克同源蛋白70与p65、NLRP3存在共定位。沉默LAMP2A基因后，与LAMP2A沉默对照+UCB组相比，LAMP2A沉默+UCB组炎症相关蛋白p65、NLRP3、caspase-1蛋白表达水平升高，炎症因子IL-1β、IL-6、TNF-α mRNA相对表达量升高以及IL-6、TNF-α水平升高（P<0.05）。结论: CMA在UCB诱导的BV2细胞损伤中被抑制，激活CMA可能通过降低p65和NLRP3蛋白水平，抑制炎症反应，拮抗胆红素神经毒性。.

RAB GTPases (RABs) control intracellular membrane trafficking with high precision. In the present study, we carried out a short hairpin RNA (shRNA) screen focused on a library of 62 RABs during infection with porcine reproductive and respiratory syndrome virus 2 (PRRSV-2), a member of the family Arteriviridae. We found that 13 RABs negatively affect the yield of PRRSV-2 progeny virus, whereas 29 RABs have a positive impact on the yield of PRRSV-2 progeny virus. Further analysis revealed that PRRSV-2 infection transcriptionally regulated RAB18 through RIG-I/MAVS-mediated canonical NF-κB activation. Disrupting RAB18 expression led to the accumulation of lipid droplets (LDs), impaired LDs catabolism, and flawed viral replication and assembly. We also discovered that PRRSV-2 co-opts chaperone-mediated autophagy (CMA) for lipolysis via RAB18, as indicated by the enhanced associations between RAB18 and perlipin 2 (PLIN2), CMA-specific lysosomal associated membrane protein 2A (LAMP2A), and heat shock protein family A (Hsp70) member 8 (HSPA8/HSC70) during PRRSV-2 infection. Knockdown of HSPA8 and LAMP2A impacted on the yield of PRRSV-2 progeny virus, implying that the virus utilizes RAB18 to promote CMA-mediated lipolysis. Importantly, we determined that the C-terminal domain (CTD) of HSPA8 could bind to the switch II domain of RAB18, and the CTD of PLIN2 was capable of associating with HSPA8, suggesting that HSPA8 facilitates the interaction between RAB18 and PLIN2 in the CMA process. In summary, our findings elucidate how PRRSV-2 hijacks CMA-mediated lipid metabolism through innate immune activation to enhance the yield of progeny virus, offering novel insights for the development of anti-PRRSV-2 treatments.

The inhibition of the unfolded protein response (UPR), which usually protects cancer cells from stress, may be exploited to potentiate the cytotoxic effect of drugs inducing ER stress. However, in this study, we found that ER stress and UPR activation by thapsigargin or tunicamycin promoted the lysosomal degradation of mutant (MUT) TP53 and that the inhibition of the UPR sensor ATF6, but not of ERN1/IRE1 or EIF2AK3/PERK, counteracted such an effect. ATF6 activation was indeed required to sustain the function of lysosomes, enabling the execution of chaperone-mediated autophagy (CMA) as well as of macroautophagy, processes involved in the degradation of MUT TP53 in stressed cancer cells. At the molecular level, by pharmacological and genetic approaches, we demonstrated that the inhibition of ATF6 correlated with the activation of MTOR and with TFEB and LAMP1 downregulation in thapsigargin-treated MUT TP53 carrying cells. We hypothesize that the rescue of MUT TP53 expression by ATF6 inhibition, could further activate MTOR and maintain lysosomal dysfunction, further inhibiting MUT TP53 degradation, in a vicious circle. The findings of this study suggest that the presence of MUT TP53, which often exerts oncogenic properties, should be considered before approaching treatments combining ER stressors with ATF6 inhibitors against cancer cells, while it could represent a promising strategy against cancer cells that harbor WT TP53.