Search Results (50)

Targeted degradation technologies, primarily referring to targeted protein degradation, have emerged as promising drug discovery strategies. In contrast to traditional “occupancy-driven” inhibition approaches, these technologies ingeniously leverage the cell’s endogenous degradation mechanisms to achieve specific elimination of disease-causing targets. Autophagy, a highly conserved cellular clearance pathway, possesses broad substrate recognition capabilities, enabling degradation of not only individual proteins but also protein aggregates, damaged organelles, and invading pathogens. Given these characteristics, researchers are actively exploring the application of autophagy mechanisms in targeted degradation technologies. Herein, we summarize recent advances in autophagy-dependent degradation approaches, including autophagosome tethering compounds (ATTEC), autophagy-targeting chimeras (AUTAC), autophagy-targeting Chimera (AUTOTAC), chaperone-mediated autophagy (CMA)-based methods, nanotechnology-based strategies, and the newly introduced autophagy-induced antibody (AUTAB) technique, highlighting their mechanisms, advantages, and potential applications in treating tumors, neurodegenerative diseases, and other challenging conditions. Full article

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder marked by neuronal loss, cognitive decline, and pathological hallmarks such as amyloid-beta (Aβ) plaques and tau neurofibrillary tangles. Recent evidence highlights autophagy as a pivotal mechanism in cellular homeostasis, mediating the clearance of misfolded proteins and damaged organelles. However, impaired autophagy contributes significantly to AD pathogenesis by disrupting proteostasis, exacerbating neuroinflammation, and promoting synaptic dysfunction. This review aims to scrutinize the intricate relationship between autophagy dysfunction and AD progression, explaining key pathways including macroautophagy, chaperone-mediated autophagy (CMA), and selective autophagy processes such as mitophagy and aggrephagy. This further extends the discussion beyond the central nervous system, evaluating the role of hepatic autophagy in Aβ clearance and systemic metabolic regulation. An understanding of autophagy’s involvement in AD pathology via various mechanisms could give rise to a novel therapeutic strategy targeting autophagic modulation to mitigate disease progression in the future. Full article

Atherosclerosis, a chronic inflammatory disease characterized by lipid accumulation and immune cell infiltration, is linked to plaque formation and cardiovascular events. While traditionally associated with lipid metabolism and endothelial dysfunction, recent research highlights the roles of autophagy and clonal hematopoiesis (CH) in its pathogenesis. Autophagy, a cellular process crucial for degrading damaged components, regulates macrophage homeostasis and inflammation, both of which are pivotal in atherosclerosis. In macrophages, autophagy influences lipid metabolism, cytokine regulation, and oxidative stress, helping to prevent plaque instability. Defective autophagy exacerbates inflammation, impairs cholesterol efflux, and accelerates disease progression. Additionally, autophagic processes in endothelial cells and smooth muscle cells further contribute to atherosclerotic pathology. Recent studies also emphasize the interplay between autophagy and CH, wherein somatic mutations in genes like TET2, JAK2, and DNMT3A drive immune cell expansion and enhance inflammatory responses in atherosclerotic plaques. These mutations modify macrophage function, intensifying the inflammatory environment and accelerating atherosclerosis. Chaperone-mediated autophagy (CMA), a selective form of autophagy, also plays a critical role in regulating macrophage inflammation by degrading pro-inflammatory cytokines and oxidized low-density lipoprotein (ox-LDL). Impaired CMA activity leads to the accumulation of these substrates, activating the NLRP3 inflammasome and worsening inflammation. Preclinical studies suggest that pharmacologically activating CMA may mitigate atherosclerosis progression. In animal models, reduced CMA activity accelerates plaque instability and increases inflammation. This review highlights the importance of autophagic regulation in macrophages, focusing on its role in inflammation, plaque formation, and the contributions of CH. Building upon current advances, we propose a hypothesis in which autophagy, programmed cell death, and clonal hematopoiesis form a critical intrinsic axis that modulates the fundamental functions of macrophages, playing a complex role in the development of atherosclerosis. Understanding these mechanisms offers potential therapeutic strategies targeting autophagy and inflammation to reduce the burden of atherosclerotic cardiovascular disease. Full article

Lysosome-associated membrane glycoproteins (LAMPs), including lysosomal membrane protein 1 (Lamp1) and lysosomal membrane protein 2 (Lamp2), are involved in phagocytosis, chaperone-mediated autophagy (CMA), and other pathways that interact with lysosomal activity. However, the role of Lamp2 in teleosts has not been clarified. In this study, we investigated the functions of lamp2 genes during Vibrio vulnificus infection. We achieved subcellular localization of the lamp2 gene at the cellular level and performed overexpression and RNA interference experiments followed by Lipopolysaccharides (LPS) stimulation to probe the expression changes of related genes. Ultrapathology analysis of the head-kidney revealed an increase in lysosomes and the formation of autophagosomal vesicles after V. vulnificus infection, suggesting that lysosomes bind to autophagosomes. The lamp2 gene, encoding 401 amino acids in Cynoglossus semilaevis, was constitutively expressed in all examined tissues of healthy half-smooth tongue sole, with the highest expression in blood. A challenge test was conducted to assess the response of half-smooth tongue sole (Cynoglossus semilaevis) to different concentrations of V. vulnificus. The results showed that the relative expression of lamp2 and its related genes—lc3, rab7, vamp8, atg14, stx17, snap29, ctsb, and ctsd—varied with time and concentration in the gill, spleen, head-kidney, blood, liver, and gut tissues. From the results of lamp2 gene overexpression and RNA interference experiments, it is hypothesized that lamp2 positively regulates lc3, rab7, vamp8, snap29, and stx17, and negatively regulates ctsd and ctsb. Our findings provide new primary data for the function of lamp2 gene in the half-smooth tongue sole., particularly its role in regulating the immune response against V. vulnificus. Full article

Glioblastoma (GB) is one of the most aggressive and treatment-resistant cancers due to its complex tumor microenvironment (TME). We previously showed that GB progression is dependent on the aberrant induction of chaperone-mediated autophagy (CMA) in pericytes (PCs), which promotes TME immunosuppression through the PC secretome. The secretion of extracellular matrix (ECM) proteins with anti-tumor (Lumican) and pro-tumoral (Osteopontin, OPN) properties was shown to be dependent on the regulation of GB-induced CMA in PCs. As biomarkers are rarely studied in TME, in this work, we aimed to validate Lumican and OPN as prognostic markers in the perivascular areas of the peritumoral niche of a cohort of GB patients. Previously, we had validated their expression in GB xenografted mice presenting GB infiltration (OPN) or GB elimination (Lumican) dependent on competent or deficient CMA PCs, respectively. Then, patient sample classification by GB infiltration into the peritumoral brain parenchyma was related to GB-induced CMA in microvasculature PCs, analyzing the expression of the lysosomal receptor, LAMP-2A. Our results revealed a correlation between GB-induced CMA activity in peritumoral PCs and GB patients’ outcomes, identifying three degrees of severity. The perivascular expression of both immune activation markers, Iba1 and CD68, was related to CMA-dependent PC immune function and determined as useful for efficient GB prognosis. Lumican expression was identified in perivascular areas of patients with less severe outcome and partially co-localizing with PCs presenting low CMA activity, while OPN was primarily found in perivascular areas of patients with poor outcome and partially co-localizing with PCs presenting high CMA activity. Importantly, we found sex differences in the incidence of middle-aged patients, being significantly higher in men but with worse prognosis in women. Our results confirmed that Lumican and OPN in perivascular areas of the GB peritumoral niche are effective predictive biomarkers for evaluating prognosis and monitoring possible therapeutic immune responses dependent on PCs in tumor progression. Full article

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. Full article

In Alzheimer’s disease (AD), tau dissociates from microtubules (MTs) due to hyperphosphorylation and misfolding. It is degraded by various mechanisms, including the 20S proteasome, chaperone-mediated autophagy (CMA), 26S proteasome, macroautophagy, and aggrephagy. Neurofibrillary tangles (NFTs) form upon the impairment of aggrephagy, and eventually, the ubiquitin chaperone valosin-containing protein (VCP) and heat shock 70 kDa protein (HSP70) are recruited to the sites of NFTs for the extraction of tau for the ubiquitin–proteasome system (UPS)-mediated degradation. However, the impairment of tau degradation in neurons allows tau to be secreted into the extracellular space. Secreted tau can be monomers, oligomers, and paired helical filaments (PHFs), which are seeding competent pathological tau that can be endocytosed/phagocytosed by healthy neurons, microglia, astrocytes, oligodendrocyte progenitor cells (OPCs), and oligodendrocytes, often causing proteotoxic stress and eventually triggers senescence. Senescent cells secrete various senescence-associated secretory phenotype (SASP) factors, which trigger cellular atrophy, causing decreased brain volume in human AD. However, the molecular mechanisms of proteotoxic stress and cellular senescence are not entirely understood and are an emerging area of research. Therefore, this comprehensive review summarizes pertinent studies that provided evidence for the sequential tau degradation, failure, and the mechanistic link between tau-driven proteotoxic stress and cellular senescence in AD. Full article

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. Full article

The p53 protein is the master regulator of cellular integrity, primarily due to its tumor-suppressing functions. Approximately half of all human cancers carry mutations in the TP53 gene, which not only abrogate the tumor-suppressive functions but also confer p53 mutant proteins with oncogenic potential. The latter is achieved through so-called gain-of-function (GOF) mutations that promote cancer progression, metastasis, and therapy resistance by deregulating transcriptional networks, signaling pathways, metabolism, immune surveillance, and cellular compositions of the microenvironment. Despite recent progress in understanding the complexity of mutp53 in neoplastic development, the exact mechanisms of how mutp53 contributes to cancer development and how they escape proteasomal and lysosomal degradation remain only partially understood. In this review, we address recent findings in the field of oncogenic functions of mutp53 specifically regarding, but not limited to, its implications in metabolic pathways, the secretome of cancer cells, the cancer microenvironment, and the regulating scenarios of the aberrant proteasomal degradation. By analyzing proteasomal and lysosomal protein degradation, as well as its connection with autophagy, we propose new therapeutical approaches that aim to destabilize mutp53 proteins and deactivate its oncogenic functions, thereby providing a fundamental basis for further investigation and rational treatment approaches for TP53-mutated cancers. Full article

Parkinson’s disease (PD) is a common movement disorder associated with the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Mutations in the PD-associated gene PARK7 alter the structure and function of the encoded protein DJ-1, and the resulting autosomal recessively inherited disease increases the risk of developing PD. DJ-1 was first discovered in 1997 as an oncogene and was associated with early-onset PD in 2003. Mutations in DJ-1 account for approximately 1% of all recessively inherited early-onset PD occurrences, and the functions of the protein have been studied extensively. In healthy subjects, DJ-1 acts as an antioxidant and oxidative stress sensor in several neuroprotective mechanisms. It is also involved in mitochondrial homeostasis, regulation of apoptosis, chaperone-mediated autophagy (CMA), and dopamine homeostasis by regulating various signaling pathways, transcription factors, and molecular chaperone functions. While DJ-1 protects neurons against damaging reactive oxygen species, neurotoxins, and mutant α-synuclein, mutations in the protein may lead to inefficient neuroprotection and the progression of PD. As current therapies treat only the symptoms of PD, the development of therapies that directly inhibit oxidative stress-induced neuronal cell death is critical. DJ-1 has been proposed as a potential therapeutic target, while oxidized DJ-1 could operate as a biomarker for PD. In this paper, we review the role of DJ-1 in the pathogenesis of PD by highlighting some of its key neuroprotective functions and the consequences of its dysfunction. Full article

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. Full article

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. Full article

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor and a substrate protein of a Cullin 4B E3 ligase complex responsible for diverse cellular processes. In the lung, this receptor is responsible for the bioactivation of benzo[a]pyrene during tumorigenesis. Realizing that the AHR function is affected by its expression level, we are interested in the degradation mechanism of AHR in the lung. Here, we have investigated the mechanism responsible for AHR degradation using human lung epithelial A549 cells. We have observed that the AHR protein levels increase in the presence of chloroquine (CQ), an autophagy inhibitor, in a dose-dependent manner. Treatment with 6-aminonicotinamide (6-AN), a chaperone-mediated autophagy (CMA) activator, decreases AHR protein levels in a concentration-dependent and time-dependent manner. This decrease suppresses the ligand-dependent activation of the AHR target gene transcription, and can be reversed by CQ but not MG132. Knockdown of lysosome-associated membrane protein 2 (LAMP2), but not autophagy-related 5 (ATG5), suppresses the chloroquine-mediated increase in the AHR protein. AHR is resistant to CMA when its CMA motif is mutated. Suppression of the epithelial-to-mesenchymal transition in A549 cells is observed when the AHR gene is knocked out or the AHR protein level is reduced by 6-AN. Collectively, we have provided evidence supporting that AHR is continuously undergoing CMA and activation of CMA suppresses the AHR function in A549 cells. Full article

Autophagy is a lysosomal degradation process known as autophagic flux, involving the engulfment of damaged proteins and organelles by double-membrane autophagosomes. It comprises microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. Macroautophagy consists of three stages: induction, autophagosome formation, and autolysosome formation. Atg8-family proteins are valuable for tracking autophagic structures and have been widely utilized for monitoring autophagy. The conversion of LC3 to its lipidated form, LC3-II, served as an indicator of autophagy. Autophagy is implicated in human pathophysiology, such as neurodegeneration, cancer, and immune disorders. Moreover, autophagy impacts urological diseases, such as interstitial cystitis /bladder pain syndrome (IC/BPS), ketamine-induced ulcerative cystitis (KIC), chemotherapy-induced cystitis (CIC), radiation cystitis (RC), erectile dysfunction (ED), bladder outlet obstruction (BOO), prostate cancer, bladder cancer, renal cancer, testicular cancer, and penile cancer. Autophagy plays a dual role in the management of urologic diseases, and the identification of potential biomarkers associated with autophagy is a crucial step towards a deeper understanding of its role in these diseases. Methods for monitoring autophagy include TEM, Western blot, immunofluorescence, flow cytometry, and genetic tools. Autophagosome and autolysosome structures are discerned via TEM. Western blot, immunofluorescence, northern blot, and RT-PCR assess protein/mRNA levels. Luciferase assay tracks flux; GFP-LC3 transgenic mice aid study. Knockdown methods (miRNA and RNAi) offer insights. This article extensively examines autophagy’s molecular mechanism, pharmacological regulation, and therapeutic application involvement in urological diseases. Full article

The heat shock factor 1 (HSF1)-mediated stress response pathway and autophagy processes play important roles in the maintenance of proteostasis. Autophagy processes are subdivided into three subtypes: macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy. Recently, molecular chaperones and co-factors were shown to be involved in the selective degradation of substrates by these three autophagy processes. This evidence suggests that autophagy processes are regulated in a coordinated manner by the HSF1-mediated stress response pathway. Recently, various studies have demonstrated that proteostasis pathways including HSF1 and autophagy are implicated in longevity. Furthermore, they serve as therapeutic targets for aging-related diseases such as cancer and neurodegenerative diseases. In the future, these studies will underpin the development of therapies against various diseases. Full article