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Rab11 family interacting protein 4 (Rab11-FIP4) regulates endocytic trafficking. A possible role for Rab11-FIP4 in the regulation of lysosomal function has been proposed, but its precise function in the regulation of cellular homeostasis is unknown. By mRNA array and protein analysis, we found that Rab11-FIP4 is downregulated in the lysosomal storage disease cystinosis, which is caused by genetic defects in the lysosomal cystine transporter, cystinosin. Rescue of Rab11-FIP4 expression in Ctns-/- fibroblasts re-established normal autophagosome levels and decreased LC3B-II expression in cystinotic cells. Furthermore, Rab11-FIP4 reconstitution increased the localization of the chaperone-mediated autophagy receptor LAMP2A at the lysosomal membrane. Treatment with genistein, a phytoestrogen that upregulates macroautophagy, or the CMA activator QX77 (CA77) restored Rab11-FIP4 expression levels in cystinotic cells supporting a cross-regulation between two independent autophagic mechanisms, lysosomal function and Rab11-FIP4. Improved cellular homeostasis in cystinotic cells rescued by Rab11-FIP4 expression correlated with decreased endoplasmic reticulum stress, an effect that was potentiated by Rab11 and partially blocked by expression of a dominant negative Rab11. Restoring Rab11-FIP4 expression in cystinotic proximal tubule cells increased the localization of the endocytic receptor megalin at the plasma membrane, suggesting that Rab11-FIP4 reconstitution has the potential to improve cellular homeostasis and function in cystinosis.

Ischemia–reperfusion is a complex brain disease involving multiple biological processes, including autophagy, oxidative stress, and mitochondria-associated apoptosis. Chaperone-mediated autophagy (CMA), a selective autophagy, is involved in the development of various neurodegenerative diseases and acute nerve injury, but its role in ischemia–reperfusion is unclear. Here, we used middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen–glucose deprivation/reoxygenation (OGD/R) models to simulate cerebral ischemic stroke in vivo and in vitro, respectively. LAMP2A (lysosome-associated membrane protein 2A), a key molecule of CMA, was dramatically downregulated in ischemia–reperfusion. Enhancement of CMA activity by LAMP2A overexpression reduced the neurological deficit, brain infarct volume, pathological features, and neuronal apoptosis of the cortex in vivo. Concomitantly, enhanced CMA activity alleviated OGD/R-induced apoptosis and mitochondrial membrane potential decline in vitro. In addition, we found that CMA inhibited the P53(Tumor protein p53) signaling pathway and reduced P53 translocation to mitochondria. The P53 activator, Nutlin-3, not only reversed the inhibitory effect of CMA on apoptosis, but also significantly weakened the protective effect of CMA on OGD/R and MCAO/R. Taken together, these results indicate that inhibition of P53-mediated mitochondria-associated apoptosis is essential for the neuroprotective effect of CMA against ischemia–reperfusion. [ABSTRACT FROM AUTHOR]

Defects in chaperone-mediated autophagy (CMA) are associated with cellular senescence, but the mechanism remains poorly understood. Here, we found that CMA inhibition induced cellular senescence in a calcium-dependent manner and identified its role in TNF-induced senescence of nucleus pulposus cells (NPC) and intervertebral disc degeneration. Based on structural and functional proteomic screens, PLCG1 (phospholipase C gamma 1) was predicted as a potential substrate for CMA deficiency to affect calcium homeostasis. We further confirmed that PLCG1 was a key mediator of CMA in the regulation of intracellular calcium flux. Aberrant accumulation of PLCG1 caused by CMA blockage resulted in calcium overload, thereby inducing NPC senescence. Immunoassays on human specimens showed that reduced LAMP2A, the rate-limiting protein of CMA, or increased PLCG1 was associated with disc senescence, and the TNF-induced disc degeneration in rats was inhibited by overexpression of Lamp2a or knockdown of Plcg1. Because CMA dysregulation, calcium overload, and cellular senescence are common features of disc degeneration and other age-related degenerative diseases, the discovery of actionable molecular targets that can link these perturbations may have therapeutic value. Abbreviation: ATRA: all-trans-retinoic acid; BrdU: bromodeoxyuridine; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; CDKN2A/p16-INK4A: cyclin dependent kinase inhibitor 2A; CMA: chaperone-mediated autophagy; DHI: disc height index; ER: endoplasmic reticulum; IP: immunoprecipitation; IP3: inositol 1,4,5-trisphosphate; ITPR/IP3R: inositol 1,4,5-trisphosphate receptor; IVD: intervertebral disc; IVDD: intervertebral disc degeneration; KD: knockdown; KO: knockout; Leu: leupeptin; MRI: magnetic resonance imaging; MS: mass spectrometry; N/L: NH4Cl and leupeptin; NP: nucleus pulposus; NPC: nucleus pulposus cells; PI: protease inhibitors; PLC: phospholipase C; PLCG1: phospholipase C gamma 1; ROS: reactive oxygen species; RT-qPCR: real-time quantitative reverse transcription PCR; SA-GLB1/β-gal: senescence-associated galactosidase beta 1; SASP: senescence-associated secretory phenotype; STV: starvation; TMT: tandem mass tag; TNF: tumor necrosis factor; TP53: tumor protein p53; UPS: ubiquitin-proteasome system. [ABSTRACT FROM AUTHOR]

Chaperone-mediated autophagy (CMA) is a selective autophagic pathway responsible for degrading cytoplasmic proteins within lysosomes. Monitoring CMA flux is essential for understanding its functions and molecular mechanisms but remains technically complex and challenging. In this study, we developed a pH-resistant probe, KFERQ-Gamillus, by screening various green fluorescent proteins. This probe is activated under conditions known to induce CMA, such as serum starvation, and relies on LAMP2A and the KFERQ motif for lysosomal localization and degradation, demonstrating its specificity for the CMA pathway. It enables the detection of CMA activity in living cells through both microscopy and image-based flow cytometry. Additionally, we created a dual-reporter system, KFERQ-Gamillus-Halo, by integrating KFERQ-Gamillus with the Halo-tag system. This probe not only distinguishes between protein synthesis and degradation but also facilitates the detection of intracellular CMA flux via immunoblotting and the rapid assessment of CMA activity using flow cytometry. Together, the KFERQ-Gamillus-Halo probe provides quantitative and time-resolved monitoring for CMA activity and flux in living cells. This tool holds promising potential for high-throughput screening and biomedical research related to CMA. [ABSTRACT FROM AUTHOR]

Autophagy is a conserved catabolic process crucial for maintaining cellular homeostasis and has emerged as a promising therapeutic target for many diseases. Mechanistically novel small-molecule autophagy regulators are highly desirable from a pharmacological point of view. Here, we report the macroautophagy-inhibitory effect of hapalindole Q, a member of the structurally intriguing but biologically understudied hapalindole family of indole terpenoids. This compound promotes the noncanonical degradation of Yes-associated protein 1 (YAP1), the downstream effector of the Hippo signaling pathway, via chaperone-mediated autophagy, disrupting proper distribution of Rab7 and suppressing autophagosome-lysosome fusion in macroautophagy. Its binding to YAP1 is further confirmed by using biophysical techniques. A preliminary structure-activity relationship study reveals that the hapalindole Q scaffold, rather than the isothiocyanate group, is essential for YAP1 binding and degradation. This work not only identifies a macroautophagy inhibitor with a distinct mechanism of action but also provided a molecular scaffold for direct targeting of YAP1, which may benefit the development of therapeutics for both autophagy-related and Hippo-YAP-related diseases. [ABSTRACT FROM AUTHOR]

Autophagy is an intracellular recycling process that maintains cellular homeostasis by degrading excess or defective macromolecules and organelles. Chaperone-mediated autophagy (CMA) is a highly selective form of autophagy in which a substrate containing a KFERQ-like motif is recognized by a chaperone protein, delivered to the lysosomal membrane, and then translocated to the lysosome for degradation with the assistance of lysosomal membrane protein 2A. Normal CMA activity is involved in the regulation of cellular proteostasis, metabolism, differentiation, and survival. CMA dysfunction disturbs cellular homeostasis and directly participates in the pathogenesis of human diseases. Previous investigations on CMA in the central nervous system have primarily focus on neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. Recently, mounting evidence suggested that brain injuries involve a wider range of types and severities, making the involvement of CMA in the bidirectional processes of damage and repair even more crucial. In this review, we summarize the basic processes of CMA and its associated regulatory mechanisms and highlight the critical role of CMA in brain injury such as cerebral ischemia, traumatic brain injury, and other specific brain injuries. We also discuss the potential of CMA as a therapeutic target to treat brain injury and provide valuable insights into clinical strategies. [ABSTRACT FROM AUTHOR]

Abstract Breast cancer remains a significant health concern, with triple-negative breast cancer (TNBC) being an aggressive subtype with poor prognosis. Epithelial-mesenchymal transition (EMT) is important in early-stage tumor to invasive malignancy progression. Snail, a central EMT component, is tightly regulated and may be subjected to proteasomal degradation. We report a novel proteasomal independent pathway involving chaperone-mediated autophagy (CMA) in Snail degradation, mediated via its cytosolic interaction with HSC70 and lysosomal targeting, which prevented its accumulation in luminal-type breast cancer cells. Conversely, Snail predominantly localized to the nucleus, thus evading CMA-mediated degradation in TNBC cells. Starvation-induced CMA activation downregulated Snail in TNBC cells by promoting cytoplasmic translocation. Evasion of CMA-mediated Snail degradation induced EMT, and enhanced metastatic potential of luminal-type breast cancer cells. Our findings elucidate a previously unrecognized role of CMA in Snail regulation, highlight its significance in breast cancer, and provide a potential therapeutic target for clinical interventions.

SORT1 (sortilin 1), a member of the the Vps10 (vacuolar protein sorting 10) family, is involved in hepatic lipid metabolism by regulating very low-density lipoprotein (VLDL) secretion and facilitating the lysosomal degradation of CES1 (carboxylesterase 1), crucial for triglyceride (TG) breakdown in the liver. This study explores whether SORT1 is targeted for degradation by chaperone-mediated autophagy (CMA), a selective protein degradation pathway that directs proteins containing KFERQ-like motifs to lysosomes via LAMP2A (lysosomal-associated membrane protein 2A). Silencing LAMP2A or HSPA8/Hsc70 with siRNA increased cytosolic SORT1 protein levels. Leupeptin treatment induced lysosomal accumulation of SORT1, unaffected by siLAMP2A co-treatment, indicating CMA-dependent degradation. Human SORT1 contains five KFERQ-like motifs (658VVTKQ662, 730VREVK734, 733VKDLK737, 734KDLKK738, and 735DLKKK739), crucial for HSPA8 recognition; mutating any single amino acid within these motifs decreased HSPA8 binding. Furthermore, compromised CMA activity resulted in elevated SORT1-mediated degradation of CES1, contributing to increased lipid accumulation in hepatocytes. Consistent with in vitro findings, LAMP2A knockdown in mice exacerbated high-fructose diet-induced fatty liver, marked by increased SORT1 and decreased CES1 levels. Conversely, LAMP2A overexpression promoted SORT1 degradation and CES1D accumulation, counteracting fasting-induced CES1D suppression through CMA activation. Our findings reveal that SORT1 is a substrate of CMA, highlighting its crucial role in directing CES1 to lysosomes. Consequently, disrupting CMA-mediated SORT1 degradation significantly affects CES1-dependent TG hydrolysis, thereby affecting hepatic lipid homeostasis.Abbreviations: APOB: apolipoprotein B; CES1: carboxylesterase 1; CMA: chaperone-mediated autophagy; HSPA8/Hsc70: heat shock protein family A (Hsp70) member 8; LAMP2A: lysosomal associated membrane protein 2A; LDL-C: low-density lipoprotein-cholesterol; PLIN: perilipin; SORT1: sortilin 1; TG: triglyceride; VLDL: very low-density lipoprotein; Vps10: vacuolar protein sorting 10. [ABSTRACT FROM AUTHOR]

Drug development for Alzheimer's disease (AD) treatment is challenging due to its complex pathogenesis. Tetradecyl 2,3-dihydroxybenzoate (ABG-001), a leading compound identified in our prior research, has shown promising NGF-mimicking activity and anti-aging properties. In the present study, both high-fat diet (HFD)-induced AD mice and naturally aging AD mice were used to evaluate anti-AD effects. Meanwhile, RNA-sequences, Western blotting, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA), cellular thermal shift assay (CETSA), drug affinity-responsive target stability (DARTS) assay, construction of expression plasmid and protein purification, surface plasmon resonance (SPR) analysis, and 16S rRNA sequence analysis were used to identify the target protein of ABG-001 and clarify the mechanism of action for this molecule. ABG-001 effectively mitigates the memory dysfunction in both HFD-induced AD mice and naturally aging AD mice. The therapeutic effect of ABG-001 is attributed to its ability to promote neurogenesis, activate chaperone-mediated autophagy (CMA), and reduce neuronal inflammation. Additionally, ABG-001 positively influenced the gut microbiota, enhancing the production of indole-3-propionic acid (IPA), which is capable of crossing the blood–brain barrier (BBB) and contributes to neuronal regeneration. Furthermore, our research revealed that IPA, linked to the anti-AD properties of ABG-001, targets the heat shock cognate 70 kDa protein (Hsc70) and regulates the Hsc70/PKM2/HK2/LC3 and FOXO3a/SIRT1 signaling pathways. ABG-001 improves the memory dysfunction of AD mice by modulating autophagy and inflammation through IPA and Hsc70 targeting. These findings offer a novel approach for treating neurodegenerative diseases, focusing on the modification of the gut microbiota and metabolites coupled with anti-aging strategies. [ABSTRACT FROM AUTHOR]

Many types of RNA viruses, including the hepatitis C virus (HCV), activate autophagy in infected cells to promote viral growth and counteract the host defense response. Autophagy acts as a catabolic pathway in which unnecessary materials are removed via the lysosome, thus maintaining cellular homeostasis. The HCV non-structural 5A (NS5A) protein is a phosphoprotein required for viral RNA replication, virion assembly, and the determination of interferon (IFN) sensitivity. Recently, increasing evidence has shown that HCV NS5A can induce autophagy to promote mitochondrial turnover and the degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) and diacylglycerol acyltransferase 1 (DGAT1). In this review, we summarize recent progress in understanding the detailed mechanism by which HCV NS5A triggers autophagy, and outline the physiological significance of the balance between host–virus interactions. [ABSTRACT FROM AUTHOR]

Breast cancer remains a significant health concern, with triple-negative breast cancer (TNBC) being an aggressive subtype with poor prognosis. Epithelial-mesenchymal transition (EMT) is important in early-stage tumor to invasive malignancy progression. Snail, a central EMT component, is tightly regulated and may be subjected to proteasomal degradation. We report a novel proteasomal independent pathway involving chaperone-mediated autophagy (CMA) in Snail degradation, mediated via its cytosolic interaction with HSC70 and lysosomal targeting, which prevented its accumulation in luminal-type breast cancer cells. Conversely, Snail predominantly localized to the nucleus, thus evading CMA-mediated degradation in TNBC cells. Starvation-induced CMA activation downregulated Snail in TNBC cells by promoting cytoplasmic translocation. Evasion of CMA-mediated Snail degradation induced EMT, and enhanced metastatic potential of luminal-type breast cancer cells. Our findings elucidate a previously unrecognized role of CMA in Snail regulation, highlight its significance in breast cancer, and provide a potential therapeutic target for clinical interventions. [ABSTRACT FROM AUTHOR]

In orchestrating cell signaling, facilitating plasma membrane repair, supervising protein secretion, managing waste elimination, and regulating energy consumption, lysosomes are indispensable guardians that play a crucial role in preserving intracellular homeostasis. Neurons are terminally differentiated post-mitotic cells. Neuronal function and waste elimination depend on normal lysosomal function. Converging data suggest that lysosomal dysfunction is a critical event in the etiology of Parkinson's disease (PD). Mutations in Glucosylceramidase Beta 1 (GBA1) and leucine-rich repeat kinase 2 (LRRK2) confer an increased risk for the development of parkinsonism. Furthermore, lysosomal dysfunction has been observed in the affected neurons of sporadic PD (sPD) patients. Given that lysosomal hydrolases actively contribute to the breakdown of impaired organelles and misfolded proteins, any compromise in lysosomal integrity could incite abnormal accumulation of proteins, including α-synuclein, the major component of Lewy bodies in PD. Clinical observations have shown that lysosomal protein levels in cerebrospinal fluid may serve as potential biomarkers for PD diagnosis and as signs of lysosomal dysfunction. In this review, we summarize the current evidence regarding lysosomal dysfunction in PD and discuss the intimate relationship between lysosomal dysfunction and pathological α-synuclein. In addition, we discuss therapeutic strategies that target lysosomes to treat PD. [ABSTRACT FROM AUTHOR]

Chaperone-mediated autophagy (CMA) is the lysosomal degradation of individually selected proteins, independent of vesicle fusion. CMA is a central part of the proteostasis network in vertebrate cells. However, CMA is also a negative regulator of anabolism, and it degrades enzymes required for glycolysis, de novo lipogenesis, and translation at the cytoplasmic ribosome. Recently, CMA has gained attention as a possible modulator of rodent aging. Two mechanistic models have been proposed to explain the relationship between CMA and aging in mice. Both of these models are backed by experimental data, and they are not mutually exclusionary. Model 1, the “Longevity Model,” states that lifespan-extending interventions that decrease signaling through the INS/IGF1 signaling axis also increase CMA, which degrades (and thereby reduces the abundance of) several proteins that negatively regulate vertebrate lifespan, such as MYC, NLRP3, ACLY, and ACSS2. Therefore, enhanced CMA, in early and midlife, is hypothesized to slow the aging process. Model 2, the “Aging Model,” states that changes in lysosomal membrane dynamics with age lead to age-related losses in the essential CMA component LAMP2A, which in turn reduces CMA, contributes to age-related proteostasis collapse, and leads to overaccumulation of proteins that contribute to age-related diseases, such as Alzheimer’s disease, Parkinson’s disease, cancer, atherosclerosis, and sterile inflammation. The objective of this review paper is to comprehensively describe the data in support of both of these explanatory models, and to discuss the strengths and limitations of each.

African swine fever (ASF) is a highly contagious, often fatal viral disease caused by African swine fever virus (ASFV), which imposes a substantial economic burden on the global pig industry. When screening for the virus replication-regulating genes in the left variable region of the ASFV genome, we observed a notable reduction in ASFV replication following the deletion of the MGF300-4L gene. However, the role of MGF300-4L in ASFV infection remains unexplored. In this study, we found that MGF300-4L could effectively inhibit the production of proinflammatory cytokines IL-1β and TNF-α, which are regulated by the NF-κB signaling pathway. Mechanistically, we demonstrated that MGF300-4L interacts with IKKβ and promotes its lysosomal degradation via the chaperone-mediated autophagy. Meanwhile, the interaction between MGF300-4L and IκBα competitively inhibits the binding of the E3 ligase β-TrCP to IκBα, thereby inhibiting the ubiquitination-dependent degradation of IκBα. Remarkably, although ASFV encodes other inhibitors of NF-κB, the MGF300-4L gene-deleted ASFV (Del4L) showed reduced virulence in pigs, indicating that MGF300-4L plays a critical role in ASFV pathogenicity. Importantly, the attenuation of Del4L was associated with a significant increase in the production of IL-1β and TNF-α early in the infection of pigs. Our findings provide insights into the functions of MGF300-4L in ASFV pathogenicity, suggesting that MGF300-4L could be a promising target for developing novel strategies and live attenuated vaccines against ASF.

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.

Objective: To investigate the role and the molecular mechanisms of apoptotic signaling in ferroptosis to regulate tumor radiosensitivity. Methods: Reactive oxygen species (ROS) and lipid peroxide levels were detected in Mouse embryonic fibroblasts(MEFs) with Bcl-xL or Mcl-1 deficiency induced by erastin. Colony formation, ROS, lipid peroxidation and the transcription/translation levels of PTGS2 were measured in Bcl-xL knockdown tumor cells induced by 5 ​Gy γ-rays or co-treated with ferrostatin-1 (Ferr-1). The protein levels of LPCAT3, ACSL4 and PEBP1 in Bcl-xL knockout MEF cells were evaluated in Bcl-xL knockout MEF cells post-radiation. Moreover, the interaction of heat shock protein 90 (HSP90) with Bcl-xL, GPX4, or LAMP2A was detected by protein mass spectrometry and immunoprecipitation assays. Results: Manipulating Bcl-xL levels facilitated radiation-induced ferroptosis by augmenting the enzymatic oxidation of polyunsaturated fatty acids (PUFAs) and enhancing chaperone-mediated autophagy (CMA) of glutathione peroxidase 4 (GPX4) (MEF cell line: t=4.540, P＜0.01; A549 ​cell line: t=56.16, P＜0.0001; t=4.885, P＜0.01; HCT116 ​cell line: t=14.75, P＜0.01; t=7.363, P＜0.05). Downregulating Bcl-xL expression promoted the activity of acyl-CoA synthetase long-chain family member 4 (ACSL4), thus increasing the enzymatic oxidation of PUFAs (t=4.258, P＜0.01). Moreover, depletion of Bcl-xL expedited the CMA process targeting GPX4 by facilitating the association of GPX4 with heat shock protein 90 (HSP90) and LAMP2A following radiation exposure. Subsequent degradation of GPX4 led to the accumulation of lipid peroxides, ultimately triggering ferroptosis. Conclusions: Our study provides initial insights into the regulatory role of Bcl-xL in ferroptosis and underscores the potential of targeting Bcl-xL as a promising therapeutic strategy for cancer by modulating both apoptotic and ferroptotic pathways.

Introduction: Aromatic (Ar)-turmerone is a bioactive component of turmeric oil obtained from Curcuma longa. We recently identified a novel analog (A2) of arturmerone that protects dopaminergic neurons from toxic stimuli by activating nuclear factor erythroid 2-related factor 2 (Nrf2). D-cysteine increases Nrf2, leading to the activation of chaperone-mediated autophagy (CMA), a pathway in the autophagy-lysosome protein degradation system, in primary cultured cerebellar Purkinje cells. In this study, we attempted to identify novel analogs of ar-turmerone that activate Nrf2 more potently and investigated whether these analogs activate CMA. Methods: Four novel analogs (A4--A7) from A2 were synthesized. We investigated the effects of A2 and novel 4 analogs on Nrf2 expression via immunoblotting and CMA activity via fluorescence observation. Results: Although all analogs, including A2, increased Nrf2 expression, only A4 activated CMA in SH-SY5Y cells. Additionally, A4-mediated CMA activation was not reversed by Nrf2 inhibition, indicating that A4 activated CMA via mechanisms other than Nrf2 activation. We focused on p38, which participates in CMA activation. Inhibition of p38 significantly prevented A4-mediated activation of CMA. Although all novel analogs significantly increased the phosphorylation of p38 6 h after drug treatment, only A4 significantly increased phosphorylation 24 h after treatment. Finally, we revealed that A4 protected SH-SY5Y cells from the cytotoxicity of rotenone, and that this protection was reversed by inhibiting p38. Conclusion: These findings suggest that the novel ar-turmerone analog, A4, activates CMA and protects SH-SY5Y cells through the persistent activation of p38. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: Acute myeloid leukemia (AML) is a cancer of the hematopoietic system characterized by hyperproliferation of undifferentiated cells of the myeloid lineage. While most of AML therapies are focused toward tumor debulking, all-trans retinoic acid (ATRA) induces neutrophil differentiation in the AML subtype acute promyelocytic leukemia (APL). Macroautophagy has been extensively investigated in the context of various cancers and is often dysregulated in AML where it can have context-dependent pro- or anti-leukemogenic effects. On the contrary, the implications of chaperone-mediated autophagy (CMA) on the pathophysiology of diseases are still being explored and its role in AML remains elusive. Methods: We took advantage of human AML primary samples and databases to analyze CMA gene expression and activity. Furthermore, we used ATRA-sensitive (NB4) and -resistant (NB4-R1) APL cells to further dissect a potential function for CMA in ATRA-mediated neutrophil differentiation. NB4-R1 cells are unique in that they do respond to retinoic acid transcriptionally but do not mature in response to retinoid signaling alone unless maturation is triggered by adding cyclic adenosine monophosphate. Results: Here, we report that CMA-related mRNA transcripts are significantly higher expressed in immature hematopoietic cells as compared to neutrophils, contrasting the macroautophagy gene expression patterns. Accordingly, lysosomal degradation of an mCherry-KFERQ CMA reporter decreases during ATRA-induced differentiation of APL cells. On the other hand, using NB4-R1 cells we found that macroautophagy flux primed ATRA-resistant NB4-R1 cells to differentiate upon ATRA treatment but reduced the association of lysosome-associated membrane protein type 2A (LAMP-2A) and heat shock protein family A (Hsp70) member 8 (HSPA8), necessary for complete neutrophil maturation. Accordingly, depletion of HSPA8 attenuated CMA activity and facilitated APL cell differentiation. In contrast, maintaining high CMA activity by ectopic expression of LAMP-2A impeded APL differentiation. Conclusion: Overall, our findings suggest that APL neutrophil differentiation requires CMA inactivation and that this pathway predominantly depends on HSPA8 and is possibly assisted by other co-chaperones. [ABSTRACT FROM AUTHOR]

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. RTqPCR and Western blotting were employed to examine the levels of HSP90, Snail, Ecadherin, 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. [ABSTRACT FROM AUTHOR]

Iron is widely linked with the onset and development of Parkinson's disease (PD). Accumulation of iron induces free radical generation and promotes α-synuclein aggregation, oxidative stress, and autophagy impairment. Deferoxamine, an iron chelator, is shown to ameliorate iron dyshomeostasis in rodents and humans. However, the role of deferoxamine in cypermethrin-induced iron accumulation is not yet known. Although an iron accumulation and impaired chaperone-mediated autophagy (CMA) contribute to PD, a link between the two is not yet widely understood. Current study is undertaken to explore the possible association between an iron accumulation and CMA in cypermethrin model of PD in the presence of deferoxamine. Level of iron, iron transporter proteins, oxidative stress, and CMA proteins along with indicators of Parkinsonism were measured. Deferoxamine attenuated cypermethrin-induced iron accumulation and number of iron-positive cells and ameliorated the demise of dopaminergic cells and dopamine content. Deferoxamine significantly normalizes cypermethrin-induced changes in iron transporter proteins, α-synuclein, lysosome-associated membrane protein-2A, and oxidative stress. The results demonstrate that deferoxamine ameliorates cypermethrin-induced iron dyshomeostasis and impairment in CMA. [ABSTRACT FROM AUTHOR]

Increased astrocytic lactoferrin (Lf) expression was observed in the brains of elderly individuals and Alzheimer's disease (AD) patients. Our previous study revealed that astrocytic Lf overexpression improved cognitive capacity by facilitating Lf secretion to neurons to inhibit β-amyloid protein (Aβ) production in APP/PS1 mice. Here, we further discovered that astrocytic Lf overexpression inhibited neuronal loss by decreasing iron accumulation and increasing glutathione peroxidase 4 (GPX4) expression in neurons within APP/PS1 mice. Furthermore, human Lf (hLf) treatment inhibited ammonium ferric citrate (FAC)-induced ferroptosis by chelating intracellular iron. Additionally, machine learning analysis uncovered a correlation between Lf and GPX4. hLf treatment boosted low-density lipoprotein receptor-related protein 1 (LRP1) internalization and facilitated its interaction with heat shock cognate 70 (HSC70), thereby inhibiting HSC70 binds to GPX4, and eventually attenuating GPX4 degradation and FAC-induced ferroptosis. Overall, astrocytic Lf overexpression inhibited neuronal ferroptosis through two pathways: reducing intracellular iron accumulation and promoting GPX4 expression via inhibiting chaperone-mediated autophagy (CMA)-mediated GPX4 degradation. Hence, upregulating astrocytic Lf expression is a promising strategy for combating AD.

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.

Abstract Chaperone‐mediated autophagy (CMA) is a lysosome‐dependent degradation pathway that eliminates proteins that are damaged, partially unfolded, or targeted for selective proteome remodeling. CMA contributes to several cellular processes, including stress response and proteostasis. Age‐associated increase in cellular stressors and decrease in CMA contribute to pathologies associated with aging in various tissues. CMA contributes to bone homeostasis in young mice. An age‐associated reduction in CMA was reported in osteoblast lineage cells; however, whether declining CMA contributes to skeletal aging is unknown. Herein we show that cellular stressors stimulate CMA in UAMS‐32 osteoblastic cells. Moreover, the knockdown of an essential component of the CMA pathway, LAMP2A, sensitizes osteoblasts to cell death caused by DNA damage, ER stress, and oxidative stress. As elevations in these stressors are thought to contribute to age‐related bone loss, we hypothesized that declining CMA contributes to the age‐associated decline in bone formation by sensitizing osteoblast lineage cells to elevated stressors. To test this, we aged male CMA‐deficient mice and controls up to 24 months of age and examined age‐associated changes in bone mass and architecture. We showed that lack of CMA did not alter age‐associated decline in bone mineral density as measured by dual x‐ray absorptiometry (DXA). Moreover, microCT analysis performed at 24 months of age showed that vertebral cancellous bone volume, cortical thickness, and porosity of CMA‐deficient and control mice were similar. Taken together, these results suggest that reduction of CMA does not contribute to age‐related bone loss.

Cell‐surface proteins are important drug targets but historically have posed big challenges for the complete elimination of their functions. Herein, we report antibody–peptide conjugates (Ab‐CMAs) in which a peptide targeting chaperone‐mediated autophagy (CMA) was conjugated with commercially available monoclonal antibodies for specific cell‐surface protein degradation by taking advantage of lysosomal degradation pathways. Unique features of Ab‐CMAs, including cell‐surface receptor‐ and E3 ligase‐independent degradation, feasibility towards different cell‐surface proteins (e.g., epidermal growth factor receptor (EGFR), programmed cell death ligand 1 (PD‐L1), human epidermal growth factor receptor 2 (HER2)) by a simple change of the antibody, and successful tumor inhibition in vivo, make them attractive protein degraders for biomedical research and therapeutic applications. As the first example employing CMA to degrade proteins from the outside in, our findings may also shed new light on CMA, a degradation pathway typically targeting cytosolic proteins. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Chaperone‐mediated autophagy (CMA) is a lysosome‐dependent degradation pathway that eliminates proteins that are damaged, partially unfolded, or targeted for selective proteome remodeling. CMA contributes to several cellular processes, including stress response and proteostasis. Age‐associated increase in cellular stressors and decrease in CMA contribute to pathologies associated with aging in various tissues. CMA contributes to bone homeostasis in young mice. An age‐associated reduction in CMA was reported in osteoblast lineage cells; however, whether declining CMA contributes to skeletal aging is unknown. Herein we show that cellular stressors stimulate CMA in UAMS‐32 osteoblastic cells. Moreover, the knockdown of an essential component of the CMA pathway, LAMP2A, sensitizes osteoblasts to cell death caused by DNA damage, ER stress, and oxidative stress. As elevations in these stressors are thought to contribute to age‐related bone loss, we hypothesized that declining CMA contributes to the age‐associated decline in bone formation by sensitizing osteoblast lineage cells to elevated stressors. To test this, we aged male CMA‐deficient mice and controls up to 24 months of age and examined age‐associated changes in bone mass and architecture. We showed that lack of CMA did not alter age‐associated decline in bone mineral density as measured by dual x‐ray absorptiometry (DXA). Moreover, microCT analysis performed at 24 months of age showed that vertebral cancellous bone volume, cortical thickness, and porosity of CMA‐deficient and control mice were similar. Taken together, these results suggest that reduction of CMA does not contribute to age‐related bone loss. [ABSTRACT FROM AUTHOR]

The dynein motor protein complex is required for retrograde transport but the functions of the intermediate-light chains that form the cargo-binding complex are not elucidated and the importance of individual subunits in maintaining cellular homeostasis is unknown. Here, using mRNA arrays and protein analysis, we show that the dynein subunit, DYNC1LI2 (dynein, cytoplasmic 1 light intermediate chain 2) is downregulated in cystinosis, a lysosomal storage disorder caused by genetic defects in CTNS (cystinosin, lysosomal cystine transporter). Reconstitution of DYNC1LI2 expression in ctns-/- cells reestablished endolysosomal dynamics. Defective vesicular trafficking in cystinotic cells was rescued by DYNC1LI2 expression which correlated with decreased endoplasmic reticulum stress manifested as decreased expression levels of the chaperone HSPA5/GRP78, and the transcription factors ATF4 and DDIT3/CHOP. Mitochondrial fragmentation, membrane potential and endolysosomal-mitochondrial association in cystinotic cells were rescued by DYNC1LI2. Survival of cystinotic cells to oxidative stress was increased by DYNC1LI2 reconstitution but not by its paralog DYNC1LI1, which also failed to decrease ER stress and mitochondrial fragmentation. DYNC1LI2 expression rescued the localization of the chaperone-mediated autophagy (CMA) receptor LAMP2A, CMA activity, cellular homeostasis and LRP2/megalin expression in cystinotic proximal tubule cells, the primary cell type affected in cystinosis. DYNC1LI2 failed to rescue phenotypes in cystinotic cells when LAMP2A was downregulated or when co-expressed with dominant negative (DN) RAB7 or DN-RAB11, which impaired LAMP2A trafficking. DYNC1LI2 emerges as a regulator of cellular homeostasis and potential target to repair underlying trafficking and CMA in cystinosis, a mechanism that is not restored by lysosomal cystine depletion therapies.Abbreviations: ACTB: actin, beta; ATF4: activating transcription factor 4; CMA: chaperone-mediated autophagy; DYNC1LI1: dynein cytoplasmic 1 light intermediate chain 1; DYNC1LI2: dynein cytoplasmic 1 light intermediate chain 2; ER: endoplasmic reticulum; LAMP1: lysosomal associated membrane protein 1; LAMP2A: lysosomal associated membrane protein 2A; LIC: light-intermediate chains; LRP2/Megalin: LDL receptor related protein 2; PTCs: proximal tubule cells; RAB: RAB, member RAS oncogene family; RAB11FIP3: RAB11 family interacting protein 3; RILP: Rab interacting lysosomal protein.

Many types of RNA viruses, including the hepatitis C virus (HCV), activate autophagy in infected cells to promote viral growth and counteract the host defense response. Autophagy acts as a catabolic pathway in which unnecessary materials are removed via the lysosome, thus maintaining cellular homeostasis. The HCV non-structural 5A (NS5A) protein is a phosphoprotein required for viral RNA replication, virion assembly, and the determination of interferon (IFN) sensitivity. Recently, increasing evidence has shown that HCV NS5A can induce autophagy to promote mitochondrial turnover and the degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) and diacylglycerol acyltransferase 1 (DGAT1). In this review, we summarize recent progress in understanding the detailed mechanism by which HCV NS5A triggers autophagy, and outline the physiological significance of the balance between host–virus interactions.

The human apolipoprotein E4 isoform (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), and lysosomal dysfunction has been implicated in AD pathogenesis. We found, by examining cells stably expressing each APOE isoform, that APOE4 increases lysosomal trafficking, accumulates in enlarged lysosomes and late endosomes, alters autophagic flux and the abundance of autophagy proteins and lipid droplets, and alters the proteomic contents of lysosomes following internalization. We investigated APOE-related lysosomal trafficking further in cell culture, and found that APOE from the post-Golgi compartment is degraded through autophagy. We found that this autophagic process requires the lysosomal membrane protein LAMP2 in immortalized neuron-like and hepatic cells, and in mouse brain tissue. Several macroautophagy-associated proteins were also required for autophagic degradation and internalization of APOE in hepatic cells. The dysregulated autophagic flux and lysosomal trafficking of APOE4 that we observed suggest a possible novel mechanism that might contribute to AD pathogenesis. This article has an associated First Person interview with the first author of the paper.

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.

Abstract In the search for alternatives to overcome the challenge imposed by drug resistance development in cancer treatment, the modulation of autophagy has emerged as a promising alternative that has achieved good results in clinical trials. Nevertheless, most of these studies have overlooked a novel and selective type of autophagy: chaperone-mediated autophagy (CMA). Following its discovery, research into CMA’s contribution to tumor progression has accelerated rapidly. Therefore, we now understand that stress conditions are the primary signal responsible for modulating CMA in cancer cells. In turn, the degradation of proteins by CMA can offer important advantages for tumorigenesis, since tumor suppressor proteins are CMA targets. Such mutual interaction between the tumor microenvironment and CMA also plays a crucial part in establishing therapy resistance, making this discussion the focus of the present review. Thus, we highlight how suppression of LAMP2A can enhance the sensitivity of cancer cells to several drugs, just as downregulation of CMA activity can lead to resistance in certain cases. Given this panorama, it is important to identify selective modulators of CMA to enhance the therapeutic response.

Chaperone-mediated autophagy (CMA) is a selective proteolytic pathway in the lysosomes. Proteins are recognized one by one through the detection of a KFERQ motif or, at least, a KFERQ-like motif, by a heat shock cognate protein 70 (Hsc70), a molecular chaperone. CMA substrates are recognized and delivered to a lysosomal CMA receptor, lysosome-associated membrane protein 2A (LAMP-2A), the only limiting component of this pathway, and transported to the lysosomal lumen with the help of another resident chaperone HSp90. Since approximately 75% of proteins are reported to have canonical, phosphorylation-generated, or acetylation-generated KFERQ motifs, CMA maintains intracellular protein homeostasis and regulates specific functions in the cells in different tissues. CMA also regulates physiologic functions in different organs, and is then implicated in disease pathogenesis related to aging, cancer, and the central nervous and immune systems. In this minireview, we have summarized the most important findings on the role of CMA in tissue homeostasis and disease pathogenesis, updating the recent advances for this Special Issue. [ABSTRACT FROM AUTHOR]

[Display omitted] • Regulated cell death (RCD) is associated with Parkinson's disease. • HSP90 participates in various RCDs by modulating clients' stability and function. • HSP90 in Parkinson's disease: Trick or treat? • HSP90 modulates proteostasis and protein quality control. • HSP90 inhibitors may be as potential therapeutic approaches of neurodegenerative disorders. Parkinson's disease (PD) is the second most common neurodegenerative disease, characterized by abnormal α-synuclein misfolding and aggregation, mitochondrial dysfunction, oxidative stress, as well as progressive death of dopaminergic neurons in the substantia nigra. Molecular chaperones play a role in stabilizing proteins and helping them achieve their proper structure. Previous studies have shown that overexpression of heat shock protein 90 (HSP90) can lead to the death of dopaminergic neurons associated with PD. Inhibiting HSP90 is considered a potential treatment approach for neurodegenerative disorders, as it may reduce protein aggregation and related toxicity, as well as suppress various forms of regulated cell death (RCD). This review provides an overview of HSP90 and its role in PD, focusing on its modulation of proteostasis and quality control of LRRK2. The review also explores the effects of HSP90 on different types of RCD, such as apoptosis, chaperone-mediated autophagy (CMA), necroptosis, and ferroptosis. Additionally, it discusses HSP90 inhibitors that have been tested in PD models. We will highlight the under-investigated neuroprotective effects of HSP90 inhibition, including modulation of oxidative stress, mitochondrial dysfunction, PINK/PARKIN, heat shock factor 1 (HSF1), histone deacetylase 6 (HDAC6), and the PHD2-HSP90 complex-mediated mitochondrial stress pathway. By examining previous literature, this review uncovers overlooked neuroprotective mechanisms and emphasizes the need for further research on HSP90 inhibitors as potential therapeutic strategies for PD. Finally, the review discusses the potential limitations and possibilities of using HSP90 inhibitors in PD therapy. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is a major health concern globally and can accumulate and cause damage in the liver for which there is no approved treatment. Baicalin and N-acetylcysteine (NAC) have been found to have protective effects against a variety of liver injuries, but it is not clear whether their combined use is effective in preventing and treating Cd-induced lipid accumulation. The study found that Cd increased the production of mitochondrial reactive oxygen species (mROS) and elevated the level of chaperone-mediated autophagy (CMA). Interestingly, mROS-mediated CMA exacerbates the Cd-induced inhibition of lipophagy. Baicalin and NAC counteracted inhibition of lipophagy by attenuating Cd-induced CMA, suggesting an interplay between CMA elevation, mitochondrial destruction, and mROS formation. Maintaining the stability of mitochondrial structure and function is essential for alleviating Cd-induced lipid accumulation in the liver. Choline is an essential component of the mitochondrial membrane and is responsible for maintaining its structure and function. Mitochondrial transcriptional factor A (TFAM) is involved in mitochondrial DNA transcriptional activation and replication. Our study revealed that the combination of baicalin and NAC can regulate choline metabolism through TFAM and thereby maintain mitochondrial structure and functionality. In summary, the combination of baicalin and NAC plays a more beneficial role in alleviating Cd-induced lipid accumulation than the drug alone, and the combination of baicalin and NAC can stabilize mitochondrial structure and function and inhibit mROS-mediated CMA through TFAM-choline, thereby promoting lipophagy to alleviate Cd-induced lipid accumulation. [ABSTRACT FROM AUTHOR]

Amyloid‐β (Aβ) protein aggregation in the brain is a pathological hallmark of Alzheimer's disease (AD) however, the underlying molecular mechanisms regulating amyloid aggregation are not well understood. Here, we studied the propitious role of E3 ubiquitin ligase Pirh2 in Aβ protein aggregation in view of its regulatory ligase activity in the ubiquitin‐proteasome system employing both cellular and sporadic rodent models of AD. Pirh2 protein abundance was significantly increased during Streptozotocin (STZ) induced AD conditions, and transient silencing of Pirh2 significantly inhibited the Aβ aggregation and modified the dendrite morphology along with the substantial decrease in choline level in the differentiated neurons. MALDI‐TOF/TOF, coimmunoprecipitation, and UbcH7‐linked in vitro ubiquitylation analysis confirmed the high interaction of Pirh2 with chaperone GRP78. Furthermore, Pirh2 silencing inhibits the STZ induced altered level of endoplasmic reticulum stress and intracellular Ca2+ levels in neuronal N2a cells. Pirh2 silencing also inhibited the AD conditions related to the altered protein abundance of HSP90 and its co‐chaperones which may collectively involve in the reduced burden of amyloid aggregates in neuronal cells. Pirh2 silencing further stabilized the nuclear translocation of phospho‐Nrf2 and inhibited the altered level of autophagy factors. Taken together, our data indicated that Pirh2 is critically involved in STZ induced AD pathogenesis through its interaction with ER‐chaperone GRP78, improves the neuronal connectivity, affects the altered level of chaperones, co‐chaperones, & autophagic markers, and collectively inhibits the Aβ aggregation. [ABSTRACT FROM AUTHOR]

Genetic code expansion enables site‐specific photo‐crosslinking by introducing photo‐reactive non‐canonical amino acids into proteins at defined positions during translation. This technology is widely used for analyzing protein–protein interactions and is applicable in mammalian cells. However, the identification of the crosslinked region still remains challenging. Here, we developed a new method to identify the crosslinked region by pre‐installing a site‐specific cleavage site, an α‐hydroxy acid (Nε‐allyloxycarbonyl‐α‐hydroxyl‐l‐lysine acid, AllocLys‐OH), into the target protein. Alkaline treatment cleaves the crosslinked complex at the position of the α‐hydroxy acid residue and thus helps to identify which side of the cleavage site, either closer to the N‐terminus or C‐terminus, the crosslinked site is located within the target protein. A series of AllocLys‐OH introductions narrows down the crosslinked region. By applying this method, we identified the crosslinked regions in lysosomal‐associated membrane protein type 2A (LAMP2A), a receptor of chaperone‐mediated autophagy, in mammalian cells. The results suggested that at least two interfaces are involved in the homophilic interaction, which requires a trimeric or higher oligomeric assembly of adjacent LAMP2A molecules. Thus, the combination of site‐specific crosslinking and site‐specific cleavage promises to be useful for revealing binding interfaces and protein complex geometries. [ABSTRACT FROM AUTHOR]

Missense mutations of ubiquilin 2 (UBQLN2) have been identified to cause X-linked amyotrophic lateral sclerosis (ALS). Proteasome-mediated protein degradation is reported to be impaired by ALS-associated mutations of UBQLN2. However, it remains unknown how these mutations affect autophagy-lysosome protein degradation, which consists of macroautophagy (MA), microautophagy (mA), and chaperone-mediated autophagy (CMA). Using a CMA/mA fluorescence reporter we found that overexpression of wild-type UBQLN2 impairs CMA. Conversely, knockdown of endogenous UBQLN2 increases CMA activity, suggesting that normally UBQLN2 negatively regulates CMA. ALS-associated mutant forms of UBQLN2 exacerbate this impairment of CMA. Using cells stably transfected with wild-type or ALS-associated mutant UBQLN2, we further determined that wild-type UBQLN2 increased the ratio of LAMP2A (a CMA-related protein) to LAMP1 (a lysosomal protein). This could represent a compensatory reaction to the impairment of CMA by wild-type UBQLN2. However, ALS-associated mutant UBQLN2 failed to show this compensation, exacerbating the impairment of CMA by mutant UBQLN2. We further demonstrated that ALS-associated mutant forms of UBQLN2 also impair MA, but wild-type UBQLN2 does not. These results support the view that ALS-associated mutant forms of UBQLN2 impair both CMA and MA which may contribute to the neurodegeneration observed in patients with UBQLN2-mediated ALS.

Disrupted homeostasis of the microtubule binding protein tau is a shared feature of a set of neurodegenerative disorders known as tauopathies. Acetylation of soluble tau is an early pathological event in neurodegeneration. In this work, we find that a large fraction of neuronal tau is degraded by chaperone-mediated autophagy (CMA) whereas, upon acetylation, tau is preferentially degraded by macroautophagy and endosomal microautophagy. Rerouting of acetylated tau to these other autophagic pathways originates, in part, from the inhibitory effect that acetylated tau exerts on CMA and results in its extracellular release. In fact, experimental blockage of CMA enhances cell-to-cell propagation of pathogenic tau in a mouse model of tauopathy. Furthermore, analysis of lysosomes isolated from brains of patients with tauopathies demonstrates similar molecular mechanisms leading to CMA dysfunction. This study reveals that CMA failure in tauopathy brains alters tau homeostasis and could contribute to aggravate disease progression.

Ferroptosis, characterized by iron accumulation and lipid peroxidation, is a form of iron-driven cell death. Mitophagy is a type of selective autophagy, where degradation of damaged mitochondria is the key mechanism for maintaining mitochondrial homeostasis. Additionally, Chaperone-mediated autophagy (CMA) is a biological process that transports individual cytoplasmic proteins to lysosomes for degradation through companion molecules such as heat shock proteins. Research has demonstrated the involvement of ferroptosis, mitophagy, and CMA in the pathological progression of Osteoarthritis (OA). Furthermore, research has indicated a significant correlation between alterations in the expression of reactive oxygen species (ROS), adenosine monophosphate (AMP)-activated protein kinase (AMPK), and hypoxia-inducible factors (HIFs) and the occurrence of OA, particularly in relation to ferroptosis and mitophagy. In light of these findings, our study aims to assess the regulatory functions of ferroptosis and mitophagy/CMA in the pathogenesis of OA. Additionally, we propose a mechanism of crosstalk between ferroptosis and mitophagy, while also examining potential pharmacological interventions for targeted therapy in OA. Ultimately, our research endeavors to offer novel insights and directions for the prevention and treatment of OA.