Kinase ULK1 Research Articles

Artesunate-driven autophagy: a shield against liver hypoxia/reoxygenation insult in rats via modulation of GLP1R, the chief metabolic kinase AMPK, mTOR, ULK1, P70S6K, cyclin D1, Akt, and GSK3β

BackgroundHepatic hypoxia/reoxygenation (H/R) insult is a critical issue in hepatic transplant and surgeries, profoundly influencing postoperative prognosis. One crucial pathomechanism in this condition is impaired autophagy flux, which disrupts liver homeostasis. Artesunate, an antimalarial drug, has shown potential in providing hepatoprotection against H/R injury; however, whether it can modulate disrupted autophagy to enhance hepatoprotection remains unclear.Purpose of the studyAccordingly, we delved into the potential mechanism(s) through which artesunate modulates the autophagy process in a hepatic H/R injury model.Methods and resultsRats were categorized into three groups, viz. sham operated, H/R, and artesunate-treated (50 mg/kg, i.p). Disease regression was evaluated microscopically, and molecular alternations were assessed biochemically using ELISA and western blotting techniques. Mechanistic analysis revealed that artesunate administration at reperfusion time significantly upregulated the gene expression of GLP1R protein expression of p-AMPK, accompanied by a downregulation in those of p-mTOR, and its target molecule p-ULK1, presenting the first trail to initiate autophagy. Additionally, artesunate reduced H/R-induced hepatic upregulated protein expression of p-mTOR/P70S6K cue, and cyclin D1 content, which positively correlated with the mTOR/P70S6K axis. Moreover, artesunate sharply upregulated active p-Akt, which in turn phosphorylated/inactivated GSK3β, a cascade that indirectly promotes autophagy. Consequently, artesunate increased the hepatic beclin-1 and LC3-II to further uphold its autophagic capacity. The hepato-therapeutic effectiveness of artesunate was further evidenced by reduced serum ALT and AST levels, along with diminished hepatic histopathological alterations.ConclusionArtesunate protected liver by triggering autophagy partly by modulating the GLP1R/AMPK/mTOR/ULK1, GLP1R/AMPK/mTOR/P70S6K, cyclin D1, and Akt/GSK3β trajectories providing a significant therapeutic potential in managing hepatic H/R insult.

The ULK1 effector BAG2 regulates autophagy initiation by modulating AMBRA1 localization

Autophagy initiation is regulated by the ULK1 kinase complex. To gain insights into functions of the holo-complex, we generated a deep interactome by combining affinity purification- and proximity labeling-mass spectrometry of all four complex members: ULK1, ATG13, ATG101, and RB1CC1/FIP200. Under starvation conditions, the ULK1 complex interacts with several protein and lipid kinases and phosphatases, implying the formation of a signalosome. Interestingly, several selective autophagy receptors also interact with ULK1, indicating the activation of selective autophagy pathways by nutrient starvation. One effector of the ULK1 complex is the HSC/HSP70 co-chaperone BAG2, which regulates the subcellular localization of the VPS34 lipid kinase complex member AMBRA1. Depending on the nutritional status, BAG2 has opposing roles. In growth conditions, the unphosphorylated form of BAG2 sequesters AMBRA1, attenuating autophagy induction. In starvation conditions, ULK1 phosphorylates BAG2 on Ser31, which supports the recruitment of AMBRA1 to the ER membrane, positively affecting autophagy.

FIP200 Phosphorylation Regulates Late Steps in Mitophagy

Mitophagy is a specific type of autophagy responsible for the selective elimination of dysfunctional or superfluous mitochondria, ensuring the maintenance of mitochondrial quality control. The initiation of mitophagy is coordinated by the ULK1 kinase complex, which engages mitophagy receptors via its FIP200 subunit. Whether FIP200 performs additional functions in the subsequent later phases of mitophagy beyond this initial step and how its regulation occurs, remains unclear. Our findings reveal that multiple phosphorylation events on FIP200 differentially control the early and late stages of mitophagy. Furthermore, these phosphorylation events influence FIP200′s interaction with ATG16L1. In summary, our results highlight the necessity for precise and dynamic regulation of FIP200, underscoring its importance in the progression of mitophagy.

Autophagy dysregulation via the USP20-ULK1 axis in the HERC2-related neurodevelopmental disorder

Sequence variants in the HERC2 gene are associated with a significant reduction in HERC2 protein levels and cause a neurodevelopmental disorder known as the HERC2-related disorder, which shares clinical features with Angelman syndrome, including global developmental delay, intellectual disability, autism, and movement disorders. Remarkably, the HERC2 gene is commonly deleted in individuals with Angelman syndrome, suggesting a potential contribution of HERC2 to the pathophysiology of this disease. Given the known critical role of autophagy in brain development and its implication in neurodevelopmental diseases, we undertook different experimental approaches to monitor autophagy in fibroblasts derived from individuals affected by the HERC2-related disorder. Our findings reveal alterations in the levels of the autophagy-related protein LC3. Furthermore, experiments with lysosomal inhibitors provide confirmation of an upregulation of the autophagy pathway in these patient-derived cells. Mechanistically, we corroborate an interaction between HERC2 and the deubiquitylating enzyme USP20; and demonstrate that HERC2 deficiency leads to increased USP20 protein levels. Notably, USP20 upregulation correlates with enhanced stability of the autophagy initiating kinase ULK1, highlighting the role of HERC2 as an autophagy regulator factor through the USP20-ULK1 axis. Moreover, we show that p38 acts as a modulator of this pathway, since p38 activation disrupts HERC2-USP20 interaction, leading to increased USP20 and LC3-II protein levels. Together, these findings uncover a previously unknown role for HERC2 in autophagy regulation and provide insights into the pathomolecular mechanisms underlying the HERC2-related disorder and Angelman syndrome.

Abstract 4322: Inhibition of ULK and KRASG12C control tumor growth in preclinical models of lung cancer

Abstract Approximately 30% of lung cancers are driven by mutationally activated KRAS. The recent FDA approval of sotorasib, a direct pharmacological inhibitor of KRASG12C, marks a critical milestone in the treatment of this important subset of lung cancer. However, a major obstacle in treating lung cancer is resistance to current therapeutic treatments. In response to cellular stresses such as RAS pathway-targeted therapeutics, RAS-mutated cancer cells have been demonstrated to increase autophagy, an intracellular recycling pathway. However, the mechanism(s) underlying treatment-induced autophagy is not well understood. Here, we demonstrate that KRASG12C-driven lung cancer cells increase autophagy in response to treatment with sotorasib. Currently, the only therapeutics in clinical use to inhibit autophagy are lysosomal inhibitors (hydroxy)chloroquine. High concentrations of these compounds are needed to achieve modest inhibition of autophagy, suggesting that the potency of these compounds may limit clinical responses. ULK kinases are serine/threonine kinases that are necessary for the initiation of autophagy. DCC-3116 is a potent inhibitor of the ULK1/2 protein kinases, master regulators of autophagy. Treatment with sotorasib and DCC-3116 led to superior anti-tumor effects in preclinical models of lung cancer. Additionally, about 30% of lung cancer patients with KRAS mutations also have deletions or inactivating mutations in LKB1, a protein involved in the regulation of nutrient sensing and autophagy. In other KRAS-driven cancers, the LKB1-AMPK-ULK1 signaling axis is a proposed mechanism as to how autophagic flux increases following KRAS pathway inhibition. However, our preliminary data demonstrates that LKB1 is dispensable for sotorasib-induced autophagy, suggesting the mechanism(s) of autophagy induction in KRASG12C-driven lung cancer may be different from those detected in other KRAS mutated cancers. Consequently, we have generated genetically engineered mouse models (GEMMs) of KRASG12C-driven lung cancer in which LKB1 is silenced to further investigate the role of LKB1 in the autophagy response of KRASG12C-driven lung cancers to pathway-targeted blockade of oncogenic KRAS signaling. We have treated KRASG12C-driven GEMMs with sotorasib and/or DCC-3116 to test the sensitivity of lung tumors to these treatment options. Citation Format: Phaedra Ghazi, Kayla O'Toole, Sanjana Boggaram, Michael Scherzer, Madhumita Bogdan, Bryan Smith, Dan Flynn, Conan Kinsey, Martin McMahon. Inhibition of ULK and KRASG12C control tumor growth in preclinical models of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4322.

Abstract 4321: The oncogenic DMTF1β splice variant promotes autophagy-dependent cancer cell motility

Abstract The oncogenic isoform DMTF1β, which is generated by alternative splicing of the DMTF1 tumor suppressor gene is associated with increased cell proliferation and tumorigenesis. Despite these correlative findings little is known about the DMTF1β mechanism of action. In order to decipher the oncogenic DMTF1β signaling pathway, we performed RNA sequencing of DMTF1β depleted prostate cancer cells and analyzed the DMTF1β-specific protein interactome in these cells. Interestingly, autophagy-related pathways belonged to the most downregulated networks in DMTF1β knockdown cells. Moreover, we found that predominantly DMTF1β, but not a protein lacking the β-specific domain interacts with the key autophagy ULK1-ATG13 protein complex. These interactions were confirmed by co-immunoprecipitation and proximity ligation assays. Importantly, DMTF1β binding to ULK1 stabilized this key autophagy protein. Accordingly, knocking down DMTF1β significantly lowered autophagic flux in breast and prostate cancer cells as assessed by LC3-dependent and independent autophagy assays. Since published data show an important role for autophagy in migration and invasion of cancer cells, we next investigated whether DMTF1β is regulating these cellular processes. Indeed, DMTF1β depleted breast and prostate cancer cells displayed significantly decreased wound closure, trans-well invasion and metastatic foci formation in a zebra fish xenograft model. Importantly, pharmacological inhibition of ULK1 kinase activity or knocking down ULK1 reversed DMTF1β-mediated cancer cell migration. In agreement, decreased cell motility in DMTF1β knockdown cancer cells could be rescued by ectopic expression of the VPS34 complex member Beclin-1. In summary, we identified a novel DMTF1β-ULK1 signaling pathway that activates aberrant cancer cell migration most probably by increasing basal cell autophagy. Targeting autophagy using ULK1 inhibitors may represent a promising strategy to lower metastatic activity of tumor cells. Citation Format: Mario P. Tschan, Jun Xu, Anna M. Bill. The oncogenic DMTF1β splice variant promotes autophagy-dependent cancer cell motility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4321.

Uncoordinated 51-like kinase 1a/b and 2 in fish Megalobrama amblycephala: Molecular cloning, functional characterization, and their potential roles in glucose metabolism

Uncoordinated (Unc) 51-like kinase (ulk1) and ulk2 are closely involved in autophagy activation, but little is known about their roles in regulating glucose homeostasis. In this study, the genes of ulk1a, ulk1b and ulk2 were cloned and characterized in fish Megalobrama amblycephala. All the three genes shared the approximate N-terminal kinase domain and the C-terminal Atg1-like_tMIT domain structure, while the amino acid sequence identity of them are different between M. amblycephala and other vertebrates. Their transcripts were widely observed in various tissues (brain, muscle, gill, heart, spleen, eye, liver, intestine, abdominal adipose and kidney), but differed in tissue expression patterns. During the glucose tolerance test and the insulin tolerance test, the up-regulated transcriptions of ulk1a, ulk1b and ulk2 were all found despite some differences in the temporal patterns. At the same time, the activities of glycolytic enzymes like hexokinase and phosphofructokinase both showed parallel increases. Furthermore, the feeding of a high-carbohydrate diet decreased the transcriptions of ulk1a, ulk1b and ulk2. Collectively, this study demonstrated that ulk1a, ulk1b and ulk2 in M. amblycephala had similar molecular characterizations, but with different conservation and tissue expression patterns. In addition, ulk1/2 might play important roles in maintaining the glucose homeostasis in fish through regulating the glycolytic pathway.

Autophagy Determines Distinct Cell Fates in Human Amnion and Chorion Cells.

Human fetal membranes (amniochorion) that line the intrauterine cavity consist of two distinct cell layers; single-layer amnion epithelial cells (AEC) and multilayer chorion trophoblast cells (CTC). These layers are connected through a collagen-rich extracellular matrix. Cellular remodeling helps support membrane growth and integrity during gestation and helps to maintain pregnancy. Preterm prelabor rupture of the human amniochorionic (fetal) membrane (pPROM) is antecedent to 40% of all spontaneous preterm birth. Oxidative stress (OS) induced activation of the p38 MAPK due to various maternal risk exposures and the amniochorion cells' senescence are reported pathological features of pPROM. Our transcriptomics analysis implicated dysregulated autophagy and epithelial-mesenchymal transition (EMT) in fetal membranes from pPROM. The molecular interplay between OS-induced p38 MAPK activation, autophagy, and EMT was investigated in AECs and CTCs to better understand the involvement of autophagy and EMT. We report the differential impact of OS on the autophagic machinery in AECs and CTCs, resulting in distinct cell fates. In AECs, OS-induced p38 MAPK activation causes autophagosome accumulation and reduced autophagic flux mediated by decreased ULK1 activity and kinase activity, leading to senescence. In CTCs, induction of autophagy has a limited effect; however, inhibition of autophagy led to SQSTM1-mediated EMT of trophoblast cells. Autophagy, EMT, and senescence were associated with proinflammatory changes. Thus, AECs and CTCs respond differently to OS via differential autophagy response, partly mediated via p38 MAPK. Besides senescence, OS-induced autophagy dysregulation in amniochorion cells may play a mechanistic role in pPROM pathophysiology.

Identification of a 5-Gene Cuproptosis Signature Predicting the Prognosis for Colon Adenocarcinoma Based on WGCNA.

Background: Colorectal cancer is a highly aggressive malignant tumor that primarily affects the digestive system. It is frequently diagnosed at an advanced stage. Cuproptosis is a copper-dependent form cell death mechanism, distinct from all other known pathways underlying cell death, tumor progression, prognosis, and immune response. Although the role of cuproptosis in colorectal cancer has been investigated over time, there is still an urgent need to explore new methods and insights to understand its potential function. Methods: The Gene Expression Omnibus and The Cancer Genome Atlas gene expression data were systematically explored to investigate the role of cuproptosis in colon adenocarcinoma. The weighted gene coexpression network analysis was used to construct a gene coexpression network and identify the critical module and cuproptosis-related genes correlated with colon adenocarcinoma prognosis. A cuproptosis-related genes prognostic signature for colon adenocarcinoma was identified and validated. To validate the identified gene signature, quantitative reverse transcription-polymerase chain reaction was performed. Cell proliferation assays were analyzed by CCK8 and cell cycle detection. In addition, reactive oxygen species assay was also analyzed. Results: Five hub cuproptosis-related genes (Dihydrolipoamide S-acetyltransferase, Cyclin-dependent kinase inhibitor 2A, ATOX1, VEGFA, and ULK1) were screened and a prognostic risk model for predicting overall survival was established based on these genes. The model was successfully tested in the validation cohort and the GEPIA database. Colon adenocarcinoma patients were categorized into high-risk and low-risk groups based on risk scores. The study revealed that patients with higher risk scores were more likely to have a poor prognosis. Moreover, Dihydrolipoamide S-acetyltransferase was a tumor suppressor gene that can induce cell death and affected the redox reactions in the colon cancer cell line. Conclusions: These findings suggest that the newly identified 5-gene signature may serve as a more reliable prognostic factor than clinical factors such as age and stage of disease. These findings offer a theoretical foundation for further investigation into potential cuproptosis-related biomarkers for predicting colon adenocarcinoma prognosis in the future.

Molecular mechanisms of the effect of standardized placental hydrolysate peptides on mitochondria functioning

Human placenta hydrolysates (HPH), the study of which was initiated by the scientific school of Vladimir P. Filatov, are currently being investigated using modern proteomic technologies. HPH is a promising tool for maintaining the function of mitochondria and regenerating tissues and organs with a high content of mitochondria (liver, heart muscle, skeletal muscles, etc.). The molecular mechanisms of action of HPH are practically not studied. Identification of mitochondrial support mitochondrial function-supporting peptides in HPH (Laennec, produced by Japan Bioproducts). Data on the chemical structure of the peptides were collected through a mass spectrometric experiment. Then, to establish the amino acid sequences of the peptides, de novo peptide sequencing algorithms based on the mathematical theory of topological and metric analysis of chemographs were applied. Bioinformatic analysis of the peptide composition of HPH was carried out using the integral protein annotation method. The biological functions of 41 peptides in the composition of HPH have been identified and described. Among the target proteins, the activity of which is regulated by the identified peptides and significantly affects the function of mitochondria, are caspases (CASP1, CASP3, CASP4) and other proteins regulating apoptosis (BCL2, CANPL1, PPARA), MAP kinases (MAPK1, MAPK3, MAPK4, MAPK8, MAPK9 , MAPK10, MAPK14), AKT1/GSK3B/MTOR cascade kinases, and a number of other target proteins (ADGRG6 receptor, inhibitor of NF-êB kinase IKKE, pyruvate dehydrogenase 2/3/4, SIRT1 sirtuin deacetylase, ULK1 kinase). HPH peptides have been identified that promote inhibition of mitochondrial pore formation, apoptosis, and excessive mitochondrial autophagy under conditions of oxidative/toxic stress, chronic inflammation, and/or hyperinsulinemia.

Ulk4 promotes Shh signaling by regulating Stk36 ciliary localization and Gli2 phosphorylation

The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis through the Gli family of transcription factors. Gli is thought to be activated at the tip of primary cilium, but the underlying mechanism has remained poorly understood. Here, we show that Unc-51-like kinase 4 (Ulk4), a pseudokinase and a member of the Ulk kinase family, acts in conjunction with another Ulk family member Stk36 to promote Gli2 phosphorylation and Hh pathway activation. Ulk4 interacts with Stk36 through its N-terminal region containing the pseudokinase domain and with Gli2 via its regulatory domain to bridge the kinase and substrate. Although dispensable for Hh-induced Stk36 kinase activation, Ulk4 is essential for Stk36 ciliary tip localization, Gli2 phosphorylation, and activation. In response to Hh, both Ulk4 and Stk36 colocalize with Gli2 at ciliary tip, and Ulk4 and Stk36 depend on each other for their ciliary tip accumulation. We further show that ciliary localization of Ulk4 depends on Stk36 kinase activity and phosphorylation of Ulk4 on Thr1023, and that ciliary tip accumulation of Ulk4 is essential for its function in the Hh pathway. Taken together, our results suggest that Ulk4 regulates Hh signaling by promoting Stk36-mediated Gli2 phosphorylation and activation at ciliary tip.

Ulk4 promotes Shh signaling by regulating Stk36 ciliary localization and Gli2 phosphorylation.

The Hedgehog (Hh) family of secreted proteins governs embryonic development and adult tissue homeostasis through the Gli family of transcription factors. Gli is thought to be activated at the tip of primary cilium, but the underlying mechanism has remained poorly understood. Here, we show that Unc-51-like kinase 4 (Ulk4), a pseudokinase and a member of the Ulk kinase family, acts in conjunction with another Ulk family member Stk36 to promote Gli2 phosphorylation and Hh pathway activation. Ulk4 interacts with Stk36 through its N-terminal region containing the pseudokinase domain and with Gli2 via its regulatory domain to bridge the kinase and substrate. Although dispensable for Hh-induced Stk36 kinase activation, Ulk4 is essential for Stk36 ciliary tip localization, Gli2 phosphorylation, and activation. In response to Hh, both Ulk4 and Stk36 colocalize with Gli2 at ciliary tip, and Ulk4 and Stk36 depend on each other for their ciliary tip accumulation. We further show that ciliary localization of Ulk4 depends on Stk36 kinase activity and phosphorylation of Ulk4 on Thr1023, and that ciliary tip accumulation of Ulk4 is essential for its function in the Hh pathway. Taken together, our results suggest that Ulk4 regulates Hh signaling by promoting Stk36-mediated Gli2 phosphorylation and activation at ciliary tip.

Decrease in α-Globin and Increase in the Autophagy-Activating Kinase ULK1 mRNA in Erythroid Precursors from β-Thalassemia Patients Treated with Sirolimus.

The β-thalassemias are hereditary monogenic diseases characterized by a low or absent production of adult hemoglobin and excess in the content of α-globin. This excess is cytotoxic for the erythroid cells and responsible for the β-thalassemia-associated ineffective erythropoiesis. Therefore, the decrease in excess α-globin is a relevant clinical effect for these patients and can be realized through the induction of fetal hemoglobin, autophagy, or both. The in vivo effects of sirolimus (rapamycin) and analogs on the induction of fetal hemoglobin (HbF) are of key importance for therapeutic protocols in a variety of hemoglobinopathies, including β-thalassemias. In this research communication, we report data showing that a decrease in autophagy-associated p62 protein, increased expression of ULK-1, and reduction in excess α-globin are occurring in erythroid precursors (ErPCs) stimulated in vitro with low dosages of sirolimus. In addition, increased ULK-1 mRNA content and a decrease in α-globin content were found in ErPCs isolated from β-thalassemia patients recruited for the NCT03877809 clinical trial and treated with 0.5-2 mg/day sirolimus. Our data support the concept that autophagy, ULK1 expression, and α-globin chain reduction should be considered important endpoints in sirolimus-based clinical trials for β-thalassemias.

Three-dimensional growth sensitizes breast cancer cells to treatment with ferroptosis-promoting drugs

Drugs causing ferroptosis, iron-mediated cell death, represent promising tools for cancer treatment. While exploring the effect of these drugs on breast cancer (BC), we found that a ferroptosis-inducing drug erastin dramatically inhibits tumorigenicity of human BC cells in mice but when used at a concentration known to effectively kill other cell types only modestly reduces such growth in 2D monolayer culture. BCs grow in vivo as 3D masses, and we found that ferroptosis inducers erastin and sulfasalazine inhibit growth of multiple human BC cell lines in 3D culture significantly stronger than in 2D culture. To understand the mechanism of this differential effect, we found that ferroptosis inducers upregulate mRNAs encoding multiple direct and indirect autophagy stimulators, such as ATG16L2, ATG9A, ATG4D, GABARAP, SQSTM/p62, SEC23A and BAX, in tumor cells growing in 2D but not in 3D culture. Furthermore, these drugs promoted autophagy of tumor cells growing in a 2D but not in a 3D manner. We observed that pharmacological inhibition of autophagy-stimulating protein kinase ULK1 or RNA interference-mediated knockdown of autophagy mediator ATG12 significantly sensitized tumor cells to erastin treatment in 2D culture. We also found that ferroptosis-promoting treatments upregulate heme oxygenase-1 (HO-1) in BC cells. HO-1 increases cellular free iron pool and can potentially promote ferroptosis. Indeed, we observed that HO-1 knockdown by RNA interference reversed the effect of ferroptosis inducers on BC cell 3D growth. Hence, the effect of these drugs on such growth is mediated by HO-1. In summary, autophagy triggered by ferroptosis-promoting drugs reduces their ability to kill BC growing in a 2D manner. This protection mechanism is inhibited in BC cells growing as a 3D mass, and ferroptosis-promoting drugs kill such cells more effectively. Moreover, this death is mediated by HO-1. Thus, ferroptosis induction represents a promising strategy for blocking 3D BC growth.

ULK phosphorylation of STX17 controls autophagosome maturation via FLNA.

Autophagy is a conserved and tightly regulated intracellular quality control pathway. ULK is a key kinase in autophagy initiation, but whether ULK kinase activity also participates in the late stages of autophagy remains unknown. Here, we found that the autophagosomal SNARE protein, STX17, is phosphorylated by ULK at residue S289, beyond which it localizes specifically to autophagosomes. Inhibition of STX17 phosphorylation prevents such autophagosome localization. FLNA was then identified as a linker between ATG8 family proteins (ATG8s) and STX17 with essential involvement in STX17 recruitment to autophagosomes. Phosphorylation of STX17 S289 promotes its interaction with FLNA, activating its recruitment to autophagosomes and facilitating autophagosome-lysosome fusion. Disease-causative mutations around the ATG8s- and STX17-binding regions of FLNA disrupt its interactions with ATG8s and STX17, inhibiting STX17 recruitment and autophagosome-lysosome fusion. Cumulatively, our study reveals an unexpected role of ULK in autophagosome maturation, uncovers its regulatory mechanism in STX17 recruitment, and highlights a potential association between autophagy and FLNA.

Acrylamide induces human chondrocyte cell death by initiating autophagy‑dependent ferroptosis.

Acrylamide (ACR) is formed during heat treatment of foodstuffs and ACR may serve as a probable malignant neoplastic disease agent in all organs and tissues of the human body. However, it is unknown if ACR is associated with ankylosing spondylitis (AS) pathogenesis. Cell viability and proliferation were determined using CCK-8 assay and EdU staining. Flow cytometry was used to determine cell death and cell cycle arrest. Intracellular lipid reactive oxygen species, Fe2+ and mitochondrial membrane potential (MMP) were analyzed using a C11-BODIPY581/591 fluorescent probe, FerroOrange staining and a JC-1 MMP Assay kit, respectively. The present study showed that ACR decreased chondrocyte cell viability in a dose-dependent manner and that ACR significantly promoted chondrocyte senescence. ACR also elevated the expression of cell cycle arrest-associated proteins, including p53, cyclin-dependent kinase inhibitor 1 and cyclin-dependent kinase inhibitor protein, in human chondrocytes. Similarly, DNA damage was also enhanced following ACR treatment in chondrocytes. In addition, the ferroptosis-specific inhibitor ferrostatin-1 (Fer-1) and the autophagy inhibitor 3-methyladenine abolished ACR-induced cell death in chondrocytes. ACR was shown to activate autophagic flux and induce mitochondrial dysfunction by increasing the MMP. Western blot analysis of ferroptosis-related proteins demonstrated that ACR decreased the expression of glutathione peroxidase 4, solute carrier family 7 member 11, transferrin receptor protein 1 and ferritin heavy chain 1 in chondrocytes whereas Fer-1 abolished these effects. ACR treatment significantly elevated the phosphorylation levels of AMP-activated protein kinase (AMPK) and serine/threonine-protein kinase ULK1 in human chondrocytes. Notably, the effect of ACR was diminished by knockdown of AMPK, as evidenced by reduced lipid reactive oxygen species accumulation and Fe2+ levels. Hence, ACR inhibited cell proliferation and contributed to cell death by inducing autophagy-dependent ferroptosis while promoting autophagy by activating AMPK-ULK1-mTOR signaling in human chondrocytes. It was hypothesized that the presence of ACR in foodstuffs may increase the risk of AS and that decreasing ACR in food products is of importance.

Mycobacterium tuberculosis protein MoxR1 enhances virulence by inhibiting host cell death pathways and disrupting cellular bioenergetics

ABSTRACT Mycobacterium tuberculosis (M. tb) utilizes the multifunctionality of its protein factors to deceive the host. The unabated global incidence and prevalence of tuberculosis (TB) and the emergence of multidrug-resistant strains warrant the discovery of novel drug targets that can be exploited to manage TB. This study reports the role of M. tb AAA+ family protein MoxR1 in regulating host-pathogen interaction and immune system functions. We report that MoxR1 binds to TLR4 in macrophage cells and further reveal how this signal the release of proinflammatory cytokines. We show that MoxR1 activates the PI3K-AKT-MTOR signalling cascade by inhibiting the autophagy-regulating kinase ULK1 by potentiating its phosphorylation at serine 757, leading to its suppression. Using autophagy-activating and repressing agents such as rapamycin and bafilomycin A1 suggested that MoxR1 inhibits autophagy flux by inhibiting autophagy initiation. MoxR1 also inhibits apoptosis by suppressing the expression of MAPK JNK1/2 and cFOS, which play critical roles in apoptosis induction. Intriguingly, MoxR1 also induced robust disruption of cellular bioenergetics by metabolic reprogramming to rewire the citric acid cycle intermediates, as evidenced by the lower levels of citric acid and electron transport chain enzymes (ETC) to dampen host defence. These results point to a multifunctional role of M. tb MoxR1 in dampening host defences by inhibiting autophagy, apoptosis, and inducing metabolic reprogramming. These mechanistic insights can be utilized to devise strategies to combat TB and better understand survival tactics by intracellular pathogens.

Targeting Unc51-like Autophagy Activating Kinase 1 (ULK1) Overcomes Adaptive Drug Resistance in Acute Myelogenous Leukemia.

ULK1 drives a cell-intrinsic adaptive resistance in AML and targeting ULK1-mediated autophagy can synergize with existing and emerging AML therapies to overcome drug-resistance and induce apoptosis.

ULK1-mediated phosphorylation regulates the conserved role of YKT6 in autophagy.

Autophagy is a catabolic process during which cytosolic material is enwrapped in a newly formed double-membrane structure called the autophagosome, and subsequently targeted for degradation in the lytic compartment of the cell. The fusion of autophagosomes with the lytic compartment is a tightly regulated step and involves membrane-bound SNARE proteins. These play a crucial role as they promote lipid mixing and fusion of the opposing membranes. Among the SNARE proteins implicated in autophagy, the essential SNARE protein YKT6 is the only SNARE protein that is evolutionarily conserved from yeast to humans. Here, we show that alterations in YKT6 function, in both mammalian cells and nematodes, produce early and late autophagy defects that result in reduced survival. Moreover, mammalian autophagosomal YKT6 is phospho-regulated by the ULK1 kinase, preventing premature bundling with the lysosomal SNARE proteins and thereby inhibiting autophagosome-lysosome fusion. Together, our findings reveal that timely regulation of the YKT6 phosphorylation status is crucial throughout autophagy progression and cell survival.

Deciphering functional roles and interplay between Beclin1 and Beclin2 in autophagosome formation and mitophagy.

The degradation of macromolecules and organelles by the process of autophagy is critical for cellular homeostasis and is often compromised during aging and disease. Beclin1 and Beclin2 are implicated in autophagy induction, and these homologs share a high degree of amino acid sequence similarity but have divergent N-terminal regions. Here, we investigated the functions of the Beclin homologs in regulating autophagy and mitophagy, a specialized form of autophagy that targets mitochondria. Both Beclin homologs contributed to autophagosome formation, but a mechanism of autophagosome formation independent of either Beclin homolog occurred in response to starvation or mitochondrial damage. Mitophagy was compromised only in Beclin1-deficient HeLa cells and mouse embryonic fibroblasts because of defective autophagosomal engulfment of mitochondria, and the function of Beclin1 in mitophagy required the phosphorylation of the conserved Ser15 residue by the kinase Ulk1. Mitochondria-ER-associated membranes (MAMs) are important sites of autophagosome formation during mitophagy, and Beclin1, but not Beclin2 or a Beclin1 mutant that could not be phosphorylated at Ser15, localized to MAMs during mitophagy. Our findings establish a regulatory role for Beclin1 in selective mitophagy by initiating autophagosome formation adjacent to mitochondria, a function facilitated by Ulk1-mediated phosphorylation of Ser15 in its distinct N-terminal region.