A mitophagy sensor PPTC7 controls BNIP3 and NIX degradation to regulate mitochondrial mass.

Mitophagy mediated by BNIP3 and NIX critically regulates mitochondrial mass. Cellular BNIP3 and NIX levels are tightly controlled by SCFFBXL4-mediated ubiquitination to prevent excessive mitochondrial loss and lethal disease. Here, we report that knockout of PPTC7, a mitochondrial matrix protein, hyperactivates BNIP3-/NIX-mediated mitophagy and causes perinatal lethality that is rescued by NIX knockout in mice. Biochemically, the PPTC7 precursor is trapped by BNIP3 and NIX to the mitochondrial outer membrane, where PPTC7 scaffolds assembly of a substrate-PPTC7-SCFFBXL4 holocomplex to degrade BNIP3 and NIX, forming a homeostatic regulatory loop. PPTC7 possesses an unusually weak mitochondrial targeting sequence to facilitate its outer membrane retention and mitophagy control. Starvation upregulates PPPTC7 expression in mouse liver to repress mitophagy, which critically maintains hepatic mitochondrial mass, bioenergetics, and gluconeogenesis. Collectively, PPTC7 functions as a mitophagy sensor that integrates homeostatic and physiological signals to dynamically control BNIP3 and NIX degradation, thereby maintaining mitochondrial mass and cellular homeostasis.

The ER membrane protein complex restricts mitophagy by controlling BNIP3 turnover.

Lysosomal degradation of autophagy receptors is a common proxy for selective autophagy. However, we find that two established mitophagy receptors, BNIP3 and BNIP3L/NIX, are constitutively delivered to lysosomes in an autophagy-independent manner. This alternative lysosomal delivery of BNIP3 accounts for nearly all its lysosome-mediated degradation, even upon mitophagy induction. To identify how BNIP3, a tail-anchored protein in the outer mitochondrial membrane, is delivered to lysosomes, we performed a genome-wide CRISPR screen for factors influencing BNIP3 flux. This screen revealed both known modifiers of BNIP3 stability as well as a pronounced reliance on endolysosomal components, including the ER membrane protein complex (EMC). Importantly, the endolysosomal system and the ubiquitin-proteosome system regulated BNIP3 independently. Perturbation of either mechanism is sufficient to modulate BNIP3-associated mitophagy and affect underlying cellular physiology. More broadly, these findings extend recent models for tail-anchored protein quality control and install endosomal trafficking and lysosomal degradation in the canon of pathways that tightly regulate endogenous tail-anchored protein localization.

FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors.

To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4 , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4-associated mtDNA depletion syndrome.

FBXL4 ubiquitin ligase deficiency promotes mitophagy by elevating NIX levels.

Selective autophagy of mitochondria, mitophagy, is linked to mitochondrial quality control and as such is critical to a healthy organism. We have used a CRISPR/Cas9 approach to screen human E3 ubiquitin ligases for influence on mitophagy under both basal cell culture conditions and upon acute mitochondrial depolarization. We identify two cullin-RING ligase substrate receptors, VHL and FBXL4, as the most profound negative regulators of basal mitophagy. We show that these converge, albeit via different mechanisms, on control of the mitophagy adaptors BNIP3 and BNIP3L/NIX. FBXL4 restricts NIX and BNIP3 levels via direct interaction and protein destabilization, while VHL acts through suppression of HIF1α-mediated transcription of BNIP3 and NIX. Depletion of NIX but not BNIP3 is sufficient to restore mitophagy levels. Our study contributes to an understanding of the aetiology of early-onset mitochondrial encephalomyopathy that is supported by analysis of a disease-associated mutation. We further show that the compound MLN4924, which globally interferes with cullin-RING ligase activity, is a strong inducer of mitophagy, thus providing a research tool in this context and a candidate therapeutic agent for conditions linked to mitochondrial dysfunction.

Nix interacts with WIPI2 to induce mitophagy.

Nix is a membrane-anchored outer mitochondrial protein that induces mitophagy. While Nix has an LC3-interacting (LIR) motif that binds to ATG8 proteins, it also contains a minimal essential region (MER) that induces mitophagy through an unknown mechanism. We used chemically induced dimerization (CID) to probe the mechanism of Nix-mediated mitophagy and found that both the LIR and MER are required for robust mitophagy. We find that the Nix MER interacts with the autophagy effector WIPI2 and recruits WIPI2 to mitochondria. The Nix LIR motif is also required for robust mitophagy and converts a homogeneous WIPI2 distribution on the surface of the mitochondria into puncta, even in the absence of ATG8s. Together, this work reveals unanticipated mechanisms in Nix-induced mitophagy and the elusive role of the MER, while also describing an interesting example of autophagy induction that acts downstream of the canonical initiation complexes.

Host cell egress of Brucella abortus requires BNIP3L-mediated mitophagy.

The facultative intracellular pathogen Brucella abortus interacts with several organelles of the host cell to reach its replicative niche inside the endoplasmic reticulum. However, little is known about the interplay between the intracellular bacteria and the host cell mitochondria. Here, we showed that B. abortus triggers substantive mitochondrial network fragmentation, accompanied by mitophagy and the formation of mitochondrial Brucella-containing vacuoles during the late steps of cellular infection. Brucella-induced expression of the mitophagy receptor BNIP3L is essential for these events and relies on the iron-dependent stabilisation of the hypoxia-inducible factor 1α. Functionally, BNIP3L-mediated mitophagy appears to be advantageous for bacterial exit from the host cell as BNIP3L depletion drastically reduces the number of reinfection events. Altogether, these findings highlight the intricate link between Brucella trafficking and the mitochondria during host cell infection.

A mitochondrial SCF-FBXL4 ubiquitin E3 ligase complex degrades BNIP3 and NIX to restrain mitophagy and prevent mitochondrial disease.

Mitophagy is a fundamental quality control mechanism of mitochondria. Its regulatory mechanisms and pathological implications remain poorly understood. Here, via a mitochondria-targeted genetic screen, we found that knockout (KO) of FBXL4, a mitochondrial disease gene, hyperactivates mitophagy at basal conditions. Subsequent counter screen revealed that FBXL4-KO hyperactivates mitophagy via two mitophagy receptors BNIP3 and NIX. We determined that FBXL4 functions as an integral outer-membrane protein that forms an SCF-FBXL4 ubiquitin E3 ligase complex. SCF-FBXL4 ubiquitinates BNIP3 and NIX to target them for degradation. Pathogenic FBXL4 mutations disrupt SCF-FBXL4 assembly and impair substrate degradation. Fbxl4-/- mice exhibit elevated BNIP3 and NIX proteins, hyperactive mitophagy, and perinatal lethality. Importantly, knockout of either Bnip3 or Nix rescues metabolic derangements and viability of the Fbxl4-/- mice. Together, beyond identifying SCF-FBXL4 as a novel mitochondrial ubiquitin E3 ligase restraining basal mitophagy, our results reveal hyperactivated mitophagy as a cause of mitochondrial disease and suggest therapeutic strategies.

PPTC7 antagonizes mitophagy by promoting BNIP3 and NIX degradation via SCFFBXL4.

Mitophagy must be carefully regulated to ensure that cells maintain appropriate numbers of functional mitochondria. The SCFFBXL4 ubiquitin ligase complex suppresses mitophagy by controlling the degradation of BNIP3 and NIX mitophagy receptors, and FBXL4 mutations result in mitochondrial disease as a consequence of elevated mitophagy. Here, we reveal that the mitochondrial phosphatase PPTC7 is an essential cofactor for SCFFBXL4-mediated destruction of BNIP3 and NIX, suppressing both steady-state and induced mitophagy. Disruption of the phosphatase activity of PPTC7 does not influence BNIP3 and NIX turnover. Rather, a pool of PPTC7 on the mitochondrial outer membrane acts as an adaptor linking BNIP3 and NIX to FBXL4, facilitating the turnover of these mitophagy receptors. PPTC7 accumulates on the outer mitochondrial membrane in response to mitophagy induction or the absence of FBXL4, suggesting a homoeostatic feedback mechanism that attenuates high levels of mitophagy. We mapped critical residues required for PPTC7-BNIP3/NIX and PPTC7-FBXL4 interactions and their disruption interferes with both BNIP3/NIX degradation and mitophagy suppression. Collectively, these findings delineate a complex regulatory mechanism that restricts BNIP3/NIX-induced mitophagy.

CTCF and BORIS-mediated autophagy regulation via alternative splicing of BNIP3L in breast cancer.

Autophagy is a pivotal regulatory and catabolic process, induced under various stressful conditions, including hypoxia. However, little is known about alternative splicing of autophagy genes in the hypoxic landscape in breast cancer. Our research unravels the hitherto unreported alternative splicing of BNIP3L, a crucial hypoxia-induced autophagic gene. We showed that BNIP3L, under hypoxic condition, forms two isoforms, a full-length isoform (BNIP3L-F) and a shorter isoform lacking exon 1 (BNIP3L-Δ1). The hypoxia-induced BNIP3L-F promotes autophagy, while under normoxia, the BNIP3L-Δ1 inhibits autophagy. We discovered a novel dimension of hypoxia-mediated epigenetic modification that regulates the alternative splicing of BNIP3L. Here, we showed differential DNA methylation of BNIP3L intron 1, causing reciprocal binding of epigenetic factor CCCTC-binding factor (CTCF) and its paralog BORIS. Additionally, we highlighted the role of CTCF and BORIS impacting autophagy in breast cancer. The differential binding of CTCF and BORIS results in alternative splicing of BNIP3L forming BNIP3L-F and BNIP3L-Δ1, respectively. The binding of CTCF on unmethylated BNIP3L intron 1 under hypoxia results in RNA Pol-II pause and inclusion of exon 1, promoting BNIP3L-F and autophagy. Interestingly, the binding of BORIS on methylated BNIP3L intron 1 under normoxia also results in RNA Pol-II pause but leads to the exclusion of exon 1 from BNIP3L mRNA. Finally, we reported the critical role of BORIS-mediated RNA Pol-II pause, which subsequently recruits SRSF6, redirecting the proximal splice-site selection, promoting BNIP3L-Δ1, and inhibiting autophagy. Our study provides novel insights into the potential avenues for breast cancer therapy by targeting autophagy regulation, specifically under hypoxic condition.

BNIP3 promotes HIF-1α-driven melanoma growth by curbing intracellular iron homeostasis.

BNIP3 is a mitophagy receptor with context-dependent roles in cancer, but whether and how it modulates melanoma growth in vivo remains unknown. Here, we found that elevated BNIP3 levels correlated with poorer melanoma patient's survival and depletion of BNIP3 in B16-F10 melanoma cells compromised tumor growth in vivo. BNIP3 depletion halted mitophagy and enforced a PHD2-mediated downregulation of HIF-1α and its glycolytic program both in vitro and in vivo. Mechanistically, we found that BNIP3-deprived melanoma cells displayed increased intracellular iron levels caused by heightened NCOA4-mediated ferritinophagy, which fostered PHD2-mediated HIF-1α destabilization. These effects were not phenocopied by ATG5 or NIX silencing. Restoring HIF-1α levels in BNIP3-depleted melanoma cells rescued their metabolic phenotype and tumor growth in vivo, but did not affect NCOA4 turnover, underscoring that these BNIP3 effects are not secondary to HIF-1α. These results unravel an unexpected role of BNIP3 as upstream regulator of the pro-tumorigenic HIF-1α glycolytic program in melanoma cells.

Hypoxia-inducible factor prolyl hydroxylase inhibitor alleviates heatstroke-induced acute kidney injury by activating BNIP3-mediated mitophagy.

Hypoxia-induced inflammation and apoptosis are important pathophysiological features of heat stroke-induced acute kidney injury (HS-AKI). Hypoxia-inducible factor (HIF) is a key protein that regulates cell adaptation to hypoxia. HIF-prolyl hydroxylase inhibitor (HIF-PHI) stabilizes HIF to increase cell adaptation to hypoxia. Herein, we reported that HIF-PHI pretreatment significantly improved renal function, enhanced thermotolerance, and increased the survival rate of mice in the context of HS. Moreover, HIF-PHI could alleviate HS-induced mitochondrial damage, inflammation, and apoptosis in renal tubular epithelial cells (RTECs) by enhancing mitophagy in vitro and in vivo. By contrast, mitophagy inhibitors Mdivi-1, 3-MA, and Baf-A1 reversed the renoprotective effects of HIF-PHI. Mechanistically, HIF-PHI protects RTECs from inflammation and apoptosis by enhancing Bcl-2 adenovirus E18 19-kDa-interacting protein 3 (BNIP3)-mediated mitophagy, while genetic ablation of BNIP3 attenuated HIF-PHI-induced mitophagy and abolished HIF-PHI-mediated renal protection. Thus, our results indicated that HIF-PHI protects renal function by upregulating BNIP3-mediated mitophagy to improve HS-induced inflammation and apoptosis of RTECs, suggesting HIF-PHI as a promising therapeutic agent to treat HS-AKI.

HIF-1 contributes to autophagy activation via BNIP3 to facilitate renal fibrosis in hypoxia in vitro and UUO in vivo.

The molecular basis of renal interstitial fibrosis, a major pathological feature of progressive kidney diseases, remains poorly understood. Autophagy has been implicated in renal fibrosis, but whether it promotes or inhibits fibrosis remains controversial. Moreover, it is unclear how autophagy is activated and sustained in renal fibrosis. The present study was designed to address these questions using the in vivo mouse model of unilateral ureteral obstruction and the in vitro model of hypoxia in renal tubular cells. Both models showed the activation of hypoxia-inducible factor-1 (HIF-1) and autophagy along with fibrotic changes. Inhibition of autophagy with chloroquine reduced renal fibrosis in unilateral ureteral obstruction model, whereas chloroquine and autophagy-related gene 7 knockdown decreased fibrotic changes in cultured renal proximal tubular cells, supporting a profibrotic role of autophagy. Notably, pharmacological and genetic inhibition of HIF-1 led to the suppression of autophagy and renal fibrosis in these models. Mechanistically, knock down of BCL2 and adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), a downstream target gene of HIF, decreased autophagy and fibrotic changes during hypoxia in BUMPT cells. Together, these results suggest that HIF-1 may activate autophagy via BNIP3 in renal tubular cells to facilitate the development of renal interstitial fibrosis.NEW & NOTEWORTHY Autophagy has been reported to participate in renal fibrosis, but its role and underlying activation mechanism is unclear. In this study, we report the role of HIF-1 in autophagy activation in models of renal fibrosis and further investigate the underlying mechanism.

N6-methyladenosine demethyltransferase FTO alleviates sepsis by upregulating BNIP3 to induce mitophagy.

N6-methyladenosine (m6A) is known to be crucial in various biological processes, but its role in sepsis-induced circulatory and cardiac dysfunction is not well understood. Specifically, mitophagy, a specialized form of autophagy, is excessively activated during lipopolysaccharide (LPS)-induced myocardial injury. This study aimed to investigate the impact of LPS-induced endotoxemia on m6A-RNA methylation and its role in regulating mitophagy in sepsis-induced myocardial dysfunction. Our research demonstrated that FTO (fat mass and obesity-associated protein), an m6A demethylase, significantly affects abnormal m6A modification in the myocardium and cardiomyocytes following LPS treatment. In mice, cardiac dysfunction and cardiomyocyte apoptosis worsened after adeno-associated virus serotype 9 (AAV9)-mediated FTO knockdown. Further analyses to uncover the cellular mechanisms improving cardiac function showed that FTO reduced mitochondrial reactive oxygen species, restored both basal and maximal respiration, and preserved mitochondrial membrane potential. We revealed that FTO plays a critical role in activating mitophagy by targeting BNIP3. Additionally, the cardioprotective effects of AAV-FTO were significantly compromised by mdivi-1, a mitophagy inhibitor. Mechanistically, FTO interacted with BNIP3 transcripts and regulated their expression in an m6A-dependent manner. Following FTO silencing, BNIP3 transcripts with elevated m6A modification levels in their coding regions were bound by YTHDF2 (YT521-B homology m6A RNA-binding protein 2), leading to mRNA destabilization and decreased BNIP3 protein levels. These findings highlight the importance of FTO-dependent cardiac m6A methylation in regulating mitophagy and enhance our understanding of this critical interplay, which is essential for developing therapeutic strategies to protect cardiac mitochondrial function, alleviate cardiac dysfunction, and improve survival during sepsis.

FoxG1/BNIP3 axis promotes mitophagy and blunts cisplatin resistance in osteosarcoma.

Cisplatin (CDDP) is a commonly used chemotherapeutic for osteosarcoma (OS) patients, and drug resistance remains as a major hurdle to undermine the treatment outcome. Here, we investigated the potential involvement of FoxG1 and BNIP3 in CDDP resistance of OS cells. FoxG1 and BNIP3 expression levels were detected in the CDDP-sensitive and CDDP-resistant OS tumors and cell lines. Mitophagy was observed through transmission electron microscope analysis. The sensitivity to CDDP in OS cells upon FoxG1 overexpression was examined in cell and animal models. We found that FoxG1 and BNIP3 showed significant downregulation in the CDDP-resistant OS tumor samples and cell lines. CDDP-resistant OS tumor specimens and cells displayed impaired mitophagy. FoxG1 overexpression promoted BNIP3 expression, enhanced mitophagy in CDDP-resistant OS cells, and resensitized the resistant cells to CDDP treatment in vitro and in vivo. Our data highlighted the role of the FoxG1/BNIP3 axis in regulating mitophagy and dictating CDDP resistance in OS cells, suggesting targeting FoxG1/BNIP3-dependent mitophagy as a potential strategy to overcome CDDP resistance in OS.

SIRT1-mediated deacetylation of FOXO3 enhances mitophagy and drives hormone resistance in endometrial cancer.

BACKGROUND: The complex interplay between Sirtuin 1 (SIRT1) and FOXO3 in endometrial cancer (EC) remains understudied. This research aims to unravel the interactions of deacetylase SIRT1 and transcription factor FOXO3 in EC, focusing on their impact on mitophagy and hormone resistance. METHODS: High-throughput sequencing, cell experiments, and bioinformatics tools were employed to investigate the roles and interactions of SIRT1 and FOXO3 in EC. Co-immunoprecipitation (Co-IP) assay was used to assess the interaction between SIRT1 and FOXO3 in RL95-2 cells. Functional assays were used to assess cell viability, proliferation, migration, invasion, apoptosis, and the expression of related genes and proteins. A mouse model of EC was established to evaluate tumor growth and hormone resistance under different interventions. Immunohistochemistry and TUNEL assays were used to assess protein expression and apoptosis in tumor tissues. RESULTS: High-throughput transcriptome sequencing revealed a close association between SIRT1, FOXO3, and EC development. Co-IP showed a protein-protein interaction between SIRT1 and FOXO3. Overexpression of SIRT1 enhanced FOXO3 deacetylation and activity, promoting BNIP3 transcription and PINK1/Parkin-mediated mitophagy, which in turn promoted cell proliferation, migration, invasion, and inhibited apoptosis in vitro, as well as increased tumor growth and hormone resistance in vivo. These findings highlighted SIRT1 as an upstream regulator and potential therapeutic target in EC. CONCLUSION: This study reveals a novel molecular mechanism underlying the functional relevance of SIRT1 in regulating mitophagy and hormone resistance through the deacetylation of FOXO3 in EC, thereby providing valuable insights for new therapeutic strategies.

Diversity of mitophagy pathways at a glance.

Mitochondria are crucial organelles that play a central role in various cell signaling and metabolic pathways. A healthy mitochondrial population is maintained through a series of quality control pathways and requires a fine-tuned balance between mitochondrial biogenesis and degradation. Defective targeting of dysfunctional mitochondria to lysosomes through mitophagy has been linked to several diseases, but the underlying mechanisms and the relative importance of distinct mitophagy pathways in vivo are largely unknown. In this Cell Science at a Glance and the accompanying poster, we describe our current understanding of how parts of, or whole, mitochondria are recognized by the autophagic machinery and targeted to lysosomes for degradation. We also discuss how this might be regulated under different physiological conditions to maintain mitochondrial and cellular health.

Antigen processing and presentation-related signature-derived BNIP3 is a novel oncogene and immunotherapy determinant in osteosarcoma based on machine learning and in vitro validation.

BACKGROUND: In recent decades, osteosarcoma has remained the most prevalent kind of malignant tumor. An important and crucial factor in immunotherapy is antigen processing and presentation (APP). The specific functions and pathogenic processes of APP in osteosarcoma have not, however, been studied. METHODS: Patients with osteosarcoma were divided into groups using APP-related genes. Machine learning was used to further build the APP-related score. Investigated in-depth were the prognostic relevance of the score, mutation features, immunological aspects, and pharmacological prediction performance. Investigations of the prognostic utility, immunological traits, drug prediction effectiveness and immunotherapy prediction of BNIP3 were performed in-depth. RESULTS: Investigations by cell counting kit-8, Transwell and 5-ethynyl-2-deoxyuridine (EdU) demonstrated that BNIP3 is an osteosarcoma tumor accelerator. The osteosarcoma gene BNIP3 may promote macrophage migration. The APP-related score shows potential for clinical use. CONCLUSIONS: It was anticipated that more in vitro and in vivo studies would confirm BNIP3's tumorigenic and immunogenic processes in osteosarcoma.

HDAC6 preserves BNIP3 expression and mitochondrial integrity by deacetylating p53 at lysine 320.

HDAC6 has been reported as a deacetylase of p53 at multiple lysine residues, associated with the canonical functions of p53, such as apoptosis and tumor suppression. We have previously reported that p53 acetylation at the lysine 320 site accumulates due to the genetic ablation of HDAC6 in mice liver. However, the biological processes affected by K320 acetylation of p53 are yet to be elucidated. In this study, we demonstrate that K320 acetylation of p53 is regulated by HDAC6 deacetylase activity. HDAC6 knockout mouse brains exhibit a significant accumulation of K320 acetylated p53 compared to other tissues. The level of K320 acetylation of p53 inversely correlates with the level of BNIP3, a direct target of p53 and essential for mitophagy. Notably, overexpressing the deacetylation mimic K320R mutant p53 restored BNIP3 expression in HDAC6 knockout MEFs. Furthermore, we observed that neurons are particularly susceptible to the genetic ablation of HDAC6, impacting BNIP3 expression, which inversely correlates with the accumulation of abnormal mitochondria characterized by swollen cristae. Our findings suggest that HDAC6 plays a crucial role in maintaining BNIP3 expression by deacetylating p53 at the K320 site, which is linked to the structural integrity of mitochondria.

AAA237, an SKP2 inhibitor, suppresses glioblastoma by inducing BNIP3-dependent autophagy through the mTOR pathway.

BACKGROUND: Glioblastoma (GBM) is the most common brain tumor with the worst prognosis. Temozolomide is the only first-line drug for GBM. Unfortunately, the resistance issue is a classic problem. Therefore, it is essential to develop new drugs to treat GBM. As an oncogene, Skp2 is involved in the pathogenesis of various cancers including GBM. In this study, we investigated the anticancer effect of AAA237 on human glioblastoma cells and its underlying mechanism. METHODS: CCK-8 assay was conducted to evaluate IC50 values of AAA237 at 48, and 72 h, respectively. The Cellular Thermal Shift Assay (CETSA) was employed to ascertain the status of Skp2 as an intrinsic target of AAA237 inside the cellular milieu. The EdU-DNA synthesis test, Soft-Agar assay and Matrigel assay were performed to check the suppressive effects of AAA237 on cell growth. To identify the migration and invasion ability of GBM cells, transwell assay was conducted. RT-qPCR and Western Blot were employed to verify the level of BNIP3. The mRFP-GFP-LC3 indicator system was utilized to assess alterations in autophagy flux and investigate the impact of AAA237 on the dynamic fusion process between autophagosomes and lysosomes. To investigate the effect of compound AAA237 on tumor growth in vivo, LN229 cells were injected into the brains of mice in an orthotopic model. RESULTS: AAA237 could inhibit the growth of GBM cells in vitro. AAA237 could bind to Skp2 and inhibit Skp2 expression and the degradation of p21 and p27. In a dose-dependent manner, AAA237 demonstrated the ability to inhibit colony formation, migration, and invasion of GBM cells. AAA237 treatment could upregulate BNIP3 as the hub gene and therefore induce BNIP3-dependent autophagy through the mTOR pathway whereas 3-MA can somewhat reverse this process. In vivo, the administration of AAA237 effectively suppressed the development of glioma tumors with no side effects. CONCLUSION: Compound AAA237, a novel Skp2 inhibitor, inhibited colony formation, migration and invasion of GBM cells in a dose-dependent manner and time-dependent manner through upregulating BNIP3 as the hub gene and induced BNIP3-dependent autophagy through the mTOR pathway therefore it might be a viable therapeutic drug for the management of GBM.

Enhancing Nix-dependent mitophagy relieves AKI by restricting TREM-1-mediated hyperactivation of inflammasome in platelets.

Platelets are highly involved in inflammation and organ injury under pathological conditions. The mitophagy in platelets may restrict hyperactivation of the inflammasome and relieve acute kidney injury (AKI). Cecal ligation puncture (CLP)/LPS-induced AKI Triggering receptor expressed on myeloid cells (TREM-1)-knockout mice models were established. Additionally, septic patients with AKI were also included. TREM-1 expression in platelets and inflammasome activation were examined. Platelet transfer assays were performed to investigate the contribution of platelet TREM-1 to renal injury. Mitophagy was evaluated in the context of inflammation. BNIP3L/Nix knockout mice were used to examine the relationship between platelet mitophagy and inflammatory activation. The results showed that the level of TREM-1 was increased and the platelet inflammasome was hyperactivated in CLP mice and septic patients, and TREM-1 activated platelet inflammasomes. TREM-1 deletion significantly abrogated hyperactivation of the platelet inflammasome and dramatically reduced AKI, whereas ablation of the mitophagy receptor BNIP3L/Nix induced the accumulation of damaged mitochondria and hyperactivation of platelet inflammasomes in CLP mice. BNIP3L/Nix controlled platelet inflammasome activation, and an amplification loop of platelet inflammasome activation and dysfunctional mitochondria controlled sepsis-related AKI. Therefore, targeting TREM-1 and NLRP3/BNIP3L in platelets may represent a novel therapeutic strategy for treating septic AKI.